20S PROTEASOME ACTIVATION AS AN INNOVATIVE THERAPEUTIC STRATEGY
                                      By
                       Taylor Joseph Martinez-Fiolek
                             A DISSERTATION
                                  Submitted to
                          Michigan State University
                  in partial fulfillment of the requirements
                               for the degree of
                     Chemistry – Doctor of Philosophy
                Pharmacology and Toxicology – Dual Major
                                     2023


                                          ABSTRACT
        Accumulation and aggregation of intrinsically disordered proteins (IDPs), such as α-
synuclein, amyloid β, and tau, is associated with the pathogenesis of several neurodegenerative
diseases, including Parkinson’s disease and Alzheimer’s disease. The 20S proteasome is the
primary protease charged with regulating cellular levels of IDPs, but as humans age these proteins
can become dysregulated resulting in their accumulation and aggregation seen in
neurodegenerative diseases. Although the pathogenesis of these neurodegenerative diseases is still
under intense investigation, it has been shown that the oligomeric forms of IDPs, including α-
synuclein and amyloid β, are toxic to neurons and can impair proteasome function. This leads to
additional accumulation of the IDPs, further promoting disease progression. Additionally, IDPs
released by degenerating neurons activate the native immune cells of the brain, microglia, and
induce their activation. These activated microglia release pro-inflammatory signaling molecules,
which when coupled with continued IDP accumulation and release results in chronic
neuroinflammation, contributing to further neuron degeneration.
        The Tepe Lab aims to develop small molecule activators of the 20S proteasome that
enhance its ability to degrade IDPs, thus assisting in the prevention of their further accumulation
and aggregation. We propose that these small molecule 20S proteasome activators represent a
novel therapeutic method by which we may impede neurodegenerative disease progression. Here,
I report the identification of novel small molecule 20S proteasome activator scaffolds that
selectively enhance 20S proteasome activity. These activators enhance 20S-mediated degradation
of IDPs that are implicated in neurodegenerative disease development. The identification of these
novel activator scaffolds will enhance the chance of success while developing this novel
therapeutic strategy and permit development of additional analogues with promising activities and


drug-like properties.
        With these activators in hand, several novel methods were developed that demonstrate the
potential of small molecule 20S proteasome activation as an innovative therapeutic strategy for
combating neurodegenerative diseases. It was demonstrated for the first time that small molecule
activators can protect against inhibition of the 20S proteasome by IDP oligomers associated with
neurodegenerative disease pathogenesis and that these oligomers can be reduced through 20S
proteasome activation in vitro. These results suggest that small molecule 20S proteasome
activation has the potential to assist in re-establishing proteostasis in diseased neurons.
Additionally, it was found that small molecule 20S proteasome activators can counteract the
accumulation of an overexpressed familial Parkinson’s disease related IDP, A53T α-synuclein, in
cells. This demonstrated that this method shows promise for translation into cellular systems. As
such, additional cellular models were conceived, and their development initiated to generate more
disease relevant models for further evaluating this method. Finally, small molecule 20S
proteasome activators were found to reduce IDP-induced release of the pro-inflammatory cytokine
TNF-α by microglia. Thus, this work begins to illuminate the great potential of small molecule
20S proteasome activators to counteract multiple IDP-driven aspects of neurodegenerative disease
pathogenesis.


                                    ACKNOWLEDGEMENTS
         I am fortunate to have many people in my life who have supported me throughout my
journey to get here, to the end of my PhD.
         First, I would like to thank my advisor, Dr. Jetze Tepe. Your support, guidance and
mentorship have helped to make me into the scientist that I am today. Thank you for encouraging
me to make my project my own, explore my own ideas and to take on a leadership role in the lab.
These things have allowed me to grow and mature as a scientist beyond my expectations.
         To my committee, Dr. Huang, Dr. Draths, and Dr. Hong. Thank you for all your feedback
and willingness to help me through this process. Whether it was to borrow equipment, problem
solve, or simply to give me a drink to calm me down after my seminar, you were each always
willing to help. Thank you to Dr. Dexheimer for your help with my high-throughput assays. Thank
you to Dr. Sortwell and her lab for collaborating with me on the primary neuron studies. Thank
you to Dr. Dorrance, Dr. Neubig, Dr. Liby and many others in the Pharmacology and Toxicology
department. I learned a great deal in classes, workshops, and gatherings while in the IPSTP
program and PharmTox department. I am so thankful that I was able to be a part of both during
my time here. Thank you to Dr. Blanchard for helping me with fellowship applications. Thank you
to all the people who make the Chemistry and PharmTox departments run and to make our lives
easier in a multitude of ways, Anna, Mary, Dawn, Eric, Bob, Tiphani, Jake, and many others. I
appreciate all that you do for these departments and for us.
         Thank you to Dr. Adam Mosey and his lab at Lake Superior State University for
collaborating with me on the Dihydroquinazoline project. Our collaboration was very enjoyable
and rewarding.
         Thank you to my previous mentors, Dr. Benjamin Swarts and Dr. Choon Lee. Your
                                                 iv


mentorship and the things I learned while working with you were instrumental to my success.
        Thank you to all my lab mates, both current and former. I have had the privilege of
watching our lab grow from 5 to 15 people and during that time it evolved into a close-knit family.
While some of the faces and personalities have changed with time, the feelings of support and
togetherness have remained. I hope that carries on for all of you because it has been invaluable to
me. To Evert and Corey, thank you for your mentorship and guidance. To Grace, we finally made
it! Thank you for always being someone with whom I could share my struggles knowing that you
understand and were often experiencing similar ones. To Katarina, Grace, Allison, Sophie,
Charles, and Konika, thank you all for becoming great friends and for encouraging me to come
out and have some fun. It was always a great time! To the biology subgroup, I am proud of what
our group has become and how we have grown together as a team. I know you all will continue to
do so. To all those mentioned above, Christi, Ayoob, Shafaat, Dare, Daniel, Evan, Kyra, Bahar,
Sydney, Shannon, and Miracle, thank you all for your support, questions, and friendship.
        To my friends, I cannot name you all here, but I appreciate all of you and thank you all for
your support. To Ankush and Emmanuel, thank you for being there to chat and go to lunch on
Fridays. It has been something to look forward to throughout our years together here. To Nate and
Kaleb, thank you for always being there to get my mind away from work and have some fun.
Kaleb, thank you for helping around the house while I’ve been busy and for doing some spelling
and grammar checking for me. To Herbert, thank you for being a source of advice, both for science
and life, and for being a friend who is ready to go out for a drink and food whenever life permits.
        To my family, I cannot express in words how thankful I am for each of you and your
undying support and encouragement. I would not have made it here without you. Thank you for
always believing in me and helping me to follow whatever path I choose in life. Thank you to my
                                                  v


pets back at home and here with me, who have served as my emotional support animals. We have
a long-standing joke in the Tepe lab about animal pictures in our slides and in zoom meetings, but
they truly have been an important part of my support system and family. To my loved ones who
are no longer with us. Thank you for helping to shape me into the man I am today. Finally, thank
you to my loving wife, Mariah. I could not have done this without you at my side. I am forever
grateful for you and your unyielding support, love, and encouragement through what has been one
of the most difficult times in my life. We have been through a lot during my time here, but we
have made it through together and will continue to do so. I love you all and I hope I have made
you proud.
                                                vi


                                                  TABLE OF CONTENTS
LIST OF ABBREVIATIONS ...................................................................................................... viii
CHAPTER ONE Proteostasis, the Proteasome and a Novel Therapeutic Strategy for
Neurodegenerative Diseases ............................................................................................................1
       1.1 Introduction ....................................................................................................................2
       1.2 The ubiquitin proteasome system ..................................................................................5
       1.3 Proteostasis disruption contributes to aging and neurodegenerative diseases .............14
       1.4 The 20S proteasome as a novel therapeutic target .......................................................24
       1.5 Conclusions ..................................................................................................................30
       REFERENCES ..................................................................................................................32
CHAPTER TWO Identification and Structure Activity Relationship of Dihydroquinazoline as a
Novel 20S Proteasome Activator Scaffold ....................................................................................55
       2.1 Introduction ..................................................................................................................56
       2.2 Results and Discussion ................................................................................................61
       2.3 Conclusions ..................................................................................................................72
       REFERENCES ..................................................................................................................74
CHAPTER THREE Repurposed Neuroleptic Agents as Novel 20S Proteasome Activator
Scaffolds ........................................................................................................................................80
       3.1 Introduction ..................................................................................................................81
       3.2 Results and Discussion ................................................................................................86
       3.3 Conclusions ................................................................................................................108
       3.4 Experimental ..............................................................................................................110
       REFERENCES ................................................................................................................123
       APPENDIX ......................................................................................................................130
CHAPTER FOUR Development of Neurodegenerative Disease Model Systems for Evaluating
20S Proteasome Activation as an Innovative Therapeutic Strategy ............................................142
       4.1 Introduction ................................................................................................................143
       4.2 Results and Discussion ..............................................................................................147
       4.3 Conclusions ................................................................................................................171
       4.4 Experimental ..............................................................................................................173
       REFERENCES ................................................................................................................176
       APPENDIX ......................................................................................................................184
CHAPTER FIVE Conclusions and Future Directions .................................................................187
       5.1 Conclusions and Future Directions ............................................................................188
       REFERENCES ................................................................................................................191
CHAPTER SIX Materials and Methods ................................................................................................... 195
       6.1 Materials ....................................................................................................................196
       6.2 Methods......................................................................................................................198
                                                                      vii


                        LIST OF ABBREVIATIONS
3D          3-dimensional
ACN         Acetonitrile
AD          Alzheimer’s disease
AFM         Atomic force microscopy
ALS         Amyotrophic lateral sclerosis
AMC         7-amino-4-methylcoumarin
APP         Amyloid precursor protein
Atm         Atmospheric pressure
ATP         Adenosine triphosphate
BBB         Blood-brain barrier
Boc-LRR-AMC Tert-butoxyl-leucyl-arginyl-arginyl-7-amino-4-methylcoumarin
BTZ         Bortezomib
°C          Degrees Celsius
Casp-L      Caspase-like
CD          Circular dichroism
CP          Core particle
CT-L        Chymotrypsin-like
DCM         Dichloromethane
DMF         Dimethylformamide
DMSO        Dimethyl sulfoxide
EC200       200% effective concentration
Equiv.      Equivalents
                                    viii


ESI      Electrospray ionization
FBS      Fetal bovine serum
FTIR     Fourier transform infrared
HbYX     Hydrophobic-tyrosine-any amino acid
HD       Huntington’s disease
HEK-293T Human embryonic kidney 293 cells containing SV40 T-antigen
HFIP     Hexafluoroisopropanol
HRMS     High-resolution mass spectrometry
HT-22    Immortalized mouse hippocampal neurons
IDP      Intrinsically disordered protein
IDR      Intrinsically disordered region
IMG      Immortalized mouse microglial cells
LB       Lysogeny broth
MHC      Major histocompatibility complex
mp       Melting point
MS       Mass spectrometry
NMR      Nuclear magnetic resonance
PA200    Proteasome activator 200 kDa
PAGE     Poly acrylamide gel electrophoresis
PBS      Phosphate-buffered saline
PD       Parkinson’s disease
Ph       Phenyl-
RP       Regulatory particle
                                    ix


Rpn          Non-ATPase regulatory particle
Rpt          ATPase regulatory particle
SDS          Sodium dodecyl sulfate
SNpc         Substantia nigra pars compacta
Suc-LLVY-AMC Succinyl-leucyl-leucyl-valyl-tyrosyl-7-amido-4-methylcoumarin
TBS          Tris-buffered saline
TEA          Triethylamine
THF          Tetrahydrofuran
TLC          Thin layer chromatography
ToF          Time of flight
Tryp-L       Trypsin-like
UCH37        Ubiquitin C-terminal hydrolase 37
UPS          Ubiquitin proteasome system
USP14        Ubiquitin specific peptidase 14
WT           Wild type
Z-LLE-AMC    Carboxylbenzyl-leucyl-leucyl-glutamyl-7-amido-4-methylcoumarin
                                       x


                                  CHAPTER ONE
Proteostasis, the Proteasome and a Novel Therapeutic Strategy for Neurodegenerative
                                     Diseases
                                         1


1.1 Introduction
1.1.1 Proteostasis
         The regulation of protein synthesis, folding, and degradation within a cell is collectively
known as protein homeostasis (proteostasis).1 Proteostasis is maintained by a wide array of cellular
machinery (~2,000 proteins) that work in tandem to ensure that proteins are present in the proper
locations, forms, and amounts to perform their required functions.2-6 These processes also serve to
prevent the accumulation of redundant, misfolded or damaged proteins, which is necessary to
avoid their cytotoxic aggregation.7 The maintenance of proteostasis in human cells is a
monumental task, considering the presence of over 10,000 proteins in human cells and significant
variations in proteome composition seen between cell types and tissues.8 This is further
complicated by large protein-to-protein variations in terms of abundance, location, function and
half-life.9-12 Additionally, the cellular proteome is in constant flux over the lifetime of a cell, which
is necessary to permit proper cellular functions. This includes the ability of a cell to progress
through the cell cycle, differentiate, respond to extracellular signals, or signal to other cells. Each
of these processes involve transient modifications to the cellular proteome.1, 12 Balance must be
maintained through this constant flux in the proteome by the various components of the
proteostasis network, including protein synthesis machinery (ribosomes), protein folding
complexes (chaperones), and proteolytic systems, including the ubiquitin proteasome system
(UPS) and lysosome-autophagy system (autophagy, Fig. 1.1). 2-6
         During protein synthesis, molecular chaperones participate in co-translational folding of
peptides as they exit the ribosome to promote proper folding into their final 3-dimensional (3D)
structure.13,  14
                  This is required for proper biological function for most proteins, apart from
intrinsically disordered proteins (IDPs), which will be discussed in more detail in a later section.
                                                    2


Similar chaperone proteins also assist in refolding of misfolded proteins when possible. These
activities help to prevent undesired interactions and aggregation of exposed hydrophobic regions
resulting from improper protein folding.15,    16
                                                  When proteins cannot be properly refolded, are
damaged beyond repair or simply become redundant, they are recycled through the proteolytic
pathways (UPS and autophagy) to regenerate amino acids and avoid their accumulation.17-20 If
damaged, misfolded, or redundant proteins are not repaired or disposed of quickly they can
accumulate, which leads to aggregation, undesired signaling events and toxicity.17, 21-23
         The three main branches of the proteostasis network (synthesis, folding and degradation)
are functionally coupled to one another, allowing for constant crosstalk and rapid adaptation to
preserve the balance of proteostasis through a variety of pathophysiological states.11, 23-27 Despite
this, the proteostasis network is not infallible. The constant flux and complexity of the cellular
proteome, coupled with the accumulation of various internal and external stresses, inevitably result
in a decline of the capacity of the proteostasis network. The result of which, is loss of proteome
integrity and dysregulation of proteostasis.2,    4, 5, 12, 28
                                                               This dysregulation of proteostasis is
responsible for many non-infectious and age-related human diseases.
         The common association between proteostasis disruption and disease development,
especially in the context of age-related diseases, makes the various components of the proteostasis
network promising targets for therapeutic intervention.1, 4, 10, 12, 28 As such, methods that allow for
modulation of the activity of proteolytic machinery have garnered much attention.24,               29-34
Specifically, the Tepe lab has developed an interest in exploring modulation of proteasome activity
as a novel route for addressing certain proteostasis-related diseases.
                                                   3


Figure 1.1: Proteostasis network breakdown. During protein synthesis by the ribosomes,
nascent peptides are assisted with folding into their native conformations by molecular chaperones.
This process can generate a range of proteins with varying degrees of disorder all of which perform
key roles within a cell. Proteins that have become redundant, damaged, or misfolded, either during
initial folding or sometime thereafter, must then be degraded via autophagy or the proteasome
system. Failure to promptly remove these undesired proteins can lead to protein aggregation. Some
of these protein aggregates can be degraded through the autophagy pathway, however their
accumulation can become toxic and contribute to disease development. Created with
BioRender.com.
                                                   4


1.2 The ubiquitin proteasome system
1.2.1 Proteasome structural features
         The UPS is the primary system responsible for selective regulation of protein levels within
a cell.35-38 The UPS plays a critical role in maintaining cellular health through regulation of cell
cycle progression, genome integrity, transcriptional regulation, apoptosis, immune responses,
overall maintenance of proteostasis and other cellular processes.30, 39, 40 As a result, decline or
dysregulation of the UPS is associated with the pathogenesis of numerous human diseases.41-47
The primary activity of the UPS centers around proteolytic cleavage of peptides by the massive
(~2,000 kDa) multi-subunit enzyme complex known as the proteasome. The proteasome exists in
multiple forms; however, the classical form is known as the 26S proteasome. The 26S proteasome
is made up of one 20S core particle (CP), often referred to as the 20S proteasome, and one or two
19S regulatory particles (RP), or caps (Fig. 1.2A).35, 48-50 The 19S, 20S and 26S forms of the
proteasome are so named due to their sedimentation coefficients, as determined by density-
gradient centrifugation analysis. While a double capped proteasome (i.e., 19S-20S-19S) has a
sedimentation coefficient of 30S and a single capped proteasome (i.e., 19S-20S) of 26S, these two
forms are both commonly referred to as the 26S proteasome in the literature.51 For the sake of
maintaining consistency with the literature and simplicity of discussions, the same naming scheme
will be used herein.
         The CP or 20S proteasome, is a 750 kDa enzyme complex made up 28 subunits organized
into four concentric heptameric rings (Fig. 1.2A), two α-rings (subunits α1–α7, Fig. 1.2B) and two
β-rings (subunits β1–β7, Fig. 1.2C).48-50, 52-54 The two β-rings of the 20S proteasome each contain
three different catalytic sites, for a total of six catalytic sites within one 20S proteasome. These
three sites provide three different threonine protease activities, one chymotrypsin-like (CT-L, β5-
                                                    5


subunit) that preferentially cleaves after hydrophobic residues, one trypsin-like (Tryp-L, β2-
subunit) that preferentially cleaves after basic residues, and one caspase-like (Casp-L, β1-subunit)
that preferentially cleaves after acidic residues (Fig. 1.2C).55-58 These catalytic sites work
congruently to enable efficient proteolytic cleavage of a massive range of protein substrates at
numerous and varied sites. The resulting small peptide fragments can be further processed by other
proteases and recycled as amino acids. The two α-rings of the 20S proteasome act as docking sites
for the 19S caps and as a gating-mechanism that can restrict substrate access to the catalytic sites
of the 20S proteasome. The gating-mechanism is facilitated through the convergence of the N-
terminal residues of the α-subunits over the pore leading into the core of the 20S proteasome.39, 59
In addition to the 19S cap, there exist other regulatory particles, like the 11S cap and PA200
(Proteasome activator 200 kDa), that also can associate with the α-rings of the 20S proteasome.60-
63
   The 20S proteasome has often been referred to as the latent state of the proteasome, because of
the action of the α-ring gates and the lack of 19S caps, which promote ubiquitin-dependent
proteolysis. However, as will be discussed in coming sections, this notion is not entirely accurate,
as the 20S proteasome performs key roles in protein regulation without the need for binding of the
19S or other regulatory particles.25, 64-66
         Upon binding to the α-rings of the 20S proteasome and formation of the 26S proteasome,
the 19S RPs are responsible for gate-opening, substrate detection, unfolding and movement into
the catalytic core for proteolytic cleavage.54, 67-69 The 19S RP is another large complex (~700 kDa)
that is composed of 19 subunits, that can be sub-divided into those associated with the base and
lid portions of the 19S. The base is made up of four non-ATPase subunits (Rpn 1, 2, 10 and 13)
and six ATPase subunits (Rpt1–6),48-50 the latter of which interact with the α-ring of the 20S
proteasome via insertion of their C termini into hydrophobic inter-subunit pockets between the α-
                                                     6


ring subunits. The C termini of the Rpt1–6 subunits each have a hydrophobic-tyrosine-any residue
(HbYX) motif that bind to conserved regions within the inter-subunit pockets and induce a
conformational change that leads to gate-opening of the 20S proteasome.67,             68, 70
                                                                                              The exact
mechanism of this gate-opening is still not clear, however. Binding of the last eight residues of
Rpt5, including its HbYX-motif, is sufficient to allosterically induce conformational changes in
the α-ring needed for gate opening. The ATPase activities of Rpt1–6 power the unfolding and
translocation of substrates into the 20S catalytic core, through coupled ATP (adenosine
triphosphate) hydrolysis.67, 68, 70 Rpn1, 10 and 13 act as substrate receptors that reversibly associate
with ubiquitin. The presence of these multiple ubiquitin binding subunits allows the 26S
proteasome to recognize and degrade a wide range of poly-ubiquitinated substrates. The lid of the
19S is made up of nine subunits (Rpn3, 5–9, 11, 12 and Sem1).71 Rpn11 functions as a
deubiquitinase, allowing for recycling of the ubiquitin tags removed from 26S proteasome
substrates as they enter the complex.72 The subunits of the lid also act as docking sites for other
proteins, like other deubiquitinating enzymes (Ubiquitin specific peptidase 14 (USP14) and
Ubiquitin C-terminal hydrolase 37 (UCH37)), that assist the 26S proteasome in substrate
recognition and trafficking.48, 50, 71, 73
                                                   7


  A
  B                                                        C
Figure 1.2: Proteasome structural overview. (A) The equilibrium between the 20S and 26S
proteasome is dictated by the ATP-dependent binding of the 19S caps. Shown are measurements
of the 20S proteasome (148 angstroms (Å) x 113 Å). (B) Cross sectional view of closed and open-
gated α-rings of the 20S proteasome, dictated by the conformation of the N-terminal tails of the α-
subunits (α1–α7). (C) Cross sectional view of a β-ring of the 20S proteasome with catalytic sites
labeled with red diamonds. Created with BioRender.com.
                                                8


1.2.2 Ubiquitin-dependent proteolysis by the 26S proteasome
        The classical proteasome pathway for regulation of protein levels within the cell involves
the 26S proteasome, a family of ubiquitin enzymes and occurs through ubiquitin-dependent
proteolysis. Ubiquitin-dependent proteolysis, as the name suggests, requires, and is initiated by the
polyubiquitination of proteins that are destined for degradation. The ubiquitin tags serve as a means
for the 26S proteasome to selectively identify, via Rpn10 and Rpn13 binding, and degrade proteins
that have become redundant or damaged. As the substrate protein is being unfolded by the ATPase
subunits of the 19S base,54, 67-69 the ubiquitin tags are removed by deubiquitinases, such as the
Rpn11, USP14 or UCH37, so that they may be recycled.74
        Ubiquitin itself is an 8 kDa protein with seven lysine residues, at positions 6, 11, 27, 29,
33, 48 and 63, that can be conjugated to other ubiquitin molecules to allow for polyubiquitination.
The K48 residue is the primary binding site when conjugated to proteins that are destined for
degradation by the 26S proteasome, whereas the other positions are used when ubiquitin is
performing other roles.75-77 The process of ubiquitination starts with activation of ubiquitin by an
E1 ubiquitin activating enzyme, which is an ATP-dependent process. Reaction between a cysteine
residue in the active site of E1 and the C-terminal end of ubiquitin results in a thioester-linkage.
Following this, the ubiquitin is passed to the E2 ubiquitin-conjugating enzyme, through formation
of a similar thioester bond with E2. Finally, the E3 ubiquitin ligase binds to both the E2-ubiquitin
complex and the protein substrate that requires ubiquitination. Together E2 and E3 catalyze the
transfer of the ubiquitin to the substrate protein, where a lysine residue of the target protein will
bind the C-terminal end of ubiquitin. Typically, for 26S proteasome degradation, several (4 or
more) ubiquitin molecules are attached to the substrate protein, in a chain, prior to its recognition
by the 26S (Fig. 1.3).75, 78-80
                                                   9


Figure 1.3: Ubiquitin-dependent proteolysis by the 26S proteasome. Ubiquitin is first activated
by E1 in an ATP-dependent step. This is followed by transfer of the activated ubiquitin to E2,
which then associates with the E3-substrate complex. E2 and E3 facilitate the transfer of ubiquitin
to the substrate protein, which is repeated to achieve polyubiquitination of the substrate prior to its
unfolding, removal of ubiquitin tags and degradation by the 26S proteasome. The resulting peptide
fragments can be further processed by other proteases and recycled for use in protein synthesis.
The ubiquitin tags are also recycled. Created with BioRender.com.
                                                  10


1.2.3 Intrinsically disordered proteins
         The classical depiction of a protein involves a well-defined 3D structure, permitting for
highly specific interactions and functions.81 While this is the case for many proteins, not all
proteins fit this depiction. A more realistic view is that proteins exist on a continuum of order and
disorder (Fig. 1.4). On this continuum, there are those classical highly structured proteins, but
there are also proteins that lack any well-defined 3D structure, as well as everything in between.82-
85
   Many proteins possess regions, known as intrinsically disordered regions (IDRs), that are unable
to fold into stable 3D structures. Other proteins, known as intrinsically disordered proteins (IDPs),
are comprised primarily of disordered regions and may have little, if any, well-defined 3D structure
or folded regions. A large portion of the proteome (~30%) is made up of IDPs or proteins with
substantial IDRs.86 IDPs and IDRs are such because of biased amino acid compositions and low
sequence complexities. For instance, they tend to have low proportions of bulky hydrophobic
amino acids and high proportions of charged and hydrophilic amino acids. This results in their
inability to fold into stable 3D structures, since the driving force for formation of those structures
is generally hydrophobic interactions between amino acids.87-90 These commonalities associated
with specific amino acid proportions within IDPs and IDRs has allowed for the development of
predictive software for protein disorder. PONDR,91 IUPred92 and SPOT-Disorder-Single93 are
examples of these types of software. Experimental confirmation of protein disorder can be
achieved through the use of nuclear magnetic resonance (NMR) spectroscopy94 and circular
dichroism (CD).95
                                                   11


Figure 1.4: Continuum of protein disorder. Proteins exist in a variety of states of order and
disorder, where some have stable and well-defined 3D structures and others have large regions
that lack any defined 3D structure or stable folding. IDPs are proteins that have few if any stable
3D structured regions when free in solution.
        Without substantial folding, IDPs and IDRs have very dynamic structures that can fluctuate
rapidly through many conformations. This flexible nature permits IDPs and IDRs to interact with
a range of binding partners and perform multiple roles within a cell or system. As such, IDPs are
often involved in, or act as, hubs in protein-interaction networks.96, 97 They are often signaling
molecules (about 75% of known IDPs) or participate in the regulation of signaling pathways. For
this reason, their cellular abundance must be tightly regulated to ensure proper signaling occurs
for important cellular processes, like in the regulation of cell cycle progression, transcription, or
translation.96-98 Their critical involvement with these pathways is evident in that dysregulation of
IDPs is associated with the development of a variety of human diseases.84, 85, 99, 100 Under healthy
conditions, the abundance of IDPs is tightly regulated through proteolytic cleavage by the 20S
proteasome.99,   101-103
                         However, in human diseases associated with IDPs, they often begin to
accumulate as some aspect of their regulation breaks down. These accumulated IDPs can cause
                                                  12


toxic signaling and are prone to aggregation, as seen in neurodegenerative diseases.7, 100, 104-110 Due
to their high degree of flexibility and lack of well-defined binding pockets, IDPs have often been
thought of as “undruggable”. As such, activation of 20S proteasome-mediated degradation of IDPs
seems like a promising target for therapeutic intervention in diseases associated with their
dysregulation.
1.2.4 20S proteasome-mediated regulation of IDPs and other unfolded peptides through
ubiquitin-independent proteolysis
        As was alluded to above, the 20S proteasome, while often considered the latent state of the
proteasome, plays a critical role in the regulation of cellular levels of IDPs, as well as some
misfolded and oxidatively damaged proteins, through proteolytic cleavage. These processes do not
require the unfolding activity of the 19S RP, nor the ubiquitination of the protein substrates as seen
with the 26S proteasome.111-113 As a result, the 20S proteasome can unremittently degrade these
substrates without the need for RPs, however some natural activators exist that can assist with this
process.61-63 The 20S proteasome exists primarily in a closed-gated conformation, by way of the
α-ring gates, which restricts access to the catalytic core.114 However, it is believed that direct
interaction of protein substrates with the α-ring can bring about a conformational change that
permits gate-opening and substrate degradation.99 Despite this, the activity of the 20S proteasome
towards its substrates can be enhanced via methods that promote open-gate conformations. Native
examples of this can be seen through the action of non-ATPase regulatory particles of the
proteasome, such as the 11S cap and PA200. Their association with the 20S proteasome leads to
open-gate conformations that can promote proteolysis of 20S proteasome substrates.61-63 In light
of these native activating factors, similar activation of the 20S proteasome, via small molecules
that induce open-gate conformations, could allow for enhancement of its activity and therapeutic
                                                 13


intervention in diseases where accumulation of its substrates, like IDPs, contribute to disease
progression.
1.3 Proteostasis disruption contributes to aging and neurodegenerative diseases
1.3.1 Aging and proteostasis decline
           Age-related cellular dysfunction and degenerative diseases are thought to be the result of
an age-dependent decline in a cells ability to maintain proteostasis. This decline in the capacity of
the proteostasis network is complex, but is likely rooted in changes in expression and activity of
proteostasis machinery and an increasing burden of oxidatively damaged and misfolded proteins.2,
4, 12, 28
          This results in an accumulation of these and other proteins, like IDPs, which are similarly
regulated by the 20S proteasome. This accumulation eventually leads to their aggregation and
further disruption of proteostasis through inhibition of the proteasome by some of these aggregate
forms.115-120 This initiates a cycle of continuing accumulation, aggregation, and disruption of
proteostasis, which ultimately results in cellular toxicity and death. These effects are especially
pronounced in nondividing, long-lived cells like neurons, where high levels of oxidative stress and
an extended duration of accumulation exacerbate the problem.3,             121, 122
                                                                                    The disruption of
proteostasis can spread from cell to cell, through seeding of aggregation, leading to widespread
degeneration of neurons.123-129 The result of this spreading disruption of proteostasis and neuron
death is the onset of neurodegenerative diseases, such as Parkinson’s disease (PD), Alzheimer’s
disease (AD), and Huntington’s disease (HD). The development of each of these diseases is
thought to be associated with the accumulation and aggregation of IDPs, such as α-synuclein,
amyloid β and huntingtin protein.104, 130-133
           To combat the declining proteostasis network seen in neurodegenerative diseases and to
help re-establish normal levels of disease related IDPs, 20S proteasome activation has been
                                                   14


proposed as a therapeutic strategy to selectively target IDPs for degradation through the native
mechanism for their disposal.25, 134-136 This proposed strategy is supported by studies done in
animal models, where genetic manipulation of the proteasome system suggests that stimulation of
its activity could aid in treatment of these diseases.41, 137 As humans age, the 20S proteasome
becomes increasingly more prevalent than its 26S counterpart, so targeting of the 20S proteasome
may prove to be more beneficial in these age-related diseases.47, 138-140 The strategy of targeting
the 20S proteasome specifically, looks even more promising when it is considered that the proteins
involved in the pathogenesis of neurodegenerative diseases, like α-synuclein and amyloid β, are
IDPs and thus likely substrates of the 20S proteasome.100, 134, 141 Additionally, selective activation
of the 20S proteasome should provide a degree of specificity towards its usual substrates,
oxidatively damaged proteins and IDPs, whereas activation of all proteasomal degradation may
result in undesired degradation of other proteins. Indeed, it has been recently demonstrated that
activation of the 20S proteasome only enhances the degradation of it normal substrates, with highly
disordered proteins being most quickly degraded.142 Furthermore, it has been demonstrated that
desirable IDPs can be protected from ubiquitin-independent degradation by the 20S proteasome
through what has come to be known as the “nanny model”.143 In this model, it was hypothesized
and later shown that protection of IDPs occurs through transient binding to nanny proteins that
protect their disordered regions from being able to be degraded by the 20S proteasome so that they
can performed necessary functions.143 In the cases of neurodegenerative diseases, the capacity of
the nanny proteins is likely quickly exceeded with the accumulating levels of pathogenic IDPs.
1.3.2 Parkinson’s disease
        PD is the second most common neurodegenerative disease overall144 and the most common
neurodegenerative disease primarily associated with decline of motor functions.145 Early
                                                15


symptoms of PD are commonly tremors, loss of balance and rigidity. As the disease progresses,
additional symptoms can arise that include cognitive changes, like mood disorders and depression,
in addition to further degeneration of motor function, including difficulty swallowing, chewing,
and speaking. These symptoms are a result of degeneration of dopaminergic neurons within the
substantia nigra pars compacta (SNpc) and resulting loss of dopamine signaling in the striatum.146,
147
        There is currently no cure for PD, nor any treatments that can hinder disease progression.
So, treatments are focused on relieving symptoms and improving quality of life. The most common
treatments for PD are related to the restoration of dopamine levels to make up for loss of dopamine
signaling, which is a major causative factor for the motor-related symptoms.146 This is often done
through treatment with a combination of Levodopa, also known as L-DOPA, and Carbidopa.
Levodopa is a dopamine precursor, that once it has entered the brain can be metabolized into
dopamine, by aromatic L-amino acid decarboxylase, and subsequently used for dopamine
signaling. Carbidopa, on the other hand, is a Levodopa mimic that cannot pass the blood-brain
barrier (BBB) and serves to boost the exposure of Levodopa to the brain and reduce side effects
through inhibition of Levodopa’s premature metabolism outside of the brain.148-150 In addition to
Levodopa/Carbidopa, there are other methods that are used to increase dopamine signaling, like
treatment with dopamine receptor agonists or dopamine metabolism inhibitors. In cases where
methods to restore dopamine signaling are not as effective, or certain symptoms, like tremors, are
persistent, other more invasive therapies can be applied. This includes things like deep brain
stimulation and lesion surgery, which can in some cases help to alleviate these persistent
symptoms. Regardless of which current treatment method is used, however, PD continues to
progress, and treatment efficacy will decline. For this reason, novel treatments are desperately
                                                  16


needed to not only combat the devastating symptoms of this disease, but also to provide hope for
disease-modifying treatments.146
         Although the pathogenesis of PD is not yet fully understood, it is widely accepted that
proteostasis disruption in the afflicted neurons is closely linked to disease development.151 The
initial disruption of proteostasis is thought to be due in part to the age-related decline in proteasome
function in dopaminergic neurons of the SNpc, as a result of decreasing subunit levels and
reduction in proteolytic activity.152, 153 This reduction in proteasome activity is thought to make
these neurons susceptible to the disruption of proteostasis by the ever-present need for clearance
of oxidatively damaged proteins and regulation of native proteins, such as the IDP α-synuclein.154
When proteostasis falls out of balance, α-synuclein and other proteins begin to accumulate and
aggregate, which is thought to lead to further disruption of proteasome activity and proteostasis in
general. This results in a vicious cycle of proteasome impairment and proteotoxicity in PD.120, 155-
159
    As such, the maintenance of proteasome function, and thus proteostasis, is critical to preventing
the constant accumulation and aggregation of unwanted proteins seen in PD progression.
         One of the major hallmarks of PD is the accumulation and aggregation of the IDP α-
synuclein. α-Synuclein is a 140 amino acid IDP that is predicted to be approximately 91%
disordered, according to the PONDR software.91, 104, 130, 146, 147, 158, 160-162 The normal function of α-
synuclein in non-diseased neurons is not well understood, despite substantial efforts to elucidate
it.163 What is known is that α-synuclein appears to play a role in membrane curvature, vesicle
trafficking and vesicle budding. This would explain its relative abundance in neuron synapses and
association with lipid membranes and SNARE complexes, but additional studies are required to
fully understand its role in these processes and any other potential roles that it may play.163-168
Regardless of its normal function, in PD α-synuclein has been implicated as a likely contributor to
                                                    17


disease progression due to the association of duplication,169 triplication170, 171 and mutations in the
SNCA gene, encoding for α-synuclein,172-176 with early onset forms of PD. Additionally, α-
synuclein accumulation, aggregation and presence as the primary protein within Lewy bodies,
which are large protein aggregates seen in the cytoplasm of neurons in a PD afflicted brain, further
implicate it in disease pathogenesis.130, 147, 158
          In PD, α-synuclein exists in multiple forms upon its accumulation, including monomers,
various oligomers, aggregates, and fibrils.109, 177, 178 While early identification of Lewy bodies and
large fibrils suggested they might be responsible for the neuron degeneration and toxicity,
mounting evidence suggests that it is the smaller soluble oligomeric forms of α-synuclein that are
the more toxic species. Whereas, the larger fibril and aggregate forms may serve a more
neuroprotective role, by sequestering the small toxic oligomers.127, 179-181 However, their presence
is still a hallmark of disease development and progression, considering in a healthy system they
should not be present.130, 147 Furthermore, it has been recently shown that some of the soluble
oligomeric forms of α-synuclein are also responsible for direct binding to and inhibition of the
proteasome, leading to further proteostasis disruption.115, 116, 155, 156 Compounding the issue, α-
synuclein is known to be a substrate of the 20S proteasome, so through proteasome inhibition
additional α-synuclein accumulation and aggregation is promoted. This leads to the vicious cycle
of proteasome inhibition and IDP accumulation, mentioned previously.155-157 The involvement of
α-synuclein with PD pathogenesis, and its place as a natural substrate for the 20S proteasome make
it a promising target for therapeutic intervention through the activation of its degradation by the
20S proteasome. Activation of the 20S proteasome could also assist in the clearance of other
accumulating disordered or damaged proteins and help to re-establish proteostasis in PD. For these
                                                   18


reasons, the Tepe lab has begun exploring small molecule activation of the 20S proteasome as a
novel therapeutic strategy for PD.
1.3.3 Alzheimer’s disease
        AD is the most common neurodegenerative disease, affecting approximately 45 million
people worldwide. Early symptoms of AD are primarily cognitive, including progressive memory
loss, difficulty multi-tasking and finding the right words, etc. Later in disease progression, these
cognitive problems continue to worsen and additional symptoms, such as behavioral changes,
impairment of mobility, hallucinations, and seizures, may develop.182, 183 Memory loss can become
extreme, to the extent that a patient cannot recognize people that they have known their entire life.
The devastating symptoms, poor prognosis, and lack of disease modifying treatments make AD an
extremely difficult disease to cope with for the patient and their loved ones. This also means that
development of novel therapeutic strategies that provide hope for disease modifying treatments is
of great importance.
        Similar to what is seen with PD, the major hallmark of AD is also IDP accumulation and
aggregation. In the case of AD, the primary IDP involved with its pathogenesis is amyloid β,
instead of α-synuclein. Amyloid plaques are extracellular aggregates that are primarily composed
of amyloid β and represent the final large aggregate form of the IDP. Amyloid β is a peptide by-
product of the metabolism of the amyloid precursor protein (APP) and commonly is either 40 or
42 amino acids, with the 42 amino acid form being the primary component of amyloid plaques,
due to a faster rate of aggregation.184-186 The leading theory of AD pathogenesis is known as the
amyloid hypothesis, which suggests that the accumulation of pathological forms of amyloid β,
resulting from the cleavage of APP by β- and γ-secretase enzymes, is caused by an imbalance
between its production and degradation.183,      184, 187
                                                          As was discussed above, a reduction in
                                                  19


proteasome activity during the aging process could be one factor that contributes to the eventual
disruption of proteostasis in regard to amyloid β peptides. Upon their accumulation, amyloid β
peptides can form a variety of aggregates, much like what is seen with α-synuclein.188-190 Also
similar to α-synuclein in PD, it is thought that soluble oligomeric forms of amyloid β are likely the
more toxic species, instead of the larger aggregates and fibrils.191-195 Additionally, amyloid β
oligomers have been shown to directly bind to and inhibit the 20S proteasome, further contributing
to amyloid β accumulation and disease progression.115, 116 The similarities between PD and AD, in
regard to IDPs and proteostasis disruption, make both promising targets for treatment through 20S
proteasome activation.
1.3.4 Other neurodegenerative diseases associated with IDP accumulation and aggregation
        Other neurodegenerative diseases, like Huntington’s disease and amyotrophic lateral
sclerosis (ALS), also share some similarities with PD and AD. They are also associated with
accumulation and aggregation of IDPs and misfolded proteins, as well as proteostasis
disruption.132, 196, 197 Oligomers of the Huntingtin protein have recently been shown to directly
inhibit the 20S proteasome, similar to α-synuclein and amyloid β.115 In some familial forms of
ALS, associated with mutations in the gene for C9orf72, disordered dipeptide repeat proteins
accumulate, aggregate and can further disrupt proteasome function.197-199 The similarities in
pathogenesis and the involvement of IDPs in these neurodegenerative diseases suggest that small
molecule 20S proteasome activation may represent a promising therapeutic strategy for multiple
neurodegenerative diseases. The studies herein will focus primarily on PD, however similar studies
can be envisioned for the other neurodegenerative diseases mentioned above. Studies focused on
other neurodegenerative diseases should be explored in the future to better understand the breadth
of potential disease targets for this proposed therapeutic method.
                                                   20


1.3.5 Neuroinflammation in neurodegenerative disease pathogenesis
         In recent years, growing evidence has been collected implicating neuroinflammation as
another major factor in the pathogenesis of neurodegenerative diseases. This has been
demonstrated in multiple neurodegenerative diseases, like PD, AD and ALS, and is thought to
result from activation of the native immune system in the brain by IDPs released from degenerating
neurons. While the degree to which neuroinflammation contributes to progression of these
neurodegenerative diseases is not fully understood, it likely plays a deleterious role through
causing further neuron degeneration via inflammatory signaling and further spreading of disease
pathology.126,   200-211
                         As such, during the exploration of novel methods for treatment of
neurodegenerative diseases the effects a novel treatment method has on this neuroinflammation
should also be evaluated, to gain a better understanding of the breadth of effects and potential
benefits. Treatments that can affect proteostasis disruption or IDP accumulation/aggregation, as
well as neuroinflammation may show greater promise for successfully modifying disease
progression, considering that each plays a part in the pathogenesis of these diseases.
         In the brain, immune responses are primarily controlled by a cell type known as microglia.
Microglia act as specialized macrophages, but differ in several ways, including but not limited to
their origin, turnover rate, gene expression while in their resting state and that they are much more
tightly regulated spatially and temporally.212 Microglial cells perform a variety of roles within the
brain that are essential to healthy brain development and function. They are central to
neurodevelopment and plasticity through extracellular signaling to neurons.213-216 They participate
in numerous extracellular signaling events related to inflammatory responses, anti-inflammatory
responses, antigen presentation, synaptic remodeling and cytotoxic signaling.212, 217 They also act
similar to macrophages by probing and sensing their surroundings constantly and when activated
                                                 21


they can scavenge and phagocytose foreign material and cellular debris.212, 217, 218 Their overall
role within the brain is to support neuron health and maintain brain homeostasis.212, 219-221
        Microglia can take on several forms associated with their degree of activation and location.
Perivascular and Juxtavascular microglia associate closely with the vasculature of the brain, as
their names suggest.222, 223 Standard microglia within the rest of the brain vary in form from their
resting, otherwise known as ramified, state through a continuum of activated forms based on the
degree of activation and type of activating stimuli.212, 224, 225 Microglia become activated following
detection of pro-inflammatory stimuli, like cytokines, chemokines, necrosis factors, foreign
materials, or potassium level changes.212, 223 During this progressive activation, which amplifies as
signals are continually detected, microglia begin to proliferate rapidly and undergo morphological
changes, becoming more ameboid-like and reducing processes used for detection.224, 225 They also
begin to upregulate Major Histocompatibility Complex (MHC) class I/II proteins and secrete
cytotoxic factors, recruitment molecules and inflammatory signaling molecules.212, 223, 226 Upon
further activation, they can transition into phagocytic microglia, where they phagocytose foreign
material, cellular debris and dead or dying cells.212, 222, 224
        Microglia can become significantly altered as humans age, resulting in what are called
“primed” microglia. Microglia can become primed via stimulation with inflammatory stimuli
throughout their lifespan. These primed microglia are associated with hyperactive inflammatory
responses upon re-exposure to activating stimuli.227,        228
                                                                  Additionally, microglia from elderly
people tend to maintain an increased active state, relative to microglia found in younger
individuals. This is a result of differences in gene expression and increased basal release of pro-
inflammatory signaling molecules by the microglia.227, 229, 230 The changes seen in these aged
microglia also result in a reduced propensity to enter their phagocytotic form, instead favoring
                                                  22


continued secretion of inflammatory signaling molecules.227, 229, 230 These effects together lead to
an overall weakening of the ability of microglia to maintain homeostasis and a propensity to cause
more severe inflammation in response to perturbations in the brain.219
        Neuroinflammation seen in neurodegenerative diseases, like PD and AD, involves
activation of microglia by IDPs released from degenerating neurons. Multiple cell surface
receptors, such as toll-like receptors (TLR2 and TLR4) and CD36, have been implicated in the
detection of IDPs in the extracellular space by microglia.126, 200-211 Following IDP detection, a pro-
inflammatory response is initiated through pro-inflammatory pathways, like the NF-κB pathway,
resulting in release of pro-inflammatory signaling molecules, like cytokines (i.e. TNF-α, IL-1β)
and chemokines (i.e. IFN-γ) and reactive oxygen species (ROS).201, 204, 219, 231 These signaling
molecules and ROS promote further neuron degeneration, which in turn leads to more release of
IDPs. This results in a vicious cycle of increasing neuron degeneration and neuroinflammation
(Fig. 1.5).126, 200-211 Under normal circumstances neuroinflammation would be resolved following
clearance of the perturbing species that activated the microglia and re-establishment of brain
homeostasis.212 However, aged and primed microglia are less capable of removal of these aberrant
proteins and cellular debris through phagocytosis and in neurodegenerative diseases neuron
degeneration and IDP release lead to persistent activation of microglia.219, 227-230 The result is
chronic neuroinflammation, which is a hallmark of and a contributor to neurodegenerative disease
pathogenesis.126,    200-211
                             As such, treatments aimed at modifying progression of these diseases
should be evaluated for their effects on microglia and neuroinflammation, in addition to neurons
and the other pathological aspects of these diseases.
                                                   23


Figure 1.5: Vicious cycle of increasing neuron degeneration and neuroinflammation seen in
Parkinson’s disease pathogenesis. Degeneration of dopaminergic neurons in PD leads to release
of accumulated α-synuclein into the extracellular space. Resting microglia detect this aberrant
protein and become activated. Activated microglia secrete various inflammatory factors, which
contribute to further neuron degeneration, thus initiating a cycle of increasing neuron degeneration
and inflammation. Created with BioRender.com.
1.4 The 20S proteasome as a novel therapeutic target
    The key involvement of the proteasome in the maintenance of proteostasis and in numerous
essential biological pathways has long garnered attention to it as a promising therapeutic target for
treatment of a variety diseases. 30, 39, 40, 134, 232-234 Specific targeting of the 20S proteasome has been
proposed as a novel strategy to counteract the accumulation of IDPs associated with
neurodegenerative disease development.25,            134-136
                                                             By targeting the 20S proteasome, IDPs and
oxidatively damaged proteins that accumulate in these diseases can be selectively targeted,
                                                         24


considering the 20S proteasome’s limited substrate scope of unfolded proteins.111-113 20S
proteasome activation could assist in re-establishing proteostasis via clearing of accumulated IDPs
and maintain its activity in neurodegenerative diseases, where proteasome activity is known to be
reduced24, 153 and even inhibited.115, 116 Additionally, the 20S proteasome becomes more prevalent
as humans age, relative to the 26S proteasome, so it may represent the more promising target in
these systems.47, 138-140 The studies outlined herein will focus on the development of small molecule
20S proteasome activators as a novel therapeutic strategy for neurodegenerative diseases. As such,
a brief summarization of the history of this technology is required to set the stage for the upcoming
chapters.
1.4.1 Small molecule 20S proteasome activators
         It was first demonstrated that the 20S proteasome could be activated using low
concentrations (0.04-0.08%) of sodium dodecyl sulfate (SDS), which proved to be a useful tool
for in vitro studies of the 20S proteasome. However, at higher concentrations SDS inhibits the
proteasome, suggesting that it is acting as a partial denaturant, allowing for easier access to the
catalytic core of the proteasome at low concentrations.55, 114 Other SDS-like activators of the
proteasome have since been identified, but due to their inability to be used in physiologically
relevant systems they are reserved for use as in vitro tools.235-237
         Some years following the discovery of SDS as a means to activate the 20S proteasome,
researchers have begun focusing on the identification and development of more drug-like small
molecule activators of the 20S proteasome. Perhaps the next small molecule 20S proteasome
activator identified was betulinic acid (Fig. 1.6A).238 Betulinic acid, a triterpenoid, was found to
enhance the CT-L activity of the 20S proteasome in vitro. However, the development of analogues
of betulinic acid and investigation of its structure activity relationships (SAR) proved to be
                                                   25


complicated due to changes in its structure resulting in inhibitors, as opposed to the desired
activators.238 Additionally, it was later found that this apparent activity did not translate to the
degradation of IDPs.239
        Following the discovery of betulinic acid as a 20S activator,238 researchers began trying to
identify novel activators that have more drug-like properties and lack the issues seen with betulinic
acid. Among the first, was a study done by Kodadek et al.239 where they developed a series of
assays designed to allow for screening of small molecules for 20S proteasome activation. Two
novel 20S proteasome activators, MK-866 (Fig. 1.6B) and AM-404 (Fig. 1.6C), were identified
in their screening. These small molecules were able to enhance the degradation of the IDP α-
synuclein in cell culture.239 Extending upon this work, Coleman and Trader240 identified additional
20S proteasome activators, including ursolic acid (Fig. 1.6D), a derivative of betulinic acid, and a
cytisine derivative (Fig. 1.6E).240
        Concurrently, the Tepe lab identified their first small molecule 20S proteasome activator
and were working to identify additional novel 20S activator scaffolds using a combination of
biochemical assays and in vitro experiments.135, 136 The first 20S proteasome activators published
by the Tepe lab were identified in a high-throughput screen of the NIH clinical collection and
Prestwick library, where the neuroleptic agent Chlorpromazine (Fig. 1.6F) and other
phenothiazine derivatives were identified as novel 20S proteasome activators.135 Several
analogues of Chlorpromazine were synthesized to diminish its dopamine D2 receptor activity and
enhance/maintain its 20S proteasome activity. These phenothiazine analogues were found to
enhance the rate of degradation of 20S proteasome substrates in biochemical and cellular assays,
and due to their drug-likeness, they represented some of the most promising candidates for drug
development at that time.135
                                                 26


         Prior to the discovery of the Chlorpromazine analogues as 20S proteasome activators, but
published thereafter, Njomen et al.136 discovered TCH-165 (Fig. 1.6G), to be a potent 20S
proteasome activator. TCH-165 was found to promote an open-gate conformation of the 20S
proteasome using atomic force microscopy (AFM) to visualize the gate-opening.136 This represents
the first and only biophysical data supporting this mechanism of small molecule 20S proteasome
activation. TCH-165 remains one of the most potent 20S proteasome activators identified and has
become a benchmark compound to which newly developed activators have been compared.136
                                                27


Figure 1.6: Selection of known small molecule 20S proteasome activators.135, 136, 238-240
1.4.2 Continuing the investigation of small molecule 20S proteasome activation as a
therapeutic strategy
        Despite the recent advances in the field of small molecule 20S proteasome activation, there
remains a great need for the identification of novel molecular classes of 20S activators, due to a
variety of limitations seen with previously identified activators. BBB permeability, low potency,
                                                  28


non-translatable activity to physiologically relevant substrates or systems and target promiscuity,
represent some of the current limitations of previously identified activators. To overcome these
limitations, the Tepe lab continues to investigate novel analogues of Chlorpromazine,135 TCH-
165136 and other molecular scaffolds, discovered during the high-throughput screen that show 20S
proteasome activity.
         Due to the field of small molecule 20S proteasome activation being relatively young, there
is still much to be discovered in terms of the utility and effects of 20S proteasome activators in
more disease relevant systems. For example, although it has been shown that these activators can
enhance the rate of degradation of IDPs in purified protein and cellular assays, it is not known
what effect these activators may have on the toxic oligomeric forms of IDPs that are associated
with many neurodegenerative diseases.83, 107, 108, 133, 186, 241-246 It is thought that these oligomeric
IDPs exist in an equilibrium with the monomeric form.188-190, 247 So, if 20S proteasome activators
can enhance clearance of monomeric IDPs in systems with various IDP forms, then they may also
reduce the toxic oligomeric forms. However, this has not yet been explored. Further complicating
this, it was recently found by Smith et al.115 that the 20S proteasome can be inhibited by some
oligomeric forms of IDPs. A study examining if 20S proteasome activators can influence IDP
oligomer concentrations and maintain 20S proteasome activity in the presence of inhibitory
oligomers may provide invaluable support for 20S proteasome activators as a potential therapeutic
strategy for the treatment of neurodegenerative diseases.
         The studies outlined herein were aimed at furthering the development of 20S proteasome
activators as a novel therapeutic strategy for treatment of neurodegenerative diseases. Chapters 2
and 3 focus on the identification of novel 20S proteasome activator scaffolds through collaboration
with Dr. Adam Mosey’s lab (Chapter 2) and from the high-throughput screen run by our lab
                                                  29


previously (Chapter 3).135 Evaluation of these scaffolds sought to ensure that they show promising
levels of activity, activate all catalytic sites of the 20S proteasome and that their activity translates
to degradation of disease relevant substrates. Furthermore, these studies sought to begin to explore
these novel scaffolds in terms of their structure activity relationship (SAR) towards 20S
proteasome activation and pave the way for the development of future analogues, with promising
drug-like properties and potent activity. Chapter 4 will focus on the development of novel
methods for evaluating the activity and utility of 20S proteasome activators in more disease
relevant model systems. Additionally, these studies sought to further our understanding of the
potential effects that small molecule 20S proteasome activators may have in diseased systems,
where IDP oligomers, 20S proteasome inhibition and neuroinflammation all play a role.
1.5 Conclusions
            The capability of a cell to maintain proteostasis is critical for it to properly perform its
functions and to its overall health.2-6 The disruption of cellular proteostasis is associated with the
development of numerous human diseases, with neurodegenerative diseases as a prime example.1,
4, 10, 12, 28, 151, 197, 248
                             As such, there is great interest in the development of therapeutics that can assist
in re-establishing proteostasis in cells and diseases where it has been disrupted.1, 4, 10, 12, 28 One of
the critical components of the proteostasis network that shows potential as a therapeutic target is
the proteasome.25, 34, 134, 233, 234, 249, 250
            The proteasome is one of the major protein degradation machineries within a cell. It is
responsible for helping to regulate protein levels to preserve proteostasis and allow for adaptation
of the proteome to the needs of a cell.30, 39, 40 It has been demonstrated that enhancing the activity
of the proteasome can alleviate the burden of proteotoxic protein accumulation and can delay
aging251-254 and extend lifespan,255-257 in cells,251,               258-263
                                                                             rodents264-266 and humans.267,  268
                                                            30


Furthermore, enhancement of proteasome activity has great therapeutic potential for treatment of
proteotoxic diseases, like neurodegenerative diseases, where accumulation and aggregation of
aberrant proteins leads to toxicity.104,  130-133
                                                  Specific targeting of the 20S proteasome should
provide an added level of selectivity, considering its activity is restricted to the degradation of
oxidatively damaged proteins and IDPs, relative to activation of all proteasomes or the 26S
proteasome.99,  103, 269
                         The pathogenesis of neurodegenerative diseases, like PD and AD, are
associated with the accumulation and aggregation of IDPs, which are natural substrates of the 20S
proteasome, so it is an especially promising target for these diseases.100, 134, 141
        The field of small molecule 20S proteasome activation is relatively new and only a handful
of bona fide activator scaffolds have been identified.25, 135, 136, 239, 270-272 As such, the identification
of novel small molecule 20S proteasome activator scaffolds would greatly enhance the likelihood
of success during the development of this novel strategy and assist in overcoming some of the
limitations associated with previous scaffolds. Additionally, much remains unexplored regarding
the potential of this therapeutic strategy and the effects that it may have on a neurodegenerative
disease afflicted brain. These diseases are complex, involving multiple cell types, various forms
of IDPs oligomers and neuroinflammation, in addition to widespread proteostasis disruption.
Novel methods to evaluate the effects of small molecule 20S proteasome activators on these
various aspects of neurodegenerative disease pathogenesis will be essential to their further
development as a therapeutic strategy.
                                                   31


                                           REFERENCES
(1) Balch, W. E.; Morimoto, R. I.; Dillin, A.; Kelly, J. W. Adapting proteostasis for disease
intervention. 2008; Vol. 319, pp 916-919.
(2) Klaips, C. L.; Jayaraj, G.; Hartl, F. Pathways of cellular proteostasis in aging and disease. The
Journal of cell biology 2018, 217 (1), 51-63. DOI: 10.1083/jcb.201709072.
(3) Powers, E. T.; Morimoto, R. I.; Dillin, A.; Kelly, J. W.; Balch, W. E. Biological and
Chemical Approaches to Diseases of Proteostasis Deficiency. Annual Review of Biochemistry
2009, 78 (1), 959-991. DOI: 10.1146/annurev.biochem.052308.114844.
(4) Labbadia, J.; Morimoto, R. I. The biology of proteostasis in aging and disease. Annual review
of biochemistry 2015, 84, 435-464. DOI: 10.1146/annurev-biochem-060614-033955.
(5) Hartl, F. U. Cellular Homeostasis and Aging. Annual Review of Biochemistry 2016, 85 (1), 1-
4. DOI: 10.1146/annurev-biochem-011116-110806.
(6) Hetz, C.; Glimcher, L. H. Protein homeostasis networks in physiology and disease. Current
opinion in cell biology 2011, 23 (2), 123-125. DOI: 10.1016/j.ceb.2011.01.004.
(7) Chiti, F.; Dobson, C. M. Protein Misfolding, Amyloid Formation, and Human Disease: A
Summary of Progress Over the Last Decade. Annual review of biochemistry 2017, 86, 27-68.
DOI: 10.1146/annurev-biochem-061516-045115.
(8) Kulak, N. A.; Geyer, P. E.; Mann, M. Loss-less Nano-fractionator for High Sensitivity, High
Coverage Proteomics. Molecular & cellular proteomics : MCP 2017, 16 (4), 694-705. DOI:
10.1074/mcp.O116.065136.
(9) Geiger, T.; Wehner, A.; Schaab, C.; Cox, J.; Mann, M. Comparative proteomic analysis of
eleven common cell lines reveals ubiquitous but varying expression of most proteins. Molecular
& cellular proteomics : MCP 2012, 11 (3). DOI: 10.1074/mcp.M111.014050.
(10) Korovila, I.; Hugo, M.; Castro, J. P.; Weber, D.; Höhn, A.; Grune, T.; Jung, T. Proteostasis,
oxidative stress and aging. Redox biology 2017, 13, 550-567. DOI: 10.1016/j.redox.2017.07.008.
(11) Tanaka, K.; Matsuda, N. Proteostasis and neurodegeneration: the roles of proteasomal
degradation and autophagy. Biochimica et biophysica acta 2014, 1843 (1), 197-204. DOI:
10.1016/j.bbamcr.2013.03.012.
(12) Hipp, M. S.; Kasturi, P.; Hartl, F. U. The proteostasis network and its decline in ageing.
Nature reviews. Molecular cell biology 2019, 20 (7), 421-435. DOI: 10.1038/s41580-019-0101-
y.
(13) Frydman, J. Folding of newly translated proteins in vivo: the role of molecular chaperones.
Annual review of biochemistry 2001, 70, 603-647. DOI: 10.1146/annurev.biochem.70.1.603.
                                                 32


(14) Hartl, F. U. Molecular chaperones in cellular protein folding. Nature 1996, 381 (6583), 571-
579. DOI: 10.1038/381571a0.
(15) Kim, Y. E.; Hipp, M. S.; Bracher, A.; Hayer-Hartl, M.; Ulrich Hartl, F. Molecular
Chaperone Functions in Protein Folding and Proteostasis. Annual Review of Biochemistry 2013,
82 (1), 323-355. DOI: 10.1146/annurev-biochem-060208-092442.
(16) Balchin, D.; Hayer-Hartl, M.; Hartl, F. U. In vivo aspects of protein folding and quality
control. Science (New York, N.Y.) 2016, 353 (6294). DOI: 10.1126/science.aac4354.
(17) Cohen-Kaplan, V.; Livneh, I.; Avni, N.; Cohen-Rosenzweig, C.; Ciechanover, A. The
ubiquitin-proteasome system and autophagy: Coordinated and independent activities. The
international journal of biochemistry & cell biology 2016, 79, 403-418. DOI:
10.1016/j.biocel.2016.07.019.
(18) Ciechanover, A. Intracellular protein degradation: From a vague idea thru the lysosome and
the ubiquitin-proteasome system and onto human diseases and drug targeting. Best practice &
research. Clinical haematology 2017, 30 (4), 341-355. DOI: 10.1016/j.beha.2017.09.001.
(19) Dikic, I. Proteasomal and Autophagic Degradation Systems. Annual review of biochemistry
2017, 86, 193-224. DOI: 10.1146/annurev-biochem-061516-044908.
(20) Varshavsky, A. The ubiquitin system, an immense realm. Annual review of biochemistry
2012, 81, 167-176. DOI: 10.1146/annurev-biochem-051910-094049.
(21) Niforou, K.; Cheimonidou, C.; Trougakos, I. P. Molecular chaperones and proteostasis
regulation during redox imbalance. Redox biology 2014, 2, 323-332. DOI:
10.1016/j.redox.2014.01.017.
(22) Douglas, P. M.; Summers, D. W.; Cyr, D. M. Molecular chaperones antagonize
proteotoxicity by differentially modulating protein aggregation pathways. Prion 2009, 3 (2), 51-
58. DOI: 10.4161/pri.3.2.8587.
(23) Ji, C. H.; Kwon, Y. T. Crosstalk and Interplay between the Ubiquitin-Proteasome System
and Autophagy. Molecules and cells 2017, 40 (7), 441-449. DOI: 10.14348/molcells.2017.0115.
(24) McNaught, K.; Olanow, C. W.; Halliwell, B.; Isacson, O.; Jenner, P. Failure of the
ubiquitin–proteasome system in Parkinson's disease. Nature Reviews Neuroscience 2001, 2 (8),
589-594. DOI: 10.1038/35086067.
(25) Njomen, E.; Tepe, J. J. Proteasome Activation as a New Therapeutic Approach to Target
Proteotoxic Disorders. Journal of Medicinal Chemistry 2019, 62 (14), 6469-6481. DOI:
10.1021/acs.jmedchem.9b00101.
(26) Korolchuk, V. I.; Mansilla, A.; Menzies, F. M.; Rubinsztein, D. C. Autophagy inhibition
compromises degradation of ubiquitin-proteasome pathway substrates. Molecular cell 2009, 33
(4), 517-527. DOI: 10.1016/j.molcel.2009.01.021.
                                                33


(27) Zheng, Q.; Su, H.; Tian, Z.; Wang, X. Proteasome malfunction activates macroautophagy in
the heart. American journal of cardiovascular disease 2011, 1 (3), 214-226.
(28) Taylor, R. C.; Dillin, A. Aging as an Event of Proteostasis Collapse. Cold Spring Harbor
Perspectives in Biology 2011, 3, a004440. DOI: 10.1101/cshperspect.a004440.
(29) Calamini, B.; Morimoto, R. Protein homeostasis as a therapeutic target for diseases of
protein conformation. Current Topics in Medicinal Chemistry 2012, 12 (22), 2623-2640. DOI:
10.2174/1568026611212220014.
(30) Voges, D.; Zwickl, P.; Baumeister, W. The 26S Proteasome: A Molecular Machine
Designed for Controlled Proteolysis. Annual Review of Biochemistry 1999, 68, 1015-1068,
review-article. DOI: 10.1146/annurev.biochem.68.1.1015.
(31) Voorhees, P.; Dees, E.; O'Neil, B.; Orlowski, R. The proteasome as a target for cancer
therapy. Clinical cancer research 2003, 9 (17), 6316-6325.
(32) Ostrowska, H. The ubiquitin-proteasome system: a novel target for anticancer and anti-
inflammatory drug research. Cellular & Molecular Biology Letters 2008, 13 (3), 353-365. DOI:
10.2478/s11658-008-0008-7.
(33) Demasi, M.; Faria, B. F. Activation of the ubiquitin-proteasome system: implications for
neurodegeneration, aging, and tumorigenesis. Journal of Neurology & Neuromedicine 2017, 2
(8), 1-4.
(34) Opattova, A.; Cente, M.; Novak, M.; Filipcik, P. The ubiquitin proteasome system as a
potential therapeutic target for treatment of neurodegenerative diseases. General physiology and
biophysics 2015, 34 (4), 337-352. DOI: 10.4149/gpb_2015024.
(35) Tanaka, K.; Mizushima, T.; Saeki, Y. The proteasome: Molecular machinery and
pathophysiological roles. 2012/03//, Vol. 393, pp 217-234. DOI: 10.1515/hsz-2011-0285.
(36) Hochstrasser, M. UBIQUITIN-DEPENDENT PROTEIN DEGRADATION. Annual Review
of Genetics 2003, 30, 405-439, review-article. DOI: 10.1146/annurev.genet.30.1.405.
(37) Tomko, R. J.; Hochstrasser, M. Molecular architecture and assembly of the eukaryotic
proteasome. Annual review of biochemistry 2013, 82, 415-445. DOI: 10.1146/annurev-biochem-
060410-150257.
(38) Wehmer, M.; Sakata, E. Recent advances in the structural biology of the 26S proteasome.
The international journal of biochemistry & cell biology 2016, 79, 437-442. DOI:
10.1016/j.biocel.2016.08.008.
(39) Thibaudeau, T. A.; Smith, D. M. A Practical Review of Proteasome Pharmacology.
Pharmacological reviews 2019, 71 (2), 170-197. DOI: 10.1124/pr.117.015370.
                                                 34


(40) Shang, F.; Taylor, A. Ubiquitin-proteasome pathway and cellular responses to oxidative
stress. Free radical biology & medicine 2011, 51 (1), 5-16. DOI:
10.1016/j.freeradbiomed.2011.03.031.
(41) Bedford, L.; Hay, D.; Devoy, A.; Paine, S.; Powe, D. G.; Seth, R.; Gray, T.; Topham, I.;
Fone, K.; Rezvani, N.; et al. Depletion of 26S Proteasomes in Mouse Brain Neurons Causes
Neurodegeneration and Lewy-Like Inclusions Resembling Human Pale Bodies. Journal of
Neuroscience 2008, 28 (33), 8189-8198. DOI: 10.1523/JNEUROSCI.2218-08.2008.
(42) Bonet-Costa, V.; Pomatto, L. C.-D.; Davies, K. J. A. The Proteasome and Oxidative Stress
in Alzheimer's Disease. Antioxidants & Redox Signaling 2016, 25 (16), 886-901. DOI:
10.1089/ars.2016.6802.
(43) Bulteau, A.-L.; Szweda, L. I.; Friguet, B. Age-Dependent Declines in Proteasome Activity
in the Heart. Archives of Biochemistry and Biophysics 2002, 397 (2), 298-304. DOI:
10.1006/abbi.2001.2663.
(44) Carrard, G.; Bulteau, A.-L.; Petropoulos, I.; Friguet, B. Impairment of proteasome structure
and function in aging. The international journal of biochemistry & cell biology 2002, 34 (11),
1461-1474. DOI: 10.1016/s1357-2725(02)00085-7.
(45) Cecarini, V.; Ding, Q.; Keller, J. N. Oxidative inactivation of the proteasome in Alzheimer's
disease. Free Radical Research 2007, 41 (6), 673-680. DOI: 10.1080/10715760701286159.
(46) Zheng, Q.; Huang, T.; Zhang, L.; Zhou, Y.; Luo, H.; Xu, H.; Wang, X. Dysregulation of
Ubiquitin-Proteasome System in Neurodegenerative Diseases. Frontiers in Aging Neuroscience
2016, 8, 303-303. DOI: 10.3389/fnagi.2016.00303.
(47) Tomaru, U.; Takahashi, S.; Ishizu, A.; Miyatake, Y.; Gohda, A.; Suzuki, S.; Ono, A.; Ohara,
J.; Baba, T.; Murata, S.; et al. Decreased Proteasomal Activity Causes Age-Related Phenotypes
and Promotes the Development of Metabolic Abnormalities. The American Journal of Pathology
2012, 180 (3), 963-972. DOI: 10.1016/j.ajpath.2011.11.012.
(48) Budenholzer, L.; Cheng, C. L.; Li, Y.; Hochstrasser, M. Proteasome Structure and
Assembly. Academic Press: 2017; Vol. 429, pp 3500-3524.
(49) Saeki, Y.; Tanaka, K. Assembly and function of the proteasome. Methods in molecular
biology (Clifton, N.J.) 2012, 832, 315-337. DOI: 10.1007/978-1-61779-474-2_22.
(50) Bard, J.; Goodall, E.; Greene, E.; Jonsson, E.; Dong, K.; Martin, A. Structure and Function
of the 26S Proteasome. Annual review of biochemistry 2018, 87, 697-724. DOI:
10.1146/annurev-biochem-062917-011931.
(51) Yoshimura, T.; Kameyama, K.; Takagi, T.; Ikai, A.; Tokunaga, F.; Koide, T.; Tanahashi,
N.; Tamura, T.; Cejka, Z.; Baumeister, W. Molecular characterization of the "26S" proteasome
complex from rat liver. Journal of structural biology 1993, 111 (3), 200-211. DOI:
10.1006/jsbi.1993.1050.
                                                 35


(52) Xie, Y. Structure, assembly and homeostatic regulation of the 26S proteasome. Journal of
molecular cell biology 2010, 2 (6), 308-317. DOI: 10.1093/jmcb/mjq030.
(53) Huang, X.; Luan, B.; Wu, J.; Shi, Y. An atomic structure of the human 26S proteasome.
Nature structural & molecular biology 2016, 23 (9), 778-785. DOI: 10.1038/nsmb.3273.
(54) da Fonseca, P. C. A.; Morris, E. P. Structure of the human 26S proteasome: subunit radial
displacements open the gate into the proteolytic core. The Journal of Biological Chemistry 2008,
283 (34), 23305-23314. DOI: 10.1074/jbc.m802716200.
(55) Tanaka, K.; Yoshimura, T.; Kumatori, A.; Ichihara, A.; Ikai, A.; Nishigai, M.; Kameyama,
K.; Takagi, T. Proteasomes (multi-protease complexes) as 20 S ring-shaped particles in a variety
of eukaryotic cells. The Journal of biological chemistry 1988, 263 (31), 16209-16217.
(56) Groll, M.; Heinemeyer, W.; Jäger, S.; Ullrich, T.; Bochtler, M.; Wolf, D. H.; Huber, R. The
catalytic sites of 20S proteasomes and their role in subunit maturation: A mutational and
crystallographic study. PNAS 1999, 20, 10976-10983, research-article. DOI:
doi.org/10.1073/pnas.96.20.10976.
(57) Groll, M.; Bochtler, M.; Brandstetter, H.; Clausen, T.; Huber, R. Molecular machines for
protein degradation. Chembiochem : a European journal of chemical biology 2005, 6 (2), 222-
256. DOI: 10.1002/cbic.200400313.
(58) Unno, M.; Mizushima, T.; Morimoto, Y.; Tomisugi, Y.; Tanaka, K.; et al. The Structure of
the Mammalian 20S Proteasome at 2.75 Å Resolution. Structure 2002, 10 (5), 609-618. DOI:
10.1016/S0969-2126(02)00748-7.
(59) Groll, M.; Bajorek, M.; Köhler, A.; Moroder, L.; Rubin, D. M.; Huber, R.; Glickman, M.
H.; Finley, D. A gated channel into the proteasome core particle. Nature Structural Biology
2000, 7 (11), 1062-1067. DOI: 10.1038/80992.
(60) Ustrell, V.; Hoffman, L.; Pratt, G.; Rechsteiner, M. PA200, a nuclear proteasome activator
involved in DNA repair. European Molecular Biology Organization Journal 2002, 21 (13),
3515-3525. DOI: 10.1093/emboj/cdf333.
(61) Stadtmueller, B.; Hill, C. Proteasome activators. Molecular cell 2011, 41 (1), 8-19. DOI:
10.1016/j.molcel.2010.12.020.
(62) Sadre-Bazzaz, K.; Whitby, F. G.; Robinson, H.; Formosa, T.; Hill, C. P. Structure of a
Blm10 complex reveals common mechanisms for proteasome binding and gate opening.
Molecular Cell 2010, 37 (5), 728-735. DOI: 10.1016/j.molcel.2010.02.002.
(63) Whitby, F. G.; Masters, E. I.; Kramer, L.; Knowlton, J. R.; Yao, Y.; Wang, C. C.; Hill, C. P.
Structural basis for the activation of 20S proteasomes by 11S regulators. Nature 2000, 408
(6808), 115-120, OriginalPaper. DOI: doi:10.1038/35040607.
                                                 36


(64) Deshmukh, F. K.; Yaffe, D.; Olshina, M. A.; Ben-Nissan, G.; Sharon, M. The Contribution
of the 20S Proteasome to Proteostasis. Biomolecules 2019, 9 (5), 190, Review. DOI:
10.3390/biom9050190.
(65) Ben-Nissan, G.; Sharon, M. Regulating the 20S proteasome ubiquitin-independent
degradation pathway. Biomolecules 2014, 4 (3), 862-884. DOI: 10.3390/biom4030862.
(66) Ding, Q.; Dimayuga, E.; Keller, J. N. Proteasome Regulation of Oxidative Stress in Aging
and Age-Related Diseases of the CNS. Antioxidants & Redox Signaling 2006, 8 (1-2), 163-172.
DOI: 10.1089/ars.2006.8.163.
(67) Smith, D. M.; Chang, S. C.; Park, S.; Finley, D.; Cheng, Y.; Goldberg, A. L. Docking of the
proteasomal ATPases' carboxyl termini in the 20S proteasome's alpha ring opens the gate for
substrate entry. Molecular cell 2007, 27 (5), 731-744. DOI: 10.1016/j.molcel.2007.06.033.
(68) Rabl, J.; Smith, D. M.; Yu, Y.; Chang, S. C.; Goldberg, A. L.; Cheng, Y. Mechanism of
Gate Opening in the 20S Proteasome by the Proteasomal ATPases. Molecular Cell 2008, 30 (3),
360-368. DOI: 10.1016/j.molcel.2008.03.004.
(69) Chu-Ping, M.; Vu, J. H.; Proske, R. J.; Slaughter, C. A.; DeMartino, G. N. Identification,
purification, and characterization of a high molecular weight, ATP-dependent activator (PA700)
of the 20 S proteasome. The Journal of biological chemistry 1994, 269 (5), 3539-3547.
(70) Ehlinger, A.; Walters, K. J. Structural insights into proteasome activation by the 19S
regulatory particle. Biochemistry 2013, 52 (21), 3618-3628. DOI: 10.1021/bi400417a.
(71) Sharon, M.; Taverner, T.; Ambroggio, X. I.; Deshaies, R. J.; Robinson, C. V. Structural
organization of the 19S proteasome lid: insights from MS of intact complexes. Plos Biology
2006, 4 (8), e267. DOI: 10.1371/journal.pbio.0040267.
(72) Verma, R.; Aravind, L.; Oania, R.; McDonald, W. H.; John R. Yates, I.; Koonin, E. V.;
Deshaies, R. J. Role of Rpn11 Metalloprotease in Deubiquitination and Degradation by the 26S
Proteasome. Science 2002, 298 (5593), 611-615, research-article. DOI:
10.1126/science.1075898.
(73) Schweitzer, A.; Aufderheide, A.; Rudack, T.; Beck, F.; Pfeifer, G.; Plitzko, J. M.; Sakata,
E.; Schulten, K.; Förster, F.; Baumeister, W. Structure of the human 26S proteasome at a
resolution of 3.9 Å. PNAS 2016, 113 (28), 7816-7821, research-article. DOI:
10.1073/pnas.1608050113.
(74) Lee, M. J.; Lee, B. H.; Hanna, J.; King, R. W.; Finley, D. Trimming of ubiquitin chains by
proteasome-associated deubiquitinating enzymes. Molecular & cellular proteomics : MCP 2011,
10 (5). DOI: 10.1074/mcp.R110.003871.
(75) Komander, D.; Rape, M. The ubiquitin code. Annual review of biochemistry 2012, 81, 203-
229. DOI: 10.1146/annurev-biochem-060310-170328.
                                                  37


(76) Vijay-Kumar, S.; Bugg, C. E.; Cook, W. J. Structure of ubiquitin refined at 1.8 A resolution.
Journal of molecular biology 1987, 194 (3), 531-544. DOI: 10.1016/0022-2836(87)90679-6.
(77) Saeki, Y. Ubiquitin recognition by the proteasome. Journal of biochemistry 2017, 161 (2),
113-124. DOI: 10.1093/jb/mvw091.
(78) Hershko, A.; Ciechanover, A. The ubiquitin system. Annual review of biochemistry 1998,
67, 425-479. DOI: 10.1146/annurev.biochem.67.1.425.
(79) Pickart, C. M. Mechanisms underlying ubiquitination. Annual review of biochemistry 2001,
70 (1), 503-533. DOI: 10.1146/annurev.biochem.70.1.503.
(80) Weissman, A. M. Themes and variations on ubiquitylation. Nature Reviews Molecular Cell
Biology 2001, 2 (3), 169-178, ReviewPaper. DOI: doi:10.1038/35056563.
(81) Uversky, V. N. A decade and a half of protein intrinsic disorder: biology still waits for
physics. Protein science : a publication of the Protein Society 2013, 22 (6), 693-724. DOI:
10.1002/pro.2261.
(82) Dunker, A. K.; Brown, C. J.; Lawson, J. D.; Iakoucheva, L. M.; Obradović, Z. Intrinsic
disorder and protein function. Biochemistry 2002, 41 (21), 6573-6582. DOI: 10.1021/bi012159+.
(83) Uversky, V. N.; Oldfield, C. J.; Dunker, A. K. Intrinsically Disordered Proteins in Human
Diseases: Introducing the D <sup>2</sup> Concept. Annual Review of Biophysics 2008, 37 (1),
215-246. DOI: 10.1146/annurev.biophys.37.032807.125924.
(84) Cheng, Y.; LeGall, T.; Oldfield, C.; Dunker, K.; Uversky, V. Abundance of Intrinsic
Disorder in Protein Associated with Cardiovascular Disease. Biochemistry 2006, 45 (35), 10448-
10460, research-article. DOI: 10.1021/bi060981d.
(85) Du, Z.; Uversky, V. N. A Comprehensive Survey of the Roles of Highly Disordered
Proteins in Type 2 Diabetes. International journal of molecular sciences 2017, 18 (10), 2010.
DOI: 10.3390/ijms18102010.
(86) Na, J. H.; Lee, W. K.; Yu, Y. G. How Do We Study the Dynamic Structure of Unstructured
Proteins: A Case Study on Nopp140 as an Example of a Large, Intrinsically Disordered Protein.
International journal of molecular sciences 2018, 19 (2), 381. DOI: 10.3390/ijms19020381.
(87) Williams, R. M.; Obradovi, Z.; Mathura, V.; Braun, W.; Garner, E. C.; Young, J.;
Takayama, S.; Brown, C. J.; Dunker, A. K. The protein non-folding problem: amino acid
determinants of intrinsic order and disorder. Pacific Symposium on Biocomputing 2001, 89-100.
DOI: 10.1142/9789814447362_0010.
(88) Romero, P.; Obradovic, Z.; Li, X.; Garner, E. C.; Brown, C. J.; Dunker, A. K. Sequence
complexity of disordered protein. Proteins 2001, 42 (1), 38-48. DOI: 10.1002/1097-
0134(20010101)42:1<38::aid-prot50>3.0.co;2-3.
                                                 38


(89) Garner, E.; Cannon, P.; Romero, P.; Obradovic, Z.; Dunker, A. K. Predicting Disordered
Regions from Amino Acid Sequence: Common Themes Despite Differing Structural
Characterization. Genome informatics. Workshop on Genome Informatics 1998, 9, 201-213.
(90) Dunker, A. K.; Garner, E.; Guilliot, S.; Romero, P.; Albrecht, K.; Hart, J.; Obradovic, Z.;
Kissinger, C.; Villafranca, J. E. Protein disorder and the evolution of molecular recognition:
theory, predictions and observations. Pacific Symposium on Biocomputing. Pacific Symposium
on Biocomputing 1998, 473-484.
(91) Xue, B.; Dunbrack, R. L.; Williams, R. W.; Dunker, A. K.; Uversky, V. N. PONDR-FIT: a
meta-predictor of intrinsically disordered amino acids. Biochimica et biophysica acta 2010, 1804
(4), 996-1010. DOI: 10.1016/j.bbapap.2010.01.011.
(92) Mészáros, B.; Erdos, G.; Dosztányi, Z. IUPred2A: context-dependent prediction of protein
disorder as a function of redox state and protein binding. Nucleic acids research 2018, 46 (W1),
329-337. DOI: 10.1093/nar/gky384.
(93) Hanson, J.; Paliwal, K.; Zhou, Y. Accurate Single-Sequence Prediction of Protein Intrinsic
Disorder by an Ensemble of Deep Recurrent and Convolutional Architectures. Journal of
chemical information and modeling 2018, 58 (11), 2369-2376. DOI: 10.1021/acs.jcim.8b00636.
(94) Goda, N.; Shimizu, K.; Kuwahara, Y.; Tenno, T.; Noguchi, T.; Ikegami, T.; Ota, M.;
Hiroaki, H. A Method for Systematic Assessment of Intrinsically Disordered Protein Regions by
NMR. International journal of molecular sciences 2015, 16 (7), 15743-15760. DOI:
10.3390/ijms160715743.
(95) Kumagai, P.; Araujo, A.; Lopes, J. Going deep into protein secondary structure with
synchrotron radiation circular dichroism spectroscopy. Biophysical reviews 2017, 9 (5), 517-527.
DOI: 10.1007/s12551-017-0314-2.
(96) Haynes, C.; Oldfield, J.; Ji, F.; Klitgord, N.; Cusick, E.; Radivojac, P.; Uversky, V.; Vidal,
M.; Iakoucheva, L. Intrinsic Disorder Is a Common Feature of Hub Proteins from Four
Eukaryotic Interactomes. PLoS Comput Biol 2006, 2 (8). DOI: 10.1371/journal.pcbi.0020100.
(97) Singh, G.; Ganapathi, M.; Dash, D. Role of intrinsic disorder in transient interactions of hub
proteins. Proteins 2007, 66 (4), 761-765. DOI: 10.1002/prot.21281.
(98) Wright, P. E.; Dyson, H. J. Intrinsically disordered proteins in cellular signalling and
regulation. Nature reviews. Molecular cell biology 2015, 16 (1), 18-29. DOI: 10.1038/nrm3920.
(99) Biran, A.; Myers, N.; Adler, J.; Broennimann, K.; Reuven, N.; Shaul, Y. A 20S proteasome
receptor for degradation of intrinsically disordered proteins. bioRxiv 2017, 210898. DOI:
10.1101/210898.
(100) Uversky, V. N. Intrinsically disordered proteins and their (disordered) proteomes in
neurodegenerative disorders. Frontiers in aging neuroscience 2015, 7, 18-18. DOI:
10.3389/fnagi.2015.00018.
                                                  39


(101) Alvarez-Castelao, B.; Goethals, M.; Vandekerckhove, J.; Castaño, J. G. Mechanism of
cleavage of alpha-synuclein by the 20S proteasome and modulation of its degradation by the
RedOx state of the N-terminal methionines. Biochimica et biophysica acta 2014, 1843 (2), 352-
365. DOI: 10.1016/j.bbamcr.2013.11.018.
(102) Asher, G.; Bercovich, Z.; Tsvetkov, P.; Shaul, Y.; Kahana, C. 20S Proteasomal
Degradation of Ornithine Decarboxylase Is Regulated by NQO1. Molecular Cell 2005, 17 (5),
645-655. DOI: 10.1016/j.molcel.2005.01.020.
(103) Asher, G.; Reuven, N.; Shaul, Y. 20S proteasomes and protein degradation “by default”.
BioEssays 2006, 28 (8), 844-849. DOI: 10.1002/bies.20447.
(104) Choi, M. L.; Gandhi, S. Crucial role of protein oligomerization in the pathogenesis of
Alzheimer's and Parkinson's diseases. The FEBS Journal 2018, 285 (19), 3631-3644. DOI:
10.1111/febs.14587.
(105) Raychaudhuri, S.; Dey, S.; Bhattacharyya, N. P.; Mukhopadhyay, D. The Role of
Intrinsically Unstructured Proteins in Neurodegenerative Diseases. PLoS ONE 2009, 4 (5),
e5566-e5566. DOI: 10.1371/journal.pone.0005566.
(106) Babu, M. M.; van der Lee, R.; de Groot, N. S.; Gsponer, J. Intrinsically disordered
proteins: Regulation and disease. Elsevier Current Trends: 2011; Vol. 21, pp 432-440.
(107) Midic, U.; Oldfield, C. J.; Dunker, A. K.; Obradovic, Z.; Uversky, V. N. Protein disorder
in the human diseasome: unfoldomics of human genetic diseases. BMC Genomics 2009, 10
(Suppl 1), S12-S12. DOI: 10.1186/1471-2164-10-S1-S12.
(108) Uversky, V. N. Wrecked regulation of intrinsically disordered proteins in diseases:
pathogenicity of deregulated regulators. Frontiers in Molecular Biosciences 2014, 1, 6-6. DOI:
10.3389/fmolb.2014.00006.
(109) Alam, P.; Bousset, L.; Melki, R.; Otzen, D. E. α-synuclein oligomers and fibrils: a
spectrum of species, a spectrum of toxicities. Journal of neurochemistry 2019, 150 (5). DOI:
10.1111/jnc.14808.
(110) Cheng, B.; Gong, H.; Xiao, H.; Petersen, R. B.; Zheng, L.; Huang, K. Inhibiting toxic
aggregation of amyloidogenic proteins: a therapeutic strategy for protein misfolding diseases.
Biochimica et biophysica acta 2013, 1830 (10), 4860-4871. DOI: 10.1016/j.bbagen.2013.06.029.
(111) Baugh, J.; Viktorova, E.; Pilipenko, E. Proteasomes Can Degrade a Significant Proportion
of Cellular Proteins Independent of Ubiquitination. Journal of Molecular Biology 2009, 386 (3),
814-827. DOI: doi.org/10.1016/j.jmb.2008.12.081.
(112) Pickering, A. M.; Davies, K. J. Degradation of damaged proteins: the main function of the
20S proteasome. Progress in molecular biology and translational science 2012, 109, 227-248.
DOI: 10.1016/B978-0-12-397863-9.00006-7.
                                                 40


(113) Erales, J.; Coffino, P. Ubiquitin-independent proteasomal degradation. Biochimica et
biophysica acta 2014, 1843 (1), 216-221. DOI: 10.1016/j.bbamcr.2013.05.008.
(114) Tanaka, K.; Yoshimura, T.; Ichihara, A. Role of substrate in reversible activation of
proteasomes (multi-protease complexes) by sodium dodecyl sulfate. Journal of biochemistry
1989, 106 (3), 495-500.
(115) Thibaudeau, T. A.; Anderson, R. T.; Smith, D. M. A common mechanism of proteasome
impairment by neurodegenerative disease-associated oligomers. Nature Communications 2018, 9
(1), 1097-1097. DOI: 10.1038/s41467-018-03509-0.
(116) Smith, D. M. Could a Common Mechanism of Protein Degradation Impairment Underlie
Many Neurodegenerative Diseases? Journal of experimental neuroscience 2018, 12,
1179069518794675-1179069518794675. DOI: 10.1177/1179069518794675.
(117) Bence, N.; Sampat, R.; Kopito, R. Impairment of the ubiquitin-proteasome system by
protein aggregation. Science (New York, N.Y.) 2001, 292 (5521). DOI:
10.1126/science.292.5521.1552.
(118) Deger, J. M.; Gerson, J. E.; Kayed, R. The interrelationship of proteasome impairment and
oligomeric intermediates in neurodegeneration. Aging cell 2015, 14 (5), 715-724. DOI:
10.1111/acel.12359.
(119) Tseng, B. P.; Green, K. N.; Chan, J. L.; Blurton-Jones, M.; LaFerla, F. M. Abeta inhibits
the proteasome and enhances amyloid and tau accumulation. Neurobiology of aging 2008, 29
(11), 1607-1618. DOI: 10.1016/j.neurobiolaging.2007.04.014.
(120) Zondler, L.; Kostka, M.; Garidel, P.; Heinzelmann, U.; Hengerer, B.; Mayer, B.;
Weishaupt, J. H.; Gillardon, F.; Danzer, K. M. Proteasome impairment by α-synuclein. PLOS
ONE 2017, 12 (9), e0184040-e0184040. DOI: 10.1371/journal.pone.0184040.
(121) Kundra, R.; Ciryam, P.; Morimoto, R. I.; Dobson, C. M.; Vendruscolo, M. Protein
homeostasis of a metastable subproteome associated with Alzheimer's disease. Proceedings of
the National Academy of Sciences of the United States of America 2017, 114 (28), E5703-E5711.
DOI: 10.1073/pnas.1618417114.
(122) Sala, A. J.; Bott, L. C.; Morimoto, R. I. Shaping proteostasis at the cellular, tissue, and
organismal level. The Journal of cell biology 2017, 216 (5), 1231-1241. DOI:
10.1083/jcb.201612111.
(123) Danzer, K. M.; Haasen, D.; Karow, A. R.; Moussaud, S.; Habeck, M.; Giese, A.;
Kretzschmar, H.; Hengerer, B.; Kostka, M. Different Species of -Synuclein Oligomers Induce
Calcium Influx and Seeding. Journal of Neuroscience 2007, 27 (34), 9220-9232. DOI:
10.1523/JNEUROSCI.2617-07.2007.
(124) Pinotsi, D.; Michel, C. H.; Buell, A. K.; Laine, R. F.; Mahou, P.; Dobson, C. M.;
Kaminski, C. F.; Kaminski Schierle, G. S. Nanoscopic insights into seeding mechanisms and
                                                 41


toxicity of α-synuclein species in neurons. Proceedings of the National Academy of Sciences
2016, 113 (14), 3815-3819. DOI: 10.1073/pnas.1516546113.
(125) Gerson, J. E.; Sengupta, U.; Kayed, R. Tau Oligomers as Pathogenic Seeds: Preparation
and Propagation In Vitro and In Vivo. Vol. 1523; 2017; pp 141-157.
(126) Garcia, P.; Jürgens-Wemheuer, W.; Huarte, O.; Michelucci, A.; Masuch, A.; Brioschi, S.;
Weihofen, A.; Koncina, E.; Coowar, D.; Heurtaux, T.; et al. Neurodegeneration and
neuroinflammation are linked, but independent of alpha-synuclein inclusions, in a
seeding/spreading mouse model of Parkinson's disease. Glia 2022, 70 (5), 935-960. DOI:
10.1002/glia.24149.
(127) Kayed, R.; Dettmer, U.; Lesné, S. E. Soluble endogenous oligomeric α-synuclein species
in neurodegenerative diseases: Expression, spreading, and cross-talk. Journal of Parkinson's
disease 2020, 10 (3), 791-818. DOI: 10.3233/JPD-201965.
(128) Lopes da Fonseca, T.; Villar-Piqué, A.; Outeiro, T. The Interplay between Alpha-
Synuclein Clearance and Spreading. Biomolecules 2015, 5 (2), 435-471. DOI:
10.3390/biom5020435.
(129) Thakur, P.; Breger, L. S.; Lundblad, M.; Wan, O. W.; Mattsson, B.; Luk, K. C.; Lee, V. M.
Y.; Trojanowski, J. Q.; Björklund, A. Modeling Parkinson's disease pathology by combination of
fibril seeds and α-synuclein overexpression in the rat brain. Proceedings of the National
Academy of Sciences of the United States of America 2017, 114 (39), E8284-E8293. DOI:
10.1073/pnas.1710442114.
(130) Alafuzoff, I.; Hartikainen, P. Alpha-synucleinopathies. Handbook of clinical neurology
2017, 145, 339-353. DOI: 10.1016/B978-0-12-802395-2.00024-9.
(131) Gammon, K. Neurodegenerative disease: brain windfall. Nature 2014, 515 (7526), 299-
300. DOI: 10.1038/nj7526-299a.
(132) Koyuncu, S.; Fatima, A.; Gutierrez-Garcia, R.; Vilchez, D. Proteostasis of Huntingtin in
Health and Disease. International journal of molecular sciences 2017, 18 (7). DOI:
10.3390/ijms18071568.
(133) Uversky, V. N.; Oldfield, C. J.; Midic, U.; Xie, H.; Xue, B.; Vucetic, S.; Iakoucheva, L.
M.; Obradovic, Z.; Dunker, A. K. Unfoldomics of human diseases: linking protein intrinsic
disorder with diseases. BMC genomics 2009, 10 Suppl 1 (Suppl 1), S7-S7. DOI: 10.1186/1471-
2164-10-S1-S7.
(134) Opoku-Nsiah, K. A.; Gestwicki, J. E. Aim for the core: suitability of the ubiquitin-
independent 20S proteasome as a drug target in neurodegeneration. Mosby Inc.: 2018; Vol. 198,
pp 48-57.
(135) Jones, C. L.; Njomen, E.; Sjögren, B.; Dexheimer, T. S.; Tepe, J. J. Small Molecule
Enhancement of 20S Proteasome Activity Targets Intrinsically Disordered Proteins. ACS
Chemical Biology 2017, 12 (9), 2240-2247. DOI: 10.1021/acschembio.7b00489.
                                                42


(136) Njomen, E.; Osmulski, P. A.; Jones, C. L.; Gaczynska, M.; Tepe, J. J. Small Molecule
Modulation of Proteasome Assembly. Biochemistry 2018, 57 (28), 4214-4224. DOI:
10.1021/acs.biochem.8b00579.
(137) Liu, Y.; Hettinger, C. L.; Zhang, D.; Rezvani, K.; Wang, X.; Wang, H. The proteasome
function reporter GFPu accumulates in young brains of the APPswe/PS1dE9 Alzheimer's disease
mouse model. Cellular and molecular neurobiology 2014, 34 (3), 315-322. DOI:
10.1007/s10571-013-0022-9.
(138) Grune, T. Oxidative stress, aging and the proteasomal system. Biogerontology 2000, 1 (1),
31-40. DOI: 10.1023/a:1010037908060.
(139) Zabel, C.; Nguyen, H. P.; Hin, S. C.; Hartl, D.; Mao, L.; Klose, J. Proteasome and
oxidative phoshorylation changes may explain why aging is a risk factor for neurodegenerative
disorders. Journal of Proteomics 2010, 73 (11), 2230-2238. DOI: 10.1016/j.jprot.2010.08.008.
(140) Vigouroux, S.; Briand, M.; Briand, Y. Linkage between the proteasome pathway and
neurodegenerative diseases and aging. Molecular neurobiology 2004, 30 (2), 201-221. DOI:
10.1385/MN:30:2:201.
(141) Coleman, R. A.; Trader, D. J. All About the Core: A Therapeutic Strategy to Prevent
Protein Accumulation with Proteasome Core Particle Stimulators. ACS Pharmacology &
Translational Science 2018, 1 (2), 140-142. DOI: 10.1021/acsptsci.8b00042.
(142) Coleman, R.; Mohallem, R.; Aryal, U.; Trader, D. Protein degradation profile reveals
dynamic nature of 20S proteasome small molecule stimulation. RSC Chemical Biology 2021, 2,
636-644. DOI: 10.1039/D0CB00191K.
(143) Tsvetkov, P.; Reuven, N.; Shaul, Y. The nanny model for IDPs. Nature Chemical Biology
2009, 5 (11), 778-781, BriefCommunication. DOI: doi:10.1038/nchembio.233.
(144) Kulkarni, A.; Preeti, K.; Tryphena, K. P.; Srivastava, S.; Singh, S. B.; Khatri, D. K.
Proteostasis in Parkinson's disease: Recent development and possible implication in diagnosis
and therapeutics. Ageing research reviews 2023, 84, 101816. DOI: 10.1016/j.arr.2022.101816.
(145) (WHO), W. H. O. Parkinson disease. 2022. (accessed 2023 April 15th).
(146) Aging, N. N. I. o. Parkinson’s Disease: Causes, Symptoms, and Treatments. @NIHAging,
2022. https://www.ncbi.nlm.nih.gov/pubmed/ (accessed 2023 April 9th).
(147) Bloem, B.; Okun, M.; Klein, C. Parkinson's disease. The Lancet 2021, 397 (10291), 12-18.
DOI: doi.org/10.1016/S0140-6736(21)00218-X.
(148) Hornykiewicz, O. A brief history of levodopa. Journal of Neurology 2010, 257 (2), 249-
252, OriginalPaper. DOI: doi:10.1007/s00415-010-5741-y.
(149) Gandhi, K. R.; Saadabadi, A. Levodopa (L-Dopa); StatPearls Publishing, 2022. DOI:
https://www.ncbi.nlm.nih.gov/books/NBK482140/.
                                                43


(150) Muller, T. Pharmacokinetics and pharmacodynamics of levodopa/carbidopa cotherapies
for Parkinson’s disease. Expert Opinion on Drug Metabolism & Toxicology 2020, 16 (5), 403-
414, review. DOI: doi.org/10.1080/17425255.2020.1750596.
(151) Lehtonen, Š.; Sonninen, T. M.; Wojciechowski, S.; Goldsteins, G.; Koistinaho, J.
Dysfunction of Cellular Proteostasis in Parkinson's Disease. Frontiers in neuroscience 2019, 13
(10), 457. DOI: 10.3389/fnins.2019.00457.
(152) McNaught, K. S. P.; Olanow, C. W.; Halliwell, B.; Isacson, O.; Jenner, P. Failure of the
ubiquitin–proteasome system in Parkinson's disease. Nature Reviews Neuroscience 2001, 2 (8),
589-594. DOI: 10.1038/35086067.
(153) McNaught, K. S.; Belizaire, R.; Isacson, O.; Jenner, P.; Olanow, C. W. Altered
proteasomal function in sporadic Parkinson's disease. Experimental neurology 2003, 179 (1), 38-
46. DOI: 10.1006/exnr.2002.8050.
(154) Kurtishi, A.; Rosen, B.; Patil, K. S.; Alves, G. W.; G., M. S. Cellular Proteostasis in
Neurodegeneration. Molecular neurobiology 2019, 56 (5), 3676-3689. DOI: 10.1007/s12035-
018-1334-z.
(155) Emmanouilidou, E.; Stefanis, L.; Vekrellis, K. Cell-produced alpha-synuclein oligomers
are targeted to, and impair, the 26S proteasome. Neurobiology of aging 2010, 31 (6), 953-968.
DOI: 10.1016/j.neurobiolaging.2008.07.008.
(156) Lindersson, E.; Beedholm, R.; Højrup, P.; Moos, T.; Gai, W.; Hendil, K. B.; Jensen, P. H.
Proteasomal inhibition by alpha-synuclein filaments and oligomers. The Journal of biological
chemistry 2004, 279 (13), 12924-12934. DOI: 10.1074/jbc.M306390200.
(157) Snyder, H.; Mensah, K.; Theisler, C.; Lee, J.; Matouschek, A.; Wolozin, B. Aggregated
and monomeric alpha-synuclein bind to the S6' proteasomal protein and inhibit proteasomal
function. The Journal of biological chemistry 2003, 278 (14), 11753-11759. DOI:
10.1074/jbc.M208641200.
(158) Ingelsson, M. Alpha-Synuclein Oligomers-Neurotoxic Molecules in Parkinson's Disease
and Other Lewy Body Disorders. Frontiers in neuroscience 2016, 10, 408-408. DOI:
10.3389/fnins.2016.00408.
(159) Winner, B.; Jappelli, R.; Maji, S. K.; Desplats, P. A.; Boyer, L.; Aigner, S.; Hetzer, C.;
Loher, T.; Vilar, M.; Campioni, S.; et al. In vivo demonstration that -synuclein oligomers are
toxic. Proceedings of the National Academy of Sciences 2011, 108 (10), 4194-4199. DOI:
10.1073/pnas.1100976108.
(160) Bengoa-Vergniory, N.; Roberts, R. F.; Wade-Martins, R.; Alegre-Abarrategui, J. Alpha-
synuclein oligomers: a new hope. Acta neuropathologica 2017, 134 (6), 819-838. DOI:
10.1007/s00401-017-1755-1.
                                                 44


(161) Conway, K. A.; Harper, J. D.; Lansbury, P. T. Accelerated in vitro fibril formation by a
mutant α-synuclein linked to early-onset Parkinson disease. Nature Medicine 1998, 4 (11), 1318-
1320. DOI: 10.1038/3311.
(162) Ibáñez, P.; Bonnet, A. M.; Débarges, B.; Lohmann, E.; Tison, F.; Agid, Y.; Dürr, A.;
Brice, A.; Pollak, P. Causal relation between α-synuclein locus duplication as a cause of familial
Parkinson's disease. The Lancet 2004, 364 (9440), 1169-1171. DOI: 10.1016/S0140-
6736(04)17104-3.
(163) Liu, C.; Zhao, Y.; Xi, H.; Jiang, J.; Yu, Y.; Dong, W. The Membrane Interaction of Alpha-
Synuclein. Frontiers in cellular neuroscience 2021, 15, 633727. DOI:
10.3389/fncel.2021.633727.
(164) Iwai, A.; Masliah, E.; Yoshimoto, M.; Ge, N.; Flanagan, L.; de Silva, H. A.; Kittel, A.;
Saitoh, T. The precursor protein of non-A beta component of Alzheimer's disease amyloid is a
presynaptic protein of the central nervous system. Neuron 1995, 14 (2), 467-475. DOI:
10.1016/0896-6273(95)90302-x.
(165) Jakes, R.; Spillantini, M. G.; Goedert, M. Identification of two distinct synucleins from
human brain. FEBS letters 1994, 345 (1), 27-32. DOI: 10.1016/0014-5793(94)00395-5.
(166) Withers, G. S.; George, J. M.; Banker, G. A.; Clayton, D. F. Delayed localization of
synelfin (synuclein, NACP) to presynaptic terminals in cultured rat hippocampal neurons. Brain
research. Developmental brain research 1997, 99 (1), 87-94. DOI: 10.1016/s0165-
3806(96)00210-6.
(167) Kahle, P. J.; Neumann, M.; Ozmen, L.; Muller, V.; Jacobsen, H.; Schindzielorz, A.;
Okochi, M.; Leimer, U.; van Der Putten, H.; Probst, A.; et al. Subcellular localization of wild-
type and Parkinson's disease-associated mutant alpha -synuclein in human and transgenic mouse
brain. The Journal of neuroscience : the official journal of the Society for Neuroscience 2000, 20
(17), 6365-6373. DOI: 10.1523/JNEUROSCI.20-17-06365.2000.
(168) Zhang, L.; Zhang, C.; Zhu, Y.; Cai, Q.; Chan, P.; Uéda, K.; Yu, S.; Yang, H. Semi-
quantitative analysis of alpha-synuclein in subcellular pools of rat brain neurons: an immunogold
electron microscopic study using a C-terminal specific monoclonal antibody. Brain research
2008, 1244, 40-52. DOI: 10.1016/j.brainres.2008.08.067.
(169) Chartier-Harlin, M. C.; Kachergus, J.; Roumier, C.; Mouroux, V.; Douay, X.; Lincoln, S.;
Levecque, C.; Larvor, L.; Andrieux, J.; Hulihan, M.; et al. Alpha-synuclein locus duplication as a
cause of familial Parkinson's disease. Lancet (London, England) 2004, 364 (9440), 1167-1169.
DOI: 10.1016/S0140-6736(04)17103-1.
(170) Miller, D. W.; Hague, S. M.; Clarimon, J.; Baptista, M.; Gwinn-Hardy, K.; Cookson, M.
R.; Singleton, A. B. α-Synuclein in blood and brain from familial Parkinson disease with SNCA
locus triplication. Neurology 2004, 62 (10). DOI: 10.1212/01.WNL.0000127517.33208.F4.
                                                 45


(171) Singleton, A. B.; Farrer, M.; Johnson, J.; Singleton, A.; Hague, S.; Kachergus, J.; Hulihan,
M.; Peuralinna, T.; Dutra, A.; Nussbaum, R.; et al. -Synuclein Locus Triplication Causes
Parkinson's Disease. Science 2003, 302 (5646), 841-841. DOI: 10.1126/science.1090278.
(172) Krüger, R.; Kuhn, W.; Müller, T.; Woitalla, D.; Graeber, M.; Kösel, S.; Przuntek, H.;
Epplen, T.; Schöls, L.; Riess, O. Ala30Pro mutation in the gene encoding alpha-synuclein in
Parkinson's disease. Nature genetics 1998, 18 (2), 106-108. DOI: 10.1038/ng0298-106.
(173) Simón-Sánchez, J.; Schulte, C.; Bras, M.; Sharma, M.; Gibbs, R.; Berg, D.; Paisan-Ruiz,
C.; Lichtner, P.; Scholz, W.; Hernandez, G.; et al. Genome-wide association study reveals
genetic risk underlying Parkinson's disease. Nature genetics 2009, 41 (12), 1308-1312. DOI:
10.1038/ng.487.
(174) Polymeropoulos, M. H.; Lavedan, C.; Leroy, E.; Ide, S. E.; Dehejia, A.; Dutra, A.; Pike,
B.; Root, H.; Rubenstein, J.; Boyer, R.; et al. Mutation in the -Synuclein Gene Identified in
Families with Parkinson's Disease. Science 1997, 276 (5321), 2045-2047. DOI:
10.1126/science.276.5321.2045.
(175) Zarranz, J.; Alegre, J.; Gómez-Esteban, C.; Lezcano, E.; Ros, R.; Ampuero, I.; Vidal, L.;
Hoenicka, J.; Rodriguez, O.; Atarés, B.; et al. The new mutation, E46K, of alpha-synuclein
causes Parkinson and Lewy body dementia. Annals of neurology 2004, 55 (2), 164-173. DOI:
10.1002/ana.10795.
(176) Conway, K. A.; Harper, J. D.; Lansbury, P. T. Accelerated in vitro fibril formation by a
mutant alpha-synuclein linked to early-onset Parkinson disease. Nature medicine 1998, 4 (11),
1318-1320. DOI: 10.1038/3311.
(177) Lashuel, H. A.; Overk, C. R.; Oueslati, A.; Masliah, E. The many faces of α-synuclein:
from structure and toxicity to therapeutic target. Nat Rev Neurosci 2013, 14 (1), 38-48. DOI:
10.1038/nrn3406.
(178) Cremades, N.; Cohen, S. I. A.; Deas, E.; Abramov, A. Y.; Chen, A. Y.; Orte, A.; Sandal,
M.; Clarke, R. W.; Dunne, P.; Aprile, F. A.; et al. Direct observation of the interconversion of
normal and toxic forms of α-synuclein. Cell 2012, 149 (5), 1048-1059. DOI:
10.1016/j.cell.2012.03.037.
(179) Brettschneider, J.; Del Tredici, K.; Lee, V. M.; Trojanowski, J. Q. Spreading of pathology
in neurodegenerative diseases: a focus on human studies. Nature reviews. Neuroscience 2015, 16
(2), 109-120. DOI: 10.1038/nrn3887.
(180) Rubinsztein, D. C. The roles of intracellular protein-degradation pathways in
neurodegeneration. Nature 2006, 443 (7113), 780-786. DOI: 10.1038/nature05291.
(181) Selkoe, D. J. Folding proteins in fatal ways. Nature 2003, 426 (6968), 900-904,
ReviewPaper. DOI: doi:10.1038/nature02264.
(182) Aging, N. N. I. o. What Is Alzheimer's Disease? NIH, 2021.
https://www.ncbi.nlm.nih.gov/pubmed/ (accessed 2023 April 9th).
                                                 46


(183) Lane, C.; Hardy, J.; Schott, J. Alzheimer's disease. European Journal of Neurology 2018,
15 (1), 59-70. DOI: 10.1111/ene.13439.
(184) Hampel, H.; Hardy, J.; Blennow, K.; Chen, C.; Perry, G.; Kim, S. H.; Villemagne, V. L.;
Aisen, P.; Vendruscolo, M.; Iwatsubo, T.; et al. The Amyloid-β Pathway in Alzheimer’s Disease.
Molecular Psychiatry 2021, 26 (10), 5481-5503, ReviewPaper. DOI: doi:10.1038/s41380-021-
01249-0.
(185) Jeong, S. Molecular and Cellular Basis of Neurodegeneration in Alzheimer's Disease.
Molecules and cells 2017, 40 (9), 613-620. DOI: 10.14348/molcells.2017.0096.
(186) Korsak, M.; Kozyreva, T. Beta Amyloid Hallmarks: From Intrinsically Disordered
Proteins to Alzheimer’s Disease. Springer, Cham, 2015; pp 401-421.
(187) Selkoe, D. J.; Hardy, J. The amyloid hypothesis of Alzheimer's disease at 25 years. EMBO
molecular medicine 2016, 8 (6), 595-608. DOI: 10.15252/emmm.201606210.
(188) Michaels, T. C. T.; Šarić, A.; Curk, S.; Bernfur, K.; Arosio, P.; Meisl, G.; Dear, A. J.;
Cohen, S. I. A.; Dobson, C. M.; Vendruscolo, M.; et al. Dynamics of oligomer populations
formed during the aggregation of Alzheimer’s Aβ42 peptide. Nature Chemistry 2020, 12 (5),
445-451, OriginalPaper. DOI: doi:10.1038/s41557-020-0452-1.
(189) Gruning, C.; Klinker, S.; Wolff, M.; Schneider, M.; Toksoz, K.; Klein, A.; Nagel-Steger,
L.; Willbold, D.; Hoyer, W. The Off-rate of Monomers Dissociating from Amyloid-β
Protofibrils. Journal of Biological Chemistry 2013, 288 (52), 37104-37111. DOI:
10.1074/jbc.M113.513432.
(190) Carulla, N.; Caddy, G. L.; Hall, D. R.; Zurdo, J.; Gairí, M.; Feliz, M.; Giralt, E.; Robinson,
C. V.; Dobson, C. M. Molecular recycling within amyloid fibrils. Nature 2023, 436 (7050), 554-
558, OriginalPaper. DOI: doi:10.1038/nature03986.
(191) Haass, C.; Selkoe, D. J. Soluble protein oligomers in neurodegeneration: lessons from the
Alzheimer's amyloid beta-peptide. Nature reviews. Molecular cell biology 2007, 8 (2), 101-112.
DOI: 10.1038/nrm2101.
(192) Shankar, M.; Li, S.; Mehta, H.; Garcia-Munoz, A.; Shepardson, E.; Smith, I.; Brett, M.;
Farrell, A.; Rowan, J.; Lemere, A.; et al. Amyloid-beta protein dimers isolated directly from
Alzheimer's brains impair synaptic plasticity and memory. Nature medicine 2008, 14 (8), 837-
842. DOI: 10.1038/nm1782.
(193) Brinkmalm, G.; Hong, W.; Wang, Z.; Liu, W.; O'Malley, T.; Sun, X.; Frosch, P.; Selkoe,
J.; Portelius, E.; Zetterberg, H.; et al. Identification of neurotoxic cross-linked amyloid-β dimers
in the Alzheimer's brain. Brain : a journal of neurology 2019, 142 (5), 1441-1457. DOI:
10.1093/brain/awz066.
(194) Walsh, M.; Klyubin, I.; Fadeeva, V.; Cullen, K.; Anwyl, R.; Wolfe, S.; Rowan, J.; Selkoe,
J. Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term
potentiation in vivo. Nature 2002, 416 (6880), 535-539. DOI: 10.1038/416535a.
                                                     47


(195) Selkoe, J. Soluble oligomers of the amyloid beta-protein impair synaptic plasticity and
behavior. Behavioural brain research 2008, 192 (1), 106-113. DOI: 10.1016/j.bbr.2008.02.016.
(196) Hardiman, O.; Al-Chalabi, A.; Chio, A.; Corr, E. M.; Logroscino, G.; Robberecht, W.;
Shaw, P. J.; Simmons, Z.; van den Berg, L. H. Amyotrophic lateral sclerosis. Nature Reviews
Disease Primers 2017, 3 (1), 1-19, ReviewPaper. DOI: doi:10.1038/nrdp.2017.71.
(197) Medinas, D.; Vicente, V.; Claudio, H. Proteostasis disturbance in amyotrophic lateral
sclerosis. Human Molecular Genetics 2017, 26 (R2). DOI: 10.1093/hmg/ddx274.
(198) Mori, K.; Weng, S.-M.; Arzberger, T.; May, S.; Rentzsch, K.; Kremmer, E.; Schmid, B.;
Kretzschmar, H. A.; Cruts, M.; Broeckhoven, C. V.; et al. The C9orf72 GGGGCC Repeat Is
Translated into Aggregating Dipeptide-Repeat Proteins in FTLD/ALS. Science 2013, 339
(6125), 1335-1338, research-article. DOI: 10.1126/science.1232927.
(199) Ruegsegger, C.; Saxena, S. Proteostasis impairment in ALS. Brain research 2016, 1648,
571-579. DOI: 10.1016/j.brainres.2016.03.032.
(200) Su, X.; Maguire-Zeiss, K.; Giuliano, R.; Prifti, L.; Venkatesh, K.; Federoff, H. Synuclein
activates microglia in a model of Parkinson's disease. Neurobiology of Aging 2007, 29 (11),
1690-1701. DOI: 10.1016/j.neurobiolaging.2007.04.006.
(201) Hoenen, C.; Gustin, A.; Birck, C.; Kirchmeyer, M.; Beaume, N.; Felten, P.; Grandbarbe,
L.; Heuschling, P.; Heurtaux, T. Alpha-Synuclein Proteins Promote Pro-Inflammatory Cascades
in Microglia: Stronger Effects of the A53T Mutant. PloS one 2016, 11 (9), e0162717. DOI:
10.1371/journal.pone.0162717.
(202) Cao, S.; Standaert, D.; Harms, A. The gamma chain subunit of Fc receptors is required for
alpha-synuclein-induced pro-inflammatory signaling in microglia. Journal of neuroinflammation
2012, 9, 259. DOI: 10.1186/1742-2094-9-259.
(203) Song, N.; Chen, L.; Xie, J. Alpha-Synuclein Handling by Microglia: Activating,
Combating, and Worsening. Neuroscience Bulletin 2021, 37 (5), 751-753, OriginalPaper. DOI:
doi:10.1007/s12264-021-00651-6.
(204) Xia, Y.; Zhang, G.; Kou, L.; Yin, S.; Han, C.; Hu, J.; Wan, F.; Sun, Y.; Wu, J.; Li, Y.; et
al. Reactive microglia enhance the transmission of exosomal α-synuclein via toll-like receptor 2.
Brain : a journal of neurology 2021, 144 (7), 2024-2037. DOI: 10.1093/brain/awab122.
(205) Heneka, M.; Carson, M.; El Khoury, J.; Landreth, G.; Brosseron, F.; Feinstein, D.; Jacobs,
A.; Wyss-Coray, T.; Vitorica, J.; Ransohoff, R.; et al. Neuroinflammation in Alzheimer's disease.
The Lancet. Neurology 2015, 14 (4), 388-405. DOI: 10.1016/S1474-4422(15)70016-5.
(206) Calsolaro, V.; Edison, P. Neuroinflammation in Alzheimer's disease: Current evidence and
future directions. Alzheimer's & dementia : the journal of the Alzheimer's Association 2016, 12
(6), 719-732. DOI: 10.1016/j.jalz.2016.02.010.
                                                48


(207) Nilson, A. N.; English, K. C.; Gerson, J. E.; Barton Whittle, T.; Nicolas Crain, C.; Xue, J.;
Sengupta, U.; Castillo-Carranza, D. L.; Zhang, W.; Gupta, P.; et al. Tau Oligomers Associate
with Inflammation in the Brain and Retina of Tauopathy Mice and in Neurodegenerative
Diseases. Journal of Alzheimer's disease : JAD 2017, 55 (3), 1083-1099. DOI: 10.3233/JAD-
160912.
(208) Onyango, I.; Jauregui, G.; Čarná, M.; Bennett, J.; Stokin, G. Neuroinflammation in
Alzheimer's Disease. Biomedicines 2021, 9 (5), 524. DOI: 10.3390/biomedicines9050524.
(209) Lall, D.; Baloh, R. Microglia and C9orf72 in neuroinflammation and ALS and
frontotemporal dementia. The Journal of clinical investigation 2017, 127 (9), 3250-3258. DOI:
10.1172/JCI90607.
(210) Liu, J.; Wang, F. Role of Neuroinflammation in Amyotrophic Lateral Sclerosis: Cellular
Mechanisms and Therapeutic Implications. Frontiers in immunology 2017, 8, 1005. DOI:
10.3389/fimmu.2017.01005.
(211) Chiot, A.; Lobsiger, C.; Boillée, S. New insights on the disease contribution of
neuroinflammation in amyotrophic lateral sclerosis. Current opinion in neurology 2019, 32 (5),
764-770. DOI: 10.1097/WCO.0000000000000729.
(212) Aloisi, F. Immune function of microglia. Glia 2001, 36 (2), 165-179. DOI:
10.1002/glia.1106.
(213) Ueno, M.; Fujita, Y.; Tanaka, T.; Nakamura, Y.; Kikuta, J.; Ishii, M.; Yamashita, T. Layer
V cortical neurons require microglial support for survival during postnatal development. Nature
neuroscience 2013, 16 (5), 543-551. DOI: 10.1038/nn.3358.
(214) Cunningham, C.; Martínez-Cerdeño, V.; Noctor, S. Microglia regulate the number of
neural precursor cells in the developing cerebral cortex. The Journal of neuroscience : the
official journal of the Society for Neuroscience 2013, 33 (10), 4216-4233. DOI:
10.1523/JNEUROSCI.3441-12.2013.
(215) Frost, J.; Schafer, D. Microglia: Architects of the Developing Nervous System. Trends in
cell biology 2016, 26 (8), 587-597. DOI: 10.1016/j.tcb.2016.02.006.
(216) Soteros, B.; Sia, G. Complement and microglia dependent synapse elimination in brain
development. WIREs mechanisms of disease 2022, 14 (3), e1545. DOI: 10.1002/wsbm.1545.
(217) Orihuela, R.; McPherson, C.; Harry, G. Microglial M1/M2 polarization and metabolic
states. British Journal of Pharmacology 2015, 173 (4), 649-665. DOI: 10.1111/bph.13139.
(218) Hickman, S. E.; Kingery, N. D.; Ohsumi, T. K.; Borowsky, M. L.; Wang, L.-c.; Means, T.
K.; El Khoury, J. The microglial sensome revealed by direct RNA sequencing. Nature
Neuroscience 2013, 16 (12), 1896-1905, OriginalPaper. DOI: doi:10.1038/nn.3554.
(219) Yang, Q.; Zhou, J. Neuroinflammation in the central nervous system: Symphony of glial
cells. Glia 2019, 67 (6), 1017-1035. DOI: 10.1002/glia.23571.
                                                 49


(220) Davis, E.; Foster, T.; Thomas, W. Cellular forms and functions of brain microglia. Brain
research bulletin 1994, 34 (1), 73-78. DOI: 10.1016/0361-9230(94)90189-9.
(221) Li, Q.; Barres, B. A. Microglia and macrophages in brain homeostasis and disease. Nature
Reviews Immunology 2017, 18 (4), 225-242, ReviewPaper. DOI: doi:10.1038/nri.2017.125.
(222) Crews, F.; Newsom, H.; Gerber, M.; Sumners, C.; Chandler, L.; Freund, G. Molecular
Mechanisms of Alcohol Neurotoxicity. In Alcohol, Cell Membranes, and Signal Transduction in
Brain, Springer, 1993.
(223) Gehrmann, J.; Matsumoto, Y.; Kreutzberg, G. Microglia: intrinsic immuneffector cell of
the brain. Brain research. Brain research reviews 1995, 20 (3), 269-287. DOI: 10.1016/0165-
0173(94)00015-h.
(224) Rawlinson, C.; Jenkins, S.; Thei, L.; Dallas, M.; Chen, R. Post-Ischaemic Immunological
Response in the Brain: Targeting Microglia in Ischaemic Stroke Therapy. Brain sciences 2020,
10 (3), 159. DOI: 10.3390/brainsci10030159.
(225) Town, T.; Nikolic, V.; Tan, J. The microglial "activation" continuum: from innate to
adaptive responses. Journal of neuroinflammation 2005, 2, 24. DOI: 10.1186/1742-2094-2-24.
(226) Walker, D.; Lue, L. Immune phenotypes of microglia in human neurodegenerative disease:
challenges to detecting microglial polarization in human brains. Alzheimer's research & therapy
2015, 7 (1), 56. DOI: 10.1186/s13195-015-0139-9.
(227) Sparkman, N.; Johnson, R. Neuroinflammation associated with aging sensitizes the brain
to the effects of infection or stress. Neuroimmunomodulation 2008, 15 (4-6), 323-330. DOI:
10.1159/000156474.
(228) Block, M. L.; Zecca, L.; Hong, J.-S. Microglia-mediated neurotoxicity: uncovering the
molecular mechanisms. Nature Reviews Neuroscience 2007, 8 (1), 57-69, ReviewPaper. DOI:
doi:10.1038/nrn2038.
(229) Grabert, K.; Michoel, T.; Karavolos, M.; Clohisey, S.; Baillie, J.; Stevens, M.; Freeman,
T.; Summers, K.; McColl, B. Microglial brain region-dependent diversity and selective regional
sensitivities to aging. Nature neuroscience 2016, 19 (3), 504-516. DOI: 10.1038/nn.4222.
(230) Deczkowska, A.; Amit, I.; Schwartz, M. Microglial immune checkpoint mechanisms.
Nature neuroscience 2018, 21 (6), 779-786. DOI: 10.1038/s41593-018-0145-x.
(231) Wu, X.; Schauss, A. Mitigation of inflammation with foods. Journal of agricultural and
food chemistry 2012, 60 (27), 6703-6717. DOI: 10.1021/jf3007008.
(232) Rastogi, N.; Mishra, D. P. Therapeutic targeting of cancer cell cycle using proteasome
inhibitors. Cell Division 2012, 7 (1), 1-10, ReviewPaper. DOI: doi:10.1186/1747-1028-7-26.
(233) Tundo, G.; Sbardella, D.; Santoro, A.; Coletta, A.; Oddone, F.; Grasso, G.; Milardi, D.;
Lacal, P.; Marini, S.; Purrello, R.; et al. The proteasome as a druggable target with multiple
                                                   50


therapeutic potentialities: Cutting and non-cutting edges. Pharmacology & therapeutics 2020,
213, 107579. DOI: 10.1016/j.pharmthera.2020.107579.
(234) Huang, X.; Dixit, V. M. Drugging the undruggables: exploring the ubiquitin system for
drug development. Cell Research 2016, 26 (4), 484-498, ReviewPaper. DOI:
doi:10.1038/cr.2016.31.
(235) Ruiz de Mena, I.; Mahillo, E.; Arribas, J.; Castaño, J. G. Kinetic mechanism of activation
by cardiolipin (diphosphatidylglycerol) of the rat liver multicatalytic proteinase. The
Biochemical journal 1993, 296 ( Pt 1) (Pt 1), 93-97.
(236) Watanabe, N.; Yamada, S. Activation of 20S proteasomes from spinach leaves by fatty
acids. Plant & cell physiology 1996, 37 (2), 147-151.
(237) Katsiki, M.; Chondrogianni, N.; Chinou, I.; Rivett, A. J.; Gonos, E. S. The Olive
Constituent Oleuropein Exhibits Proteasome Stimulatory Properties <i>In Vitro</i> and Confers
Life Span Extension of Human Embryonic Fibroblasts. Rejuvenation Research 2007, 10 (2),
157-172. DOI: 10.1089/rej.2006.0513.
(238) Huang, L.; Ho, P.; Chen, C.-H. Activation and inhibition of the proteasome by betulinic
acid and its derivatives. FEBS Letters 2007, 581 (25), 4955-4959. DOI:
10.1016/j.febslet.2007.09.031.
(239) Trader, D. J.; Simanski, S.; Dickson, P.; Kodadek, T. Establishment of a suite of assays
that support the discovery of proteasome stimulators. Biochimica et Biophysica Acta (BBA) -
General Subjects 2017, 1861 (4), 892-899. DOI: 10.1016/J.BBAGEN.2017.01.003.
(240) Coleman, R. A.; Trader, D. J. Development and Application of a Sensitive Peptide
Reporter to Discover 20S Proteasome Stimulators. ACS Combinatorial Science 2018, 20 (5),
269-276. DOI: 10.1021/acscombsci.7b00193.
(241) Uversky, V. N. Targeting intrinsically disordered proteins in neurodegenerative and
protein dysfunction diseases: another illustration of the D(2) concept. Expert review of
proteomics 2010, 7 (4), 543-564. DOI: 10.1586/epr.10.36.
(242) Uversky, V. N. Synuclein Misfolding and Neurodegenerative Diseases; 2008.
https://pdfs.semanticscholar.org/c720/c58ef79cceaf1ceba137bb5208f5ba0df28d.pdf.
(243) Levine, Z. A.; Larini, L.; LaPointe, N. E.; Feinstein, S. C.; Shea, J.-E. Regulation and
aggregation of intrinsically disordered peptides. Proceedings of the National Academy of
Sciences of the United States of America 2015, 112 (9), 2758-2763. DOI:
10.1073/pnas.1418155112.
(244) Raychaudhuri, S.; Majumder, P.; Sarkar, S.; Giri, K.; Mukhopadhyay, D.; Bhattacharyya,
N. P. Huntingtin interacting protein HYPK is intrinsically unstructured. Proteins: Structure,
Function, and Bioinformatics 2007, 71 (4), 1686-1698. DOI: 10.1002/prot.21856.
                                                 51


(245) Uversky, V. N. Intrinsic Disorder in Proteins Associated with Neurodegenerative Diseases.
Springer Netherlands, 2009; pp 21-75.
(246) Longhena, F.; Spano, P.; Bellucci, A. Targeting of Disordered Proteins by Small
Molecules in Neurodegenerative Diseases. Springer, Cham, 2017; pp 85-110.
(247) Cremades, N.; Cohen, S. I. A.; Deas, E.; Abramov, A. Y.; Chen, A. Y.; Orte, A.; Sandal,
M.; Clarke, R. W.; Dunne, P.; Aprile, F. A.; et al. Direct observation of the interconversion of
normal and toxic forms of α-synuclein. Cell 2012, 149 (5), 1048-1059. DOI:
10.1016/j.cell.2012.03.037.
(248) Cozachenco, D.; Ribeiro, F.; Ferreira, S. Defective proteostasis in Alzheimer's disease.
Ageing research reviews 2023, 85, 101862. DOI: 10.1016/j.arr.2023.101862.
(249) Leestemaker, Y.; de Jong, A.; Witting, K. F.; Penning, R.; Schuurman, K.; Rodenko, B.;
Zaal, E. A.; van de Kooij, B.; Laufer, S.; Heck, A. J. R.; et al. Proteasome Activation by Small
Molecules. Cell Chemical Biology 2017, 24 (6), 725-736.e727. DOI:
10.1016/j.chembiol.2017.05.010.
(250) Trippier, P. C.; Zhao, K. T.; Fox, S. G.; Schiefer, I. T.; Benmohamed, R.; Moran, J.;
Kirsch, D. R.; Morimoto, R. I.; Silverman, R. B. Proteasome activation is a mechanism for
pyrazolone small molecules displaying therapeutic potential in amyotrophic lateral sclerosis.
ACS Chemical Neuroscience 2014, 5 (9), 823-829. DOI: 10.1021/cn500147v.
(251) Chondrogianni, N.; Sakellari, M.; Lefaki, M.; Papaevgeniou, N.; Gonos, E. S. Proteasome
activation delays aging in vitro and in vivo. Free Radical Biology and Medicine 2014, 71, 303-
320. DOI: 10.1016/j.freeradbiomed.2014.03.031.
(252) Gonos, E. Proteasome Activation Delays Aging and Protects Against Proteotoxicity in
Neurodegenerative Disease. Vol. 821; 2015; pp 7-7.
(253) Chondrogianni, N.; Voutetakis, K.; Kapetanou, M.; Delitsikou, V.; Papaevgeniou, N.;
Sakellari, M.; Lefaki, M.; Filippopoulou, K.; Gonos, E. S. Proteasome activation: An innovative
promising approach for delaying aging and retarding age-related diseases. Ageing Research
Reviews 2015, 23 (Pt A), 37-55. DOI: 10.1016/j.arr.2014.12.003.
(254) Chondrogianni, N.; Gonos, E. S. Proteasome activation as a novel antiaging strategy.
IUBMB Life 2008, 60 (10), 651-655. DOI: 10.1002/iub.99.
(255) Vilchez, D.; Morantte, I.; Liu, Z.; Douglas, P. M.; Merkwirth, C.; Rodrigues, A. P. C.;
Manning, G.; Dillin, A. RPN-6 determines C. elegans longevity under proteotoxic stress
conditions. Nature 2012, 489 (7415), 263-268. DOI: 10.1038/nature11315.
(256) Kruegel, U.; Robison, B.; Dange, T.; Kahlert, G.; Delaney, J. R.; Kotireddy, S.; Tsuchiya,
M.; Tsuchiyama, S.; Murakami, C. J.; Schleit, J.; et al. Elevated Proteasome Capacity Extends
Replicative Lifespan in Saccharomyces cerevisiae. PLoS Genetics 2011, 7 (9), e1002253-
e1002253. DOI: 10.1371/journal.pgen.1002253.
                                                 52


(257) Mayor, T.; Sharon, M.; Glickman, M. H. Tuning the proteasome to brighten the end of the
journey. American Journal of Physiology-Cell Physiology 2016, 311 (5), C793-C804. DOI:
10.1152/ajpcell.00198.2016.
(258) Chondrogianni, N.; Gonos, E. S. Proteasome Function Determines Cellular Homeostasis
and the Rate of Aging. 2010; pp 38-46.
(259) Kapeta, S.; Chondrogianni, N.; Gonos, E. S. Nuclear Erythroid Factor 2-mediated
Proteasome Activation Delays Senescence in Human Fibroblasts. Journal of Biological
Chemistry 2010, 285 (11), 8171-8184. DOI: 10.1074/jbc.M109.031575.
(260) Kapetanou, M.; Chondrogianni, N.; Petrakis, S.; Koliakos, G.; Gonos, E. S. Proteasome
activation enhances stemness and lifespan of human mesenchymal stem cells. Free Radical
Biology and Medicine 2017, 103, 226-235. DOI: 10.1016/j.freeradbiomed.2016.12.035.
(261) Chondrogianni, N.; Georgila, K.; Kourtis, N.; Tavernarakis, N.; Gonos Efstathios, S.
Enhanced proteasome degradation extends Caenorhabditis elegans lifespan and alleviates
aggregation-related pathologies. Free Radical Biology and Medicine 2014, 75, S18-S18. DOI:
10.1016/j.freeradbiomed.2014.10.632.
(262) Chondrogianni, N.; Gonos, E. S. Overexpression of hUMP1/POMP proteasome accessory
protein enhances proteasome-mediated antioxidant defence. Experimental Gerontology 2007, 42
(9), 899-903. DOI: 10.1016/j.exger.2007.01.012.
(263) Voutetakis, K.; Delitsikou, V.; Magouritsas, M. G.; Gonos, E. S. Anti-ageing properties of
Khelma Longevity™: treatment of human fibroblasts increases proteasome levels and decreases
the levels of oxidized proteins. New Biotechnology 2017, 38 (Pt A), 36-39. DOI:
10.1016/j.nbt.2017.03.002.
(264) Rodriguez, K. A.; Edrey, Y. H.; Osmulski, P.; Gaczynska, M.; Buffenstein, R. Altered
Composition of Liver Proteasome Assemblies Contributes to Enhanced Proteasome Activity in
the Exceptionally Long-Lived Naked Mole-Rat. PLoS ONE 2012, 7 (5), e35890-e35890. DOI:
10.1371/journal.pone.0035890.
(265) Rodriguez, K. A.; Osmulski, P. A.; Pierce, A.; Weintraub, S. T.; Gaczynska, M.;
Buffenstein, R. A cytosolic protein factor from the naked mole-rat activates proteasomes of other
species and protects these from inhibition. Biochimica et Biophysica Acta (BBA) - Molecular
Basis of Disease 2014, 1842 (11), 2060-2072. DOI: 10.1016/j.bbadis.2014.07.005.
(266) Medina, D. X.; Caccamo, A.; Oddo, S. Methylene Blue Reduces Aβ Levels and Rescues
Early Cognitive Deficit by Increasing Proteasome Activity. Brain Pathology 2011, 21 (2), 140-
149. DOI: 10.1111/j.1750-3639.2010.00430.x.
(267) Chondrogianni, N.; Petropoulos, I.; Franceschi, C.; Friguet, B.; Gonos, E. S. Fibroblast
cultures from healthy centenarians have an active proteasome. Experimental gerontology 2000,
35 (6-7), 721-728. DOI: 10.1016/s0531-5565(00)00137-6.
                                                53


(268) Cabreiro, F.; Perichon, M.; Jatje, J.; Malavolta, M.; Mocchegiani, E.; Friguet, B.;
Petropoulos, I. Zinc supplementation in the elderly subjects: Effect on oxidized protein
degradation and repair systems in peripheral blood lymphocytes. Experimental Gerontology
2008, 43 (5), 483-487. DOI: 10.1016/j.exger.2007.10.007.
(269) Tsvetkov, P.; Asher, G.; Paz, A.; Reuven, N.; Sussman, J.; Silman, I.; Shaul, Y.
Operational definition of intrinsically unstructured protein sequences based on susceptibility to
the 20S proteasome. Proteins 2008, 70 (4), 1357-1366. DOI: 10.1002/prot.21614.
(270) Jones, C. L.; Tepe, J. J. Proteasome Activation to Combat Proteotoxicity. Molecules 2019,
24 (15), 2841, Review. DOI: 10.3390/molecules24152841.
(271) Coleman, R. A.; Muli, C. S.; Zhao, Y.; Bhardwaj, A.; Newhouse, T. R.; Trader, D. J.
Analysis of chain length, substitution patterns, and unsaturation of AM-404 derivatives as 20S
proteasome stimulators. Bioorganic & Medicinal Chemistry Letters 2019, 29 (3), 420-423. DOI:
10.1016/J.BMCL.2018.12.030.
(272) George, D.; Tepe, J. Advances in Proteasome Enhancement by Small Molecules.
Biomolecules 2021, 11 (12), 1789. DOI: 10.3390/biom11121789.
                                                 54


                                                       CHAPTER TWO
 Identification and Structure Activity Relationship of Dihydroquinazoline as a Novel 20S
                                            Proteasome Activator Scaffold
    Reproduced in part with permission from Fiolek, T.; Magyar, C.; Wall, T.; Davies, S.; Campbell, M.; Savich, C.; Tepe, J.; Mosey, R.
Dihydroquinazolines enhance 20S proteasome activity and induce degradation of α-synuclein, an intrinsically disordered protein associated with
              neurodegeneration. Bioorganic & Medicinal Chemistry Letters 2021, 36, 127821. Copyright 2021 Elsevier Ltd.
                                                                  55


2.1 Introduction
2.1.1 Background
      The proteasome has long been considered a promising target for drug intervention in a variety
of disease states involving disruptions of normal protein expression and regulation. This is due to
the proteasomes deep involvement in maintaining protein homeostasis (proteostasis) and in
numerous essential biological pathways.1-8 The proteasome has been theorized as a potential drug
target for the treatment of cancer,9-11 neurodegenerative diseases,12-14 and age-related diseases.15-
17
   Modulation of proteasome activity with small molecule drugs was first approached via the use
of proteasome inhibitors.9, 18-20
      Proteasome inhibition has been proven as an effective therapeutic method for the treatment
of certain cancers, specifically multiple myeloma (MM).9, 18-20 The treatment of MM, a malignant
tumor of differentiated B-cells,21,    22
                                          via proteasome inhibition is made possible due to the
production of large quantities of immature immunoglobin by these cells.18 This makes them highly
reliant upon the proteasome to clear these non-functional proteins and allow for the recycling of
the amino acids. Partial inhibition of the proteasome leads to build up of these redundant proteins
and disruption of proteostasis in these cancer cells.18, 19, 23 This disruption of proteostasis induces
cell cycle arrest, inhibition of angiogenesis, and eventual apoptosis of the MM cells preferentially
over healthy cells.11
      Several competitive small molecule inhibitors of the proteasome have been approved by the
FDA, the first of which was bortezomib (BTZ) in 2003 (Fig. 2.1A).24-26 BTZ is a boronate peptide-
based competitive proteasome inhibitor that covalently binds to the active site threonine of the
proteasome to prevent substrate binding.20 These drugs, while effective in many cases at treating
MM and related cancers, suffer from problems associated with resistance and off-target toxicity.27-
                                                  56


30
     These shortcomings led to efforts to develop non-competitive inhibitors of the proteasome,
which would hopefully avoid the development of resistance due to active site mutations or the
over-expression of the β-5 catalytic subunit.31-33
         During the Tepe lab’s exploration of non-competitive inhibitors of the proteasome, it was
discovered that imidazolines, like TCH-165 (Fig. 2.1B),34, 35 were actually able to enhance 20S
proteasome-mediated degradation of fluorogenic peptide substrates in vitro (Fig. 2.1C).36 These
finding, while unexpected, were very exciting and warranted a detailed study to elucidate the
mechanism of TCH-165 mediated 20S proteasome activation.
                                A
     B                                        C                  15
                                               20S activity      10
                                             (fold of control)   5
                                                                 0
                                                                      -8   -7      -6    -5       -4   -3
                                                                                log[M], TCH-165
Figure 2.1: (A) Structure of Bortezomib. (B) Structure of TCH-165. (C) Concentration-
dependent induction of the proteolytic activity of the 20S proteasome by TCH-165. These
data were collected in triplicate (n=3). Error bars denote standard deviation.36
                                                          57


       Several experiments were conducted to explore TCH-165’s mechanism of action on the 20S
proteasome.36 Firstly, the Tepe lab collaborated with Prof. Maria Gaczynska to perform atomic
force microscopy (AFM) imaging experiments of the 20S proteasome. In these experiments, 20S
proteasome particles were repeatedly scanned and imaged by oscillating (tapping) mode AFM in
liquid. Under these conditions the proteasomes constantly change between open and closed forms,
with a ratio of about 3:1 favoring the more stable close-gate conformation.37, 38 Interestingly, it
was found that TCH-165 treatment resulted in a concentration-dependent increase in open-gate
20S proteasomes (Fig. 2.2A/B).36
      To begin exploring how this increase in open-gated conformations might be taking place, in
silico molecular docking models were employed by Dr. Corey Jones in the Tepe lab. The resulting
preferred docking poses suggested that TCH-165 is binding in the α1-2 inter-subunit pocket within
the α-rings of the 20S proteasome (Fig. 2.2C).36 These pockets are typically occupied by the Hb-
Y-X motifs of the C-terminal peptide tails, referred to as the Rpt peptides, of the 19S caps when
assembled into the 26S form of the proteasome.39, 40 These peptide tails allow for docking of the
19S caps and contribute to their ability to induce an open gate conformation of the 20S core particle
upon binding.40 The α1-2 inter-subunit pocket is typically occupied by the Rpt3 peptide of the 19S
cap.
      It was hypothesized that TCH-165 binds in a similar manner to that of the Rpt3 peptide in the
α1-2 inter-subunit pocket and induces a conformational change that promotes α-ring gate-opening
or stabilization of the open-gate conformation. To further test this hypothesis, a competition
experiment was performed by Prof. Maria Gaczynska with TCH-165 and the Rpt3 peptide.
Treatment of the 20S with Rpt3 on its own does not lead to an increase in activity. 40 This study
supported the hypothesis that TCH-165 is binding in the inter-subunit pocket when it was found
                                                  58


that activation of the 20S proteasome by TCH-165 could be partially inhibited through the addition
of the Rpt-3 peptide (Fig. 2.2D).36
    A                                                 B
    C                                                      D
Figure 2.2: TCH-165 stabilizes the open-gate conformation of the 20S proteasome,
presumably through interaction with the α1-2 inter-subunit pocket. (A) Tilted top-view AFM
images of standing 20S particles with closed- and open-gate conformations. (B) Percent of open-
gate 20S particles in populations of the 20S proteasome treated with TCH-165. Data are mean ±
standard deviation of at least four fields with 120–260 particles per field (one-way ANOVA; *p <
0.05, **p < 0.01, ***p < 0.001). (C) Top view of the 20S α-ring showing the preferred docking
                                                  59


site of TCH-165 utilizing Autodock Vina. (D) TCH-165 competition experiment with the Rpt3
peptide (which normally binds in the α1-2 pocket) for 20S proteolysis (n = 3, *p<0.05).36
      The discovery and subsequent mechanistic studies of TCH-165 as a 20S proteasome activator
by the Tepe lab36 initiated an expanded investigation by our group to identify additional small
molecular activators. Despite the several literature reports demonstrating the clinical relevance of
20S proteasome activation for treatment of a variety of diseases,9-17 there are very few molecules
that had been identified as direct or indirect activators of the 20S proteasome. Additionally, many
of these activators suffer from limitations, such as low potency, off-target effects, and poor drug-
like properties.36, 41-47 The continued exploration of 20S proteasome activation as a therapeutic
method will require additional molecular scaffolds to be explored to identify new lead molecules
for testing in disease model systems.
      As part of this effort to identify novel scaffolds of 20S proteasome activators, I began a
collaboration with Prof. Adam Mosey and his lab at Lake Superior State University. The novel
chemistry developed by Prof. Mosey’s lab allowed for rapid generation of a small library of
compounds with analogous structures to that seen in Fig. 2.3.48 I screened the library of
dihydroquinazoline analogues for 20S proteasome activity using a fluorogenic peptide degradation
assay and evaluated the structure activity relationship (SAR) of the dihydroquinazoline scaffold.49-
51
Figure 2.3: General structure of compounds synthesized by the Mosey group.
                                                   60


2.1.2 Objective
        The goals of this project were (1) to further explore the robustness of allosteric small
molecule 20S proteasome activation by evaluating a novel 20S activator scaffold, (2) to explore
the SAR of the dihydroquinazoline 20S activator scaffold and (3) demonstrate the translation of
20S activity seen by dihydroquinazoline analogues to the enhancement of intrinsically disordered
protein (IDP) digestion.
2.2 Results and Discussion
2.2.1 Synthesis of a small library of dihydroquinazoline analogues for exploration as 20S
proteasome activators
        Synthesis of the dihydroquinazoline analogues was accomplished via Prof. Adam Mosey’s
recently reported one-pot multicomponent reaction of amides, amines and aldehydes (Scheme
2.1).48 This method involves in situ imine formation from an amine and an aldehyde in the presence
of molecular sieves, followed by tandem assembly of the heterocyclic ring through successive
Tf2O-mediated amide dehydration, imine insertion, and Pictet-Spengler-like cyclization. The
multicomponent nature of the method permits the construction of highly diverse
dihydroquinazolines due to the compatibility of a wide range of simple starting materials.
Scheme 2.1: Multicomponent synthesis of dihydroquinazoline analogues.
        A small library of dihydroquinazolines (Fig. 2.4) was generated using this multicomponent
method to begin probing the ability of members of this class of compounds to activate the 20S
                                                61


proteasome. Compounds synthesized to populate the library used for this study differed in their
structural features at the 7-, 2- and 3-positions (R1, R2 and R3, respectively) of the heterocyclic
scaffold (Fig. 2.3). Variation at the 7- and 2-positions was accomplished using select amides (e.g.
1 – 9), while the substituents at the 3-position were introduced using chosen amines (e.g. 10 – 24).
R1 and R2 groups introduced from the starting amides provided preliminary SAR information
which was utilized for the construction of the remaining members of the compound library in
which the R3 group was varied. Simple alkyl and alkoxy substituents were explored at R1, along
with the absence of any additional group at this location (e.g. 1 – 4), and the investigated R2
substituents included alkyl and cycloalkyl groups to compare them to the aromatic counterpart
(e.g. 5 – 9 vs 2). A range of R3 substituents were installed to include aryl and heteroaryl groups
(e.g. 10 – 13), tethered heteroaryl groups (e.g. 14 – 15), and alkyl groups with varying ring and
heteroatom placement (e.g. 16 – 24).
Figure 2.4: Structurally related dihydroquinazoline analogues analyzed in this SAR study.
A set of dihydroquinazoline analogues were synthesized with a variety of functionalities on their
periphery to decipher a SAR for the activation of the 20S proteasome by this scaffold.
                                                  62


2.2.2 Screening and SAR of related dihydroquinazoline analogues as 20S proteasome
activators
        Screening of this small library of dihydroquinazolines (Fig 2.4) was performed in two
stages. In the first stage, each compound was screened at 3 concentrations (3, 10 and 30 µM) to
select lead agents. Secondly, lead agents were further analysed using full concentration responses
(6-point titration ranging from 1.25 µM-40 µM) for each of the three proteolytic activities of the
20S proteasome and a combination thereof.
        The proteolytic activity of the 20S proteasome can be monitored in vitro by measuring the
increase in 7-amino-4-methylcoumarin (AMC) fluorescence over time, following the cleavage of
fluorogenic peptide substrates for each of the different catalytic sites of the proteasome.49,      51
Proteolysis of the peptide substrates is enhanced if an open-gate conformation of the 20S
proteasome is stabilized.36 A combination of chymotrypsin-like (CT-L, Suc-LLVY-AMC),
trypsin-like (Tryp-L, Boc-LRR-AMC) and caspase-like (Casp-L, Z-LLE-AMC) peptide
substrates, one for each of the three different catalytic sites of the 20S, were used in equal amounts
to provide a comprehensive look at the activation potential of each compound. In the initial screen,
pure human 20S proteasome was pre-treated with 3, 10 or 30 μM of the analogues or DMSO
(vehicle control) in 96-well plates for 15 minutes at 37 °C. To each sample was then added a
mixture of the three substrates (each at a concentration of 13.3 μM). The release of AMC was
measured every 5 minutes, over the course of 1 hour, and the resulting 20S activity changes were
determined by comparing to the DMSO treated 20S. Relative changes in activity were quantified
by calculating the fold-increase in activity over the vehicle control for each analogue at a given
concentration (Table 2.1).
                                                   63


Table 2.1: 20S proteasome activity analysis of structurally related dihydroquinazoline analogues
(compounds 1–24).
                                          30 μM      10 μM       3 μM
                            Compound
                                                (fold of control)
                                  1          4         1.9        1.5
                                  2         7.9        3.5        2.2
                                  3         8.1        2.8        1.6
                                  4         2.8        2.8        2.6
                                  5         6.5        3.8        1.8
                                  6         2.7        1.5        1.3
                                  7         2.3        1.5        1.4
                                  8         1.8        2.8        1.8
                                  9         1.8        1.1        0.7
                                 10         9.5        4.8        2.1
                                 11         6.9        3.5        1.3
                                 12         5.9        2.4        1.2
                                 13         5.3        4.2        2.8
                                 14          2         1.4        1.3
                                 15         2.8        1.7        1.3
                                 16         6.7        3.2        1.6
                                 17          1          1         0.9
                                 18          7         4.1        2.9
                                 19         1.1        0.9        0.8
                                 20         1.6        1.4        1.2
                                 21         8.2         3         1.6
                                 22         2.7        1.3        0.6
                                 23         2.3        1.3        0.9
                                 24         1.3        1.5        1.6
        The resulting data (Table 2.1) show a few insightful trends in the SAR of the
dihydroquinazolines. Small changes in substitution at the 7-position appear to have a significant
effect on activity of the dihydroquinazolines. Compound 1, which displays 4-fold (i.e. 400%)
increase over background 20S activity, lacks a substituent at the 7-position but is otherwise
identical to compounds 2 (7.9-fold increase) and 3 (8.1-fold increase). This small change results
in a reduction in 20S activity from 8-fold enhancement down to a 4-fold enhancement at 30 μM.
Similarly, the addition of a longer alkyl chain on compound 4 resulted in a steep drop in 20S
activity to 2.8-fold at 30 μM. Changes at the 2-position show similar effects to that of the 7-
                                                64


position, where most substitutions other than a phenyl group (compounds 5–9) caused marked
decreases in 20S activity. All have less than 3-fold activation at 30 μM, apart from compound 5
(6.5-fold increase).
        Substitutions at the 3-position showed more flexibility to changes than either the 7- or 2-
positions, while still having a significant effect on the relative 20S activities of the analogues.
Many of the most active analogues, like compounds 2, 3, 5 and 10 (7.9, 8.1, 6.5 and 9.5-fold
increase of 20S activity, respectively), contain a phenyl or benzyl functionality at the 3-position.
Other similarly sized and shaped substituents like cyclohexane (compound 16 (6.7-fold)) and
pyridine compounds 11 (6.9-fold) and 12 (5.9-fold)) also provided some of the most active
analogues. Interestingly, larger substituents at the 3-position as seen in compounds 18 (7-fold) and
13 (5.3-fold) also yielded highly active analogues, suggesting that additional functionalities may
be incorporated here for further optimization if necessary. The substitution of the phenyl or benzyl
groups for some other heterocycles such as N-methyl piperidine (compounds 17 (1-fold) and 19
(1.2-fold)), tetrahydropyran (compound 20 (1.6-fold)) or even a pyridine linked by a methyl group
in compound 14 (2-fold) lead to significant decreases in 20S activity. This suggests that placement
of heteroatoms at the 3-position may have the potential to disrupt hydrophobic interactions in that
region. The difference in activity shown between 17 and 18 could be caused by a disruption of
hydrophobic interactions with the addition of the piperidine nitrogen, which could then be
reinstated or replaced by new interactions made by the phenyl group in 18. The addition of non-
cyclic substituents at the 3-position (compounds 22, 23 and 24) resulted in very little 20S activity
(2.7, 2.3 and 1.6-fold increase in 20S activity, respectively) in all cases suggesting that larger
hydrophobic groups at the 3-position are likely required for 20S activity.
                                                   65


2.2.3 Select dihydroquinazoline analogues activate all catalytic sites of the 20S proteasome
in a concentration-dependent manner
         After analysing the results in Table 2.1, three of the most promising analogues were
selected for further studies into their 20S activity. Compounds 2, 10, and 18 were selected to be
carried forward due to their relatively high max-fold increases and more promising concentration-
response at lower concentrations. Compound 17 was also carried forward to use as an inactive
control since it had no discernible activity towards the 20S and was structurally similar to
compound 18.
         Compounds 2, 10, 18 and 17 were further evaluated to obtain a full concentration-response
(Fig. 2.5) of their activities towards the 20S proteasome using each of the three substrates for the
three catalytic sites individually and the combination of the three substrates. This was done to
ensure that each of the selected compounds activate the 20S proteasome at all three catalytic sites,
which is critical for effective IDP degradation, as these proteins are likely to contain multiple
cleavage sites for each. Previously identified 20S proteasome activators that were only able to
activate a single catalytic site showed relatively poor enhancement of IDP degradation in vitro
when compared to those that activated all three catalytic sites.44 By fitting the relative fluorescent
units and concentrations into a four-parameter dose-response curve the resulting data were
analysed. Through this more thorough testing of the selected dihydroquinazoline analogue leads
(compounds 2, 10 and 18), it was found that each was able to effectively enhance the degradation
of all three different peptide substrates of the 20S proteasome in a concentration-dependent manner
(Fig. 2.5). Whereas the inactive control (compound 17) was unable to enhance the degradation of
any of the peptide substrates in these experiments.
                                                   66


                           20                                                                          20
                                                                   Combo                                                                                             Combo
 20S Proteasome Activity                                                     20S Proteasome Activity
                           15                                      CT-L                                15
                                                                                                                                                                     CT-L
                                                                   Casp-L                                                                                            Casp-L
                           10                                      Tryp-L                              10                                                            Tryp-L
     (fold of control)                                                           (fold of control)
                           5                                                                            5
                           0                                                                            0
                           -6.0    -5.5     -5.0     -4.5   -4.0                                        - 6 .0   - 5 .5          - 5 .0            - 4 .5   - 4 .0
                                     log[M], Compound 2                                                             lo g [ M ] , C o m p o u n d   10
                           20                                                                          20
                                                                   Combo                                                                                             Combo
 20S Proteasome Activity                                                     20S Proteasome Activity
                                                                   CT-L                                                                                              CTL
                           15                                                                          15
                                                                   Casp-L                                                                                            Casp-L
                           10                                      Tryp-L                              10                                                            Tryp-L
     (fold of control)                                                           (fold of control)
                           5                                                                            5
                           0                                                                            0
                           -6.0    -5.5     -5.0     -4.5   -4.0                                        -6.0     -5.5             -5.0             -4.5     -4.0
                                    log[M], Compound 18                                                             log[M], Compound 17
Figure 2.5: Select dihydroquinazoline analogues activate all catalytic sites of the 20S
proteasome in a dose dependent manner. Concentration–response curves of compounds 2, 10,
18 and 17 for CT-L, Casp-L, Tryp -L, and the combination of the three sites of the 20S. These data
were collected in triplicate (n=3). Error bars denote standard deviation.
                            From the data in Fig. 2.5, the concentration at which 20S activity was doubled (EC200) was
calculated for each compound using each substrate and the combination of the three substrates
(Table 2.2). Because of variations in the maximum fold enhancement achieved by different 20S
enhancers, EC200 values allow for easy comparisons to be made between activators. Additionally,
these EC200 values may provide better insight into the potency of the activators, relative to their
maximum achieved activity. Through these calculations, it was found that each of the active
compounds (2, 10 and 18) achieved high maximum fold increases in 20S activity towards each of
the peptide substrates, as well as the combination of the three substrates (Table 2.2). This
                                                                            67


demonstrates that these activators and this scaffold show potential to enhance the degradation of
full length IDPs, for the reasons mentioned above. Additionally, it was found that each of the active
analogues (compounds 2, 10 and 18) had EC200’s for 20S enhancement in the low μM range (1 –
2.5 µM) when using the combination of the 3 peptide substrates (Table 2.2). This level of potency
is on par with some of the most potent small activators discovered by the Tepe lab to date.36, 44
Table 2.2: Detailed analysis of 20S activation by select dihydroquinazoline analogues.
                       Combo                 CT-L                Casp-L                Tryp-L
                  EC200      Max      EC200       Max      EC200       Max       EC200       Max
 Compound
                  (μM)       Fold      (μM)       Fold     (μM)        Fold      (μM)        Fold
       2           2.0       10.1       12.9       5.6       5.6       13.4        5.0       11.7
       10          2.3       11.1        8.1       6.7       5.8       15.3        2.5       15.0
       18          1.3        5.5       10.5       3.8       2.8       10.6        1.7        8.7
       17          N/A        1.4       N/A        1.1      N/A         0.8       N/A         1.0
2.2.4 Compound 18 enhances the degradation of the IDP α-synuclein in vitro
         Although these three analogues showed near equipotent activities, compound 18 was
selected as the lead molecule to move forward with due to its lowest overall EC 200 value (Table
2.2: combo, EC200 1.3 μM) and relatively low individual site activities for the 20S. The activity of
compound 18 was further evaluated by observing its ability to enhance 20S mediated degradation
of the intrinsically disordered protein (IDP) α-synuclein (Fig. 2.6A). This was done to determine
if the activity seen by dihydroquinazoline analogues translates to more relevant full length protein
targets of the 20S.52-54 The IDP α-synuclein was selected for these studies due to its predicted
highly disordered nature (Fig. 2.6A) and its known association with the development of
Parkinson’s disease, as well as other synucleinopathies.55-59
         Briefly, the 20S proteasome was incubated with compound 18, followed by addition of
pure human α-synuclein. This mixture was then incubated for 4 hours. The digestions were
                                                 68


analysed using silver stain (Fig. 2.6C) and densitometry was performed using image J software
(Fig. 2.6B) to explore the ability of compound 18 to enhance α-synuclein degradation by the 20S.
It was found that compound 18 effectively enhanced the rate of degradation of α-synuclein by the
20S in vitro in a concentration-dependant manner (Fig. 2.6B and C). As a control, the proteasome
inhibitor, bortezomib, which prevented α-synuclein degradation, was used to confirm that the
clearance of α-synuclein is a proteasome mediated event. The resulting data show that the prior
fluorogenic peptide substrate-based results for the dihydroquinazoline scaffold are translatable to
the degradation of IDPs.
Figure 2.6: The most potent dihydroquinazoline analogue (compound 18) enhances 20S-
mediated degradation of the IDP α-synuclein in vitro. (A) Intrinsic disorder in α-synuclein as
predicted with PONDR VSL2 software. (B) Densitometry of C using image J software. (C) Silver
                                                 69


stain of α-synuclein digestion with the 20S proteasome pretreated with dihydroquinazoline
analogue (compound 18) or the proteasome inhibitor bortezomib. These data were collected in
triplicate (n=3). Error bars denote standard deviation. Ordinary one-way ANOVA statistical
analysis was used to determine statistical significance (*p<0.05, ***p<0.001, ****p<0.0001).
          These data demonstrate that dihydroquinazolines represent a promising scaffold from
which potent 20S activators that enhance IDP degradation can be developed can be developed.
Additionally, recently developed synthetic methods allow for access to a broad scope of
dihydroquinazoline analogues, permitting the exploration of a variety of different substituents and
substitution patterns. Among the analogues tested, several active compounds were identified, as
well as a few of the most potent 20S activators identified to date. Further optimization and testing
of dihydroquinazoline analogues may yield even more potent and drug-like leads, which can assist
in the continued exploration of 20S activation as a novel therapeutic method.
2.2.5 Screening of an extended dihydroquinazoline library shows numerous 20S proteasome
active analogues, but did not yield improved leads
          In an effort to explore a larger library of dihydroquinazoline analogues and to identify even
more potent lead molecules, the Mosey lab provided nearly 80 dihydroquinazoline analogues
synthesized using their methodology.48 To screen this larger library of dihydroquinazoline
analogues economically and more rapidly, our standard 3-substrate combination fluorogenic
peptide assay was modified slightly by using reduced volumes required for running the assay and
fit it in 384-well plates. This was done following protocols used by our lab previously in a high
throughput screen to identify 20S activators, that will be discussed in more detail in Chapter 3.44
This study was done with assistance from Dr. Tomas Dexheimer and MSU’s assay development
and drug repurposing core (ADDRC). Human 20S proteasome was pretreated with various
                                                      70


concentrations of each analogue followed by addition of the 3-substrate combination. Proteasome
activity was quantified by measuring fluorescence intensity associated with AMC release at time
= 0 min and then again at time = 60 min. The level of activity for each treatment was determined
by subtracting signal from time = 0 from that found at time = 60 min. The resulting difference in
AMC fluorescence was compared to that of the DMSO vehicle control. This was done at 8-
concentrations for each analogue to obtain concentration response curves. From these data, the
maximum fold increase in activity over the vehicle control, as well as an EC200 for each compound
was determined, as described above. The resulting data is summarized in a graph showing the
maximum fold increase over the vehicle (y-axis) and EC200 (x-axis) of each compound (Figure
2.7).
                                  8
                                  7
 Max fold increase over vehicle
                                  6
                                  5
                                  4
                                  3
                                  2
                                  1
                                  0
                                      0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
                                                                         EC200 (µM)
Figure 2.7: Screening of an extended library of dihydroquinazoline analogues using
fluorogenic peptide substrates shows numerous active compounds. Graph summarizing EC200
values (x-axis) and maximum fold increase over vehicle (30 μM). The top performing compounds
                                                                           71


were those with the highest maximum fold increases, as well as those with the lowest EC200 values.
A combination of high max fold increase and low EC200 value represents the ideal lead compounds.
        Through the screening of this larger library, it was found that many of these
dihydroquinazoline analogues activated the 20S proteasome to various degrees. However, none of
the additional dihydroquinazoline analogues tested in this high throughput manner showed
improvement relative to the most active analogues explored in the initial SAR study. After analysis
of this data and discussions with Prof. Mosey, he and his lab have begun synthesizing additional
dihydroquinazoline analogues to explore different substitutions at the 4-position to further
diversify the scaffold.
2.3 Conclusions
      In summary, a small library of dihydroquinazoline analogues were screened for 20S
proteasome activity. It was found that several of these analogues effectively enhanced the activity
of the 20S proteasome towards the degradation of fluorogenic peptide substrates for each of the
catalytic sites of the proteasome. Comparison of the SAR of these dihydroquinazoline analogues
identified structural motifs that, when used in combination, yielded some of the most potent 20S
proteasome activators to date. Detailed evaluation of top dihydroquinazoline analogues
(compounds 2, 10 and 18) from this library demonstrated that each was capable of activating all 3
catalytic sites of the 20S proteasome in a concentration-dependent manner. It was also
demonstrated that the activity of compound 18 translates well to enhance the 20S mediated
degradation of a natural IDP substrate of the 20S proteasome, α-synuclein. Continued screening
of dihydroquinazoline analogues has yet to yield substantial improvements in activity relative to
the top compounds identified in the aforementioned SAR study. However, ongoing efforts are
being made to diversify this scaffold further and identify more potent leads, by making use of
                                                 72


recently developed synthetic methods that allow for rapid generation of dihydroquinazolines with
varied substitutions and substitution patterns.
This work:
     (1) Further demonstrated the robustness of allosteric small molecule 20S proteasome
          activators through the identification of dihydroquinazolines as a novel 20S activator
          scaffold.
     (2) Explored the SAR of the dihydroquinazoline scaffolds 20S proteasome activity, and
          identified analogues with potencies on par with the most potent activators established
          thus far.
     (3) Demonstrated that the activity of the dihydroquinazoline scaffold translates to enhance
          20S proteasome-mediated degradation of the natural IDP substrate α-synuclein in vitro.
                                                73


                                            REFERENCES
(1) Voges, D.; Zwickl, P.; Baumeister, W. The 26S Proteasome: A Molecular Machine Designed
for Controlled Proteolysis. Annual Review of Biochemistry 1999, 68, 1015-1068, review-article.
DOI: 10.1146/annurev.biochem.68.1.1015.
(2) McNaught, K. S. P.; Olanow, C. W.; Halliwell, B.; Isacson, O.; Jenner, P. Failure of the
ubiquitin–proteasome system in Parkinson's disease. Nature Reviews Neuroscience 2001, 2 (8),
589-594. DOI: 10.1038/35086067.
(3) Voorhees, P.; Dees, E.; O'Neil, B.; Orlowski, R. The proteasome as a target for cancer
therapy. Clinical cancer research 2003, 9 (17), 6316-6325.
(4) Ostrowska, H. The ubiquitin-proteasome system: a novel target for anticancer and anti-
inflammatory drug research. Cellular & Molecular Biology Letters 2008, 13 (3), 353-365. DOI:
10.2478/s11658-008-0008-7.
(5) Calamini, B.; Morimoto, R. Protein homeostasis as a therapeutic target for diseases of protein
conformation. Current Topics in Medicinal Chemistry 2012, 12 (22), 2623-2640. DOI:
10.2174/1568026611212220014.
(6) Huang, X.; Dixit, V. M. Drugging the undruggables: exploring the ubiquitin system for drug
development. Cell Research 2016, 26 (4), 484-498, ReviewPaper. DOI: doi:10.1038/cr.2016.31.
(7) Njomen, E.; Tepe, J. J. Proteasome Activation as a New Therapeutic Approach to Target
Proteotoxic Disorders. Journal of Medicinal Chemistry 2019, 62 (14), 6469-6481. DOI:
10.1021/acs.jmedchem.9b00101.
(8) Tundo, G.; Sbardella, D.; Santoro, A.; Coletta, A.; Oddone, F.; Grasso, G.; Milardi, D.;
Lacal, P.; Marini, S.; Purrello, R.; et al. The proteasome as a druggable target with multiple
therapeutic potentialities: Cutting and non-cutting edges. Pharmacology & therapeutics 2020,
213, 107579. DOI: 10.1016/j.pharmthera.2020.107579.
(9) Mitsiades, N.; Mitsiades, C. S.; Poulaki, V.; Chauhan, D.; Fanourakis, G.; Gu, X.; Bailey, C.;
Joseph, M.; Libermann, T. A.; Treon, S. P.; et al. Molecular sequelae of proteasome inhibition in
human multiple myeloma cells. PNAS 2002, 99 (22), 14374-14379, research-article. DOI:
10.1073/pnas.202445099.
(10) D'Arcy, P.; Brnjic, S.; Olofsson, M. H.; Fryknäs, M.; Lindsten, K.; De Cesare, M.; Perego,
P.; Sadeghi, B.; Hassan, M.; Larsson, R.; et al. Inhibition of proteasome deubiquitinating activity
as a new cancer therapy. Nature Medicine 2011, 17 (12), 1636-1640, OriginalPaper. DOI:
doi:10.1038/nm.2536.
(11) Rastogi, N.; Mishra, D. P. Therapeutic targeting of cancer cell cycle using proteasome
inhibitors. Cell Division 2012, 7 (1), 1-10, ReviewPaper. DOI: doi:10.1186/1747-1028-7-26.
(12) Bedford, L.; Hay, D.; Devoy, A.; Paine, S.; Powe, D. G.; Seth, R.; Gray, T.; Topham, I.;
Fone, K.; Rezvani, N.; et al. Depletion of 26S Proteasomes in Mouse Brain Neurons Causes
                                                   74


Neurodegeneration and Lewy-Like Inclusions Resembling Human Pale Bodies. Journal of
Neuroscience 2008, 28 (33), 8189-8198. DOI: 10.1523/JNEUROSCI.2218-08.2008.
(13) Demasi, M.; Faria, B. F. Activation of the ubiquitin-proteasome system: implications for
neurodegeneration, aging, and tumorigenesis. Journal of Neurology & Neuromedicine 2017, 2
(8), 1-4.
(14) Boland, B.; Yu, W. H.; Corti, O.; Mollereau, B.; Henriques, A.; Bezard, E.; Pastores, G. M.;
Rubinsztein, D. C.; Nixon, R. A.; Duchen, M. R.; et al. Promoting the clearance of neurotoxic
proteins in neurodegenerative disorders of ageing. Nature Reviews Drug Discovery 2018, 17 (9),
660-688. DOI: 10.1038/nrd.2018.109.
(15) Chondrogianni, N.; Gonos, E. S. Proteasome activation as a novel antiaging strategy.
IUBMB Life 2008, 60 (10), 651-655. DOI: 10.1002/iub.99.
(16) Chondrogianni, N.; Sakellari, M.; Lefaki, M.; Papaevgeniou, N.; Gonos, E. S. Proteasome
activation delays aging in vitro and in vivo. Free Radical Biology and Medicine 2014, 71, 303-
320. DOI: 10.1016/j.freeradbiomed.2014.03.031.
(17) Chondrogianni, N.; Voutetakis, K.; Kapetanou, M.; Delitsikou, V.; Papaevgeniou, N.;
Sakellari, M.; Lefaki, M.; Filippopoulou, K.; Gonos, E. S. Proteasome activation: An innovative
promising approach for delaying aging and retarding age-related diseases. Ageing Research
Reviews 2015, 23 (Pt A), 37-55. DOI: 10.1016/j.arr.2014.12.003.
(18) Meister, S.; Schubert, U.; Neubert, K.; Herrmann, K.; Burger, R.; Gramatzki, M.; Hahn, S.
S. C.; Schreiber, S.; Wilhelm, S.; Herrmann, M.; et al. Extensive immunoglobulin production
sensitizes myeloma cells for proteasome inhibition. Cancer Research 2007, 67 (4), 1783-1792.
DOI: 10.1158/0008-5472.CAN-06-2258.
(19) Bianchi, G.; Oliva, L.; Cascio, P.; Pengo, N.; Fontana, F.; Cerruti, F.; Orsi, A.; Pasqualetto,
E.; Mezghrani, A.; Calbi, V.; et al. The proteasome load versus capacity balance determines
apoptotic sensitivity of multiple myeloma cells to proteasome inhibition | Blood | American
Society of Hematology. Blood 2009, 113 (13), 3040-3049. DOI: 10.1182/blood-2008-08-
172734.
(20) Kisselev, A.; Linden, W.; Overkleeft, H. Proteasome Inhibitors: An Expanding Army
Attacking a Unique Target. Chemistry & Biology 2012, 19 (1), 99-115. DOI:
10.1016/j.chembiol.2012.01.003.
(21) Chiang, M.; Stadtmauer, E. NF-kappaB, IL-6 and myeloma cell growth: making the
connection. Cancer biology & therapy 2004, 3 (10), 1018-1020. DOI: 10.4161/cbt.3.10.1326.
(22) Anderson, K.; Alsina, M.; Bensinger, W.; Biermann, J.; Chanan-Khan, A.; Cohen, A.;
Devine, S.; Djulbegovic, B.; Faber, E.; Gasparetto, C.; et al. Multiple myeloma. Journal of the
National Comprehensive Cancer Network : JNCCN 2011, 9 (10), 1146-1183. DOI:
10.6004/jnccn.2011.0095.
                                                 75


(23) Cenci, S.; Mezghrani, A.; Cascio, P.; Bianchi, G.; Cerruti, F.; Fra, A.; Lelouard, H.;
Masciarelli, S.; Mattioli, L.; Oliva, L.; et al. Progressively impaired proteasomal capacity during
terminal plasma cell differentiation. The EMBO Journal 2006, 25 (5), 1104-1113. DOI:
10.1038/sj.emboj.7601009.
(24) Kane, R.; Bross, P.; Farrell, A.; Pazdur, R. Velcade®: U.S. FDA Approval for the
Treatment of Multiple Myeloma Progressing on Prior Therapy. The Oncologist 2003, 8 (6), 508-
513. DOI: 10.1634/theoncologist.8-6-508.
(25) Kirk, C. Discovery and Development of Second-Generation Proteasome Inhibitors.
Seminars in Hematology 2012, 49 (3), 207-214. DOI: 10.1053/j.seminhematol.2012.04.007.
(26) Kuhn, D.; Orlowski, R.; Bjorklund, C. Second generation proteasome inhibitors:
carfilzomib and immunoproteasome-specific inhibitors (IPSIs). Current cancer drug targets
2011, 11 (3), 285-295. DOI: 10.2174/156800911794519725.
(27) Murray, M.; Auger, M.; Bowles, K. Overcoming bortezomib resistance in multiple
myeloma. Biochemical Society transactions 2014, 42 (4), 804-808. DOI: 10.1042/BST20140126.
(28) Lü, S.; Wang, J. The resistance mechanisms of proteasome inhibitor bortezomib. Biomarker
research 2013, 1, 13. DOI: 10.1186/2050-7771-1-13.
(29) Wu, Y.; Yang, J.; Saitsu, H. Bortezomib-resistance is associated with increased levels of
proteasome subunits and apoptosis-avoidance. Oncotarget 2016, 7 (47), 77622-77634. DOI:
10.18632/oncotarget.12731.
(30) Carozzi, V.; Renn, C.; Bardini, M.; Fazio, G.; Chiorazzi, A.; Meregalli, C.; Oggioni, N.;
Shanks, K.; Quartu, M.; Serra, M.; et al. Bortezomib-Induced Painful Peripheral Neuropathy: An
Electrophysiological, Behavioral, Morphological and Mechanistic Study in the Mouse. PLoS
One 2013, 8 (9). DOI: 10.1371/journal.pone.0072995.
(31) Anbanandam, A.; Albarado, D.; Tirziu, D.; Simons, M.; Veeraraghavan, S. Molecular basis
for proline- and arginine-rich peptide inhibition of proteasome. Journal of Molecular Biology
2008, 384 (1), 219-227. DOI: 10.1016/j.jmb.2008.09.021.
(32) Ruschak, A.; Slassi, M.; Kay, L.; Schimmer, A. Novel proteasome inhibitors to overcome
bortezomib resistance. Journal of the National Cancer Institute 2011, 103 (13). DOI:
10.1093/jnci/djr160.
(33) Gaczynska, M.; Osmulski, P.; Gao, Y.; Post, M.; Simons, M. Proline- and Arginine-Rich
Peptides Constitute a Novel Class of Allosteric Inhibitors of Proteasome Activity†. Biochemistry
2003, 42 (29), 8663-8670, rapid-communication. DOI: 10.1021/bi034784f.
(34) Lansdell, T. A.; Hurchla, M. A.; Xiang, J.; Hovde, S.; Weilbaecher, K. N.; Henry, R. W.;
Tepe, J. J. Noncompetitive Modulation of the Proteasome by Imidazoline Scaffolds Overcomes
Bortezomib Resistance and Delays MM Tumor Growth in Vivo. ACS Chemical Biology 2012, 8
(3), 578-587, research-article. DOI: 10.1021/cb300568r.
                                                    76


(35) Azevedo, L. M.; Lansdell, T. A.; Ludwig, J. R.; Mosey, R. A.; Woloch, D. K.; Cogan, D. P.;
Patten, G. P.; Kuszpit, M. R.; Fisk, J. S.; Tepe, J. J. Inhibition of the Human Proteasome by
Imidazoline Scaffolds. Journal of Medicinal Chemistry 2013, 56 (14), 5974-5978. DOI:
10.1021/jm400235r.
(36) Njomen, E.; Osmulski, P. A.; Jones, C. L.; Gaczynska, M.; Tepe, J. J. Small Molecule
Modulation of Proteasome Assembly. Biochemistry 2018, 57 (28), 4214-4224. DOI:
10.1021/acs.biochem.8b00579.
(37) Gaczynska, M.; Osmulski, P. A. Atomic Force Microscopy of Proteasome Assemblies. Vol.
736; 2011; pp 117-132.
(38) Osmulski, P. A.; Hochstrasser, M.; Gaczynska, M. A Tetrahedral Transition State at the
Active Sites of the 20S Proteasome Is Coupled to Opening of the α-Ring Channel. Structure
2009, 17 (8), 1137-1147. DOI: 10.1016/j.str.2009.06.011.
(39) Schweitzer, A.; Aufderheide, A.; Rudack, T.; Beck, F.; Pfeifer, G.; Plitzko, J. M.; Sakata,
E.; Schulten, K.; Förster, F.; Baumeister, W. Structure of the human 26S proteasome at a
resolution of 3.9 Å. PNAS 2016, 113 (28), 7816-7821, research-article. DOI:
10.1073/pnas.1608050113.
(40) Smith, D. M.; Chang, S. C.; Park, S.; Finley, D.; Cheng, Y.; Goldberg, A. L. Docking of the
proteasomal ATPases' carboxyl termini in the 20S proteasome's alpha ring opens the gate for
substrate entry. Molecular cell 2007, 27 (5), 731-744. DOI: 10.1016/j.molcel.2007.06.033.
(41) Huang, L.; Ho, P.; Chen, C.-H. Activation and inhibition of the proteasome by betulinic acid
and its derivatives. FEBS Letters 2007, 581 (25), 4955-4959. DOI:
10.1016/j.febslet.2007.09.031.
(42) Lee, B.-H.; Lee, M. J.; Park, S.; Oh, D.-C.; Elsasser, S.; Chen, P.-C.; Gartner, C.; Dimova,
N.; Hanna, J.; Gygi, S. P.; et al. Enhancement of proteasome activity by a small-molecule
inhibitor of USP14. Nature 2010, 467 (7312), 179-184. DOI: 10.1038/nature09299.
(43) Trippier, P. C.; Zhao, K. T.; Fox, S. G.; Schiefer, I. T.; Benmohamed, R.; Moran, J.; Kirsch,
D. R.; Morimoto, R. I.; Silverman, R. B. Proteasome activation is a mechanism for pyrazolone
small molecules displaying therapeutic potential in amyotrophic lateral sclerosis. ACS Chemical
Neuroscience 2014, 5 (9), 823-829. DOI: 10.1021/cn500147v.
(44) Jones, C. L.; Njomen, E.; Sjögren, B.; Dexheimer, T. S.; Tepe, J. J. Small Molecule
Enhancement of 20S Proteasome Activity Targets Intrinsically Disordered Proteins. ACS
Chemical Biology 2017, 12 (9), 2240-2247. DOI: 10.1021/acschembio.7b00489.
(45) Leestemaker, Y.; de Jong, A.; Witting, K. F.; Penning, R.; Schuurman, K.; Rodenko, B.;
Zaal, E. A.; van de Kooij, B.; Laufer, S.; Heck, A. J. R.; et al. Proteasome Activation by Small
Molecules. Cell Chemical Biology 2017, 24 (6), 725-736.e727. DOI:
10.1016/j.chembiol.2017.05.010.
                                                  77


(46) Trader, D. J.; Simanski, S.; Dickson, P.; Kodadek, T. Establishment of a suite of assays that
support the discovery of proteasome stimulators. Biochimica et Biophysica Acta (BBA) - General
Subjects 2017, 1861 (4), 892-899. DOI: 10.1016/J.BBAGEN.2017.01.003.
(47) Coleman, R. A.; Muli, C. S.; Zhao, Y.; Bhardwaj, A.; Newhouse, T. R.; Trader, D. J.
Analysis of chain length, substitution patterns, and unsaturation of AM-404 derivatives as 20S
proteasome stimulators. Bioorganic and Medicinal Chemistry Letters 2019, 29 (3), 420-423.
DOI: 10.1016/j.bmcl.2018.12.030.
(48) Magyar, C. L.; Wall, T. J.; Davies, S. B.; Campbell, M. V.; Barna, H. A.; Smith, S. R.;
Savich, C. J.; Mosey, R. A. Triflic anhydride mediated synthesis of 3,4-dihydroquinazolines: A
three-component one-pot tandem procedure. Organic and Biomolecular Chemistry 2019, 17
(34), 7995-8000. DOI: 10.1039/c9ob01596e.
(49) Kisselev, A. F.; Akopian, T. N.; Castillo, V.; Goldberg, A. L. Proteasome active sites
allosterically regulate each other, suggesting a cyclical bite-chew mechanism for protein
breakdown. Molecular Cell 1999, 4 (3), 395-402. DOI: 10.1016/S1097-2765(00)80341-X.
(50) Gaczynska, M.; Osmulski, P. A. Characterization of noncompetitive regulators of
proteasome activity. Academic Press Inc.: 2005; Vol. 398, pp 425-438.
(51) Kisselev, A.; Goldberg, A. Monitoring Activity and Inhibition of 26S Proteasomes with
Fluorogenic Peptide Substrates. Methods in Enzymology 2005, 398, 364-378. DOI:
10.1016/S0076-6879(05)98030-0.
(52) Bennett, M.; Bishop, J.; Leng, Y.; Chock, P.; Chase, T.; Mouradian, M. Degradation of α-
Synuclein by Proteasome. Journal of Biological Chemistry 1999, 274 (48), 33855-33858. DOI:
10.1074/jbc.274.48.33855.
(53) Webb, J.; Ravikumar, B.; Atkins, J.; Skepper, J.; Rubinsztein, D. Alpha-Synuclein is
degraded by both autophagy and the proteasome. The Journal of biological chemistry 2003, 278
(27), 25009-25013. DOI: 10.1074/jbc.M300227200.
(54) Alvarez-Castelao, B.; Goethals, M.; Vandekerckhove, J.; Castaño, J. G. Mechanism of
cleavage of alpha-synuclein by the 20S proteasome and modulation of its degradation by the
RedOx state of the N-terminal methionines. Biochimica et Biophysica Acta (BBA) - Molecular
Cell Research 2014, 1843 (2), 352-365. DOI: 10.1016/j.bbamcr.2013.11.018.
(55) Wills, J.; Jones, J.; Haggerty, T.; Duka, V.; Joyce, J. N.; Sidhu, A. Elevated tauopathy and
alpha-synuclein pathology in postmortem Parkinson's disease brains with and without dementia.
Experimental Neurology 2010, 225 (1), 210-218. DOI: 10.1016/j.expneurol.2010.06.017.
(56) Alafuzoff, I.; Hartikainen, P. Alpha-synucleinopathies. Handbook of clinical neurology
2017, 145, 339-353. DOI: 10.1016/B978-0-12-802395-2.00024-9.
(57) Berrocal, R.; Vasquez, V.; Krs, S. R.; Gadad, B. S.; Ks, R. α-Synuclein Misfolding Versus
Aggregation Relevance to Parkinson’s Disease: Critical Assessment and Modeling. Humana
Press Inc.: 2015; Vol. 51, pp 1417-1431.
                                                  78


(58) Martí, M.; Tolosa, E.; Campdelacreu, J. Clinical overview of the synucleinopathies.
Movement disorders : official journal of the Movement Disorder Society 2003, 18 (6). DOI:
10.1002/mds.10559.
(59) Lehtonen, Š.; Sonninen, T. M.; Wojciechowski, S.; Goldsteins, G.; Koistinaho, J.
Dysfunction of Cellular Proteostasis in Parkinson's Disease. Frontiers in neuroscience 2019, 13
(10), 457. DOI: 10.3389/fnins.2019.00457.
                                               79


                                                      CHAPTER THREE
          Repurposed Neuroleptic Agents as novel 20S Proteasome Activator Scaffolds
     Reproduced in part with permission from Fiolek J. Taylor, Keel L. Katarina and Tepe J. Jetze. Fluspirilene Analogs Activate the 20S
Proteasome and Overcome Proteasome Impairment by Intrinsically Disordered Protein Oligomers. ACS Chem. Neurosci. 2021. Copyright 2021
                                                        American Chemical Society
                                                                   80


3.1 Introduction
3.1.1 Background
      The regulation of protein synthesis, degradation and folding within a cell is collectively
known as proteostasis. Proteostasis is maintained by a wide array of cellular machinery that works
to ensure that proteins are present in the proper location and amounts to perform their required
functions.1-6 However, as humans age, dysregulation of the proteostasis network is inevitable.
When this dysregulation of proteostasis occurs, there can be disastrous effects on the cell and even
on neighboring cells due to interference with critical signaling pathways. These effects are
associated with a variety of human diseases. One increasingly prevalent example of this is seen in
neurodegenerative diseases, such as Parkinson’s disease, Alzheimer’s disease, Huntington’s
disease, and amyotrophic lateral sclerosis.7-19 In these neurodegenerative diseases, accumulation
of specific IDPs leads to toxic signaling and disruption of proteostasis caused by their uncontrolled
aggregation and oligomerization.20-27 As a result, novel therapeutic methods that have the potential
to assist in reestablishing proper levels of these IDPs are of great interest.
      Recently, it has been recognized that the 20S proteasome plays a critical role in maintaining
proteostasis through the direct degradation of oxidatively damaged and intrinsically disordered
proteins.6, 28-35 The 20S proteasome may therefore serve as the default protease to unremittently
maintain low levels of IDPs, without the need for post-translational modifications, including
protein ubiquitination.6, 28 Especially highly disordered proteins appear to be the main target of the
20S proteasome.36 Unfortunately, when dysregulation of IDPs leads to production that outpaces
their proteasomal clearance, the IDPs accumulate, and subsequently oligomerize and aggregate.
The oligomeric forms of the IDPs in turn can inhibit the 20S proteasome, resulting in a downward
spiral of IDP accumulation, oligomerization and disease progression.37-41
                                                   81


      These findings have sparked the search for small molecules capable of enhancing the
proteolytic activity of the 20S proteasome.42-48 Small molecule proteasome enhancers are still
relatively scarce, with only a few bona fide examples reported in the literature.49-55 The Tepe lab
developed an interest in this field of 20S proteasome activation following their discovery of the
small molecule 20S activator TCH-165 and related imidazoline analogues.50 Since then, the Tepe
lab has pursued novel 20S proteasome activator scaffolds through a variety of strategies, including
design and synthesis of novel scaffolds, through collaborations (Chapter 2)49 and through the
repurposing and modification of established drugs (this Chapter).51
      Repurposing of existing drugs was considered a promising strategy for the discovery of novel
20S proteasome activators, because it should allow for the identification of small molecule
activator scaffolds with good drug-like properties. Our hypothesized strategy for targeting
neurodegenerative diseases through 20S proteasome activation would require activators that can
effectively cross the blood-brain barrier (BBB). So, by identifying 20S activator scaffolds amongst
preexisting drugs, we could greatly increase our chances of success by starting with molecules that
can cross the BBB.56 This would allow for a much more streamlined drug development process
and help to avoid issues with bioavailability in in vivo models, should this technology progress to
that point.57, 58
                  This was also important because of the relatively few previously identified
activators in the literature,49-55 several are not considered to be bona fide activators due to poor
drug-like properties, detergent-like behavior, and a lack of translation of activity to physiologically
relevant substrates and systems. This includes detergents, such as sodium dodecyl sulfate (SDS)
and natural products, like oleuropein,59 and betulinic acid.60 These, as well as others, are
questionable as true 20S agonists, since their activity does not translate under more physiologically
                                                   82


relevant conditions and may result in disruption of proteasome subunits.54 This drug repurposing
strategy should help to avoid future issues with other similar nondrug-like molecules.
      To begin identifying 20S proteasome activator scaffolds amongst known drugs, the Tepe lab
performed a high-throughput screen (HTPS) of the NIH Clinical Collection and Prestwick
libraries.51 For this screen, purified 20S proteasome was exposed to the compounds (10 μM) in the
presence of the fluorogenic chymotrypsin-like (CT-L) peptide substrate (Suc-LLVY-AMC). In
this activity assay, proteolysis of the peptide substrate is enhanced if a compound induces an open-
gate conformation of the α-ring, or through other allosteric interactions, to allow for more rapid
proteolytic cleavage inside the core particle. This enhanced proteolysis of the substrates is
indicated by the release of AMC from the peptide substrate (as discussed in Chapter 2).61 The
compounds were deemed inactive if the CT-L activity was <2 fold (at 10 μM) over the DMSO
vehicle control. From this screen, several novel small molecule activators of the 20S proteasome
were identified. This included several neuroleptic agents, such as Chlorpromazine,62 other
phenothiazines (Fig. 3.1A), Aripiprazole63 and Fluspirilene64, 65 (Fig. 3.2), which could represent
promising starting points for the development of BBB permeable 20S activators as therapeutics
for neurodegenerative diseases.51
      Chlorpromazine and related phenothiazine analogues were the first of these neuroleptic
agents to be explored more thoroughly by the Tepe lab (Fig. 3.1A).51 It was found that
Chlorpromazine could effectively enhance the proteolytic cleavage of the CT-L fluorogenic
peptide (Fig. 3.1B) and IDP substrates (Fig. 3.1C) of the 20S proteasome in vitro.51 In silico
docking done on Chlorpromazine suggests that it, similar to TCH-165, may be acting on the 20S
proteasome by binding in an inter-subunit pocket of the α-ring (Fig. 3.1D).50 Thus, promoting an
                                                   83


open-gate conformation of the 20S proteasome and allowing for easier substrate access to the
catalytic core, where proteolytic cleavage can occur.49-51
  A                                  B
                                                                      D
                                                                      Chlorpromazine
                                                                                         α3
                                                                                               α2
                                     C
Figure 3.1: Phenothiazines, like Chlorpromazine, enhance 20S mediated degradation of
peptide and IDP substrates, presumably through allosteric interactions with the α-ring. (A)
Structures of phenothiazine analogues identified in a HTPS as 20S activators. (B) Concentration
response (0–80 μM) of phenothiazine analogues in fluorogenic peptide (CT-L) digestions with
purified human 20S proteasome. Error bars denote standard deviation. (C) In vitro enhancement
of purified human 20S proteasome-mediated α-synuclein degradation by Chlorpromazine,
visualized using western blot.51 (D) In silico docking model suggests Chlorpromazine likely binds
in the α2-3 pocket of the α-ring of the 20S proteasome.
     The exciting results obtained with Chlorpromazine encouraged additional studies,51 wherein
I sought to validate the activation of the 20S proteasome by other neuroleptic agents identified in
our HTPS, like Aripiprazole63 and Fluspirilene64, 65 (Fig. 3.2), so that they may be repurposed. This
would allow for the identification of other novel 20S activator scaffolds with promising drug-like
                                                 84


properties for use in studying the effects of 20S proteasome activation in neurodegenerative
disease models. With a larger pool of drug-like small molecule scaffolds that activate the 20S
proteasome, we increase our chances of success when developing them as therapeutics, learn more
about what motifs can be used to generate novel activators, and can address some of the limitations
of previously discovered activators. These limitations include poor drug-like properties, lack of
BBB permeability, non-translatable activity to native IDP substrates, detergent-like behavior,
troublesome synthesis, and off-target effects.51, 54, 59, 60
      In these studies, I explored the neuroleptic agents Aripiprazole63 and Fluspirilene64, 65 (Fig.
3.2), and analogues thereof, as 20S proteasome activators. I evaluated their activity in detail
using fluorogenic peptide and IDP substrates. Then, I employed in silico docking to assist in
analogue design. In the case of Aripiprazole, I synthesized and evaluated the analogues.
Whereas, in the case of Fluspirilene, I collaborated with Dr. Katarina Keel, who performed the
synthesis of Fluspirilene and its analogues, and with whom I worked together on the docking and
analogue design.
Figure 3.2: The neuroleptic agents Chlorpromazine, Aripiprazole and Fluspirilene were
identified as potential 20S activator scaffolds in a HTPS.
                                                  85


3.1.2 Objective
        The goals of these studies were (1) to validate the neuroleptic agents Aripiprazole and
Fluspirilene as bona fide 20S activator scaffolds, (2) design, synthesize and evaluate novel
analogues of these scaffolds to diminish their neuroleptic activity and maintain/enhance 20S
activity, and (3) identify promising lead molecules for use in the development of novel assays
exploring the potential of this strategy for targeting neurodegenerative diseases.
3.2 Results and Discussion
3.2.1 The neuroleptic agent Aripiprazole is a small molecule activator of the 20S proteasome
        Aripiprazole (Fig. 3.2) was identified as a novel 20S activator in the HTPS performed by
our lab previously, in which Chlorpromazine was also identified.51 Aripiprazole is a FDA approved
neuroleptic agent with dopamine D2 receptor partial agonist activity used for the treatment of
schizophrenia.63 The studies outlined here were undertaken to verify Aripiprazole’s 20S
proteasome activity, develop analogues of Aripiprazole and to assess whether its activity translates
to a more relevant IDP substrate. This was done as part of an effort to identify a promising activator
for use in the development of novel methods for exploring the potential of this 20S activation
strategy for combating neurodegenerative diseases.
        First, I set out to evaluate Aripiprazole’s activity utilizing fluorogenic peptide substrates
for the 20S proteasome that are conjugated to AMC, as discussed in Chapter 2. This assay was
similar to what was utilized in the HTPS that originally identified Aripiprazole among other
compounds as 20S activators.51 Here, this assay was used to further evaluate Aripiprazole’s
activity at each of the 3 catalytic sites (CT-L, Tryp-L and Casp-L) of the proteasome, determine if
this activity occurs in a concentration-dependent manner and to determine its relative potency. It
should be noted that at the time that Aripiprazole was being tested, we did not yet utilize a
                                                   86


combination of the 3 fluorogenic peptide substrates to obtain a more comprehensive look at the
activity of activators and to compare them. That practice began with my testing of Fluspirilene
(Fig. 3.6) and the dihydroquinazoline analogues (Chapter 2).49 From these data, it was found that
Aripiprazole effectively activates each of the 3 catalytic sites in a concentration-dependent manner
(Fig. 3.3A). It was able to achieve an EC200 for each catalytic site at low μM concentrations and
led to high maximum fold increases over the DMSO vehicle control at higher concetrations (Fig.
3.3B). These initial results suggested that Aripiprazole may represent a good scaffold from which
other analogues may be developed and used to develop novel methods for exploring the potential
of this therapeutic method.
                                                    20
                      A                                           CT-like
                          20S proteasome activity
                                                    15            Tryp-like
                                                                  Casp-like
                                                    10
                              (fold of control)
                                                    5
                                                    0
                                                         -7          -6            -5          -4     -3
                                                                         log[M], Aripiprazole
        B
              Catalytic site                                      CT-L                     Tryp-L           Casp-L
                Substrate                                     Suc-LLVY-AMC              Boc-LRR-AMC        Z-LLE-AMC
               EC200 (μM)                                       2.8 ± 0.1                 5.0 ± 0.7        2.4 ± 0.3
            Max Fold Increase                                  10.4 ± 0.4                 7.9 ± 0.8        15.1 ± 0.6
Figure 3.3: Aripiprazole enhances 20S mediated degradation of peptide substrates. (A)
Concentration response (0–80 μM) from fluorogenic peptide digestions with Aripiprazole. These
data were collected in triplicate. Error bars denote standard deviation. (B) Calculated EC200 and
max fold increases in activity over the vehicle control. Shown with calculated standard deviations.
                                                                              87


3.2.2 Aripiprazole enhances the degradation of the IDP α-synuclein in vitro, but lacks
translation in terms of potency
        After confirming that Aripiprazole has promising activity on the 20S proteasome in
fluorogenic peptide assays, relative to other activators that our lab has studied,50, 51 I then utilized
α-synuclein, an IDP associated with Parkinson’s disease, as a substrate.7, 10, 17-19, 22, 26 This was
done to determine whether the activity of Aripiprazole translates well to the degradation of natural
substrates for the 20S, not just small peptides.66 For these experiments pure human 20S proteasome
was incubated together with Aripiprazole (3, 10, 30 or 100 µM), TCH-165 (10 µM), the
proteasome inhibitor Epoxomicin (1 µM),67 or DMSO (Vehicle, Fig. 3.4). Then, pure human α-
synuclein substrate was introduced and the resulting mixture was incubated for 4 hours. The
remaining α-synuclein, as well as 20S proteasome subunits, were subjected to denaturing
conditions and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used
to resolve the proteins. The resulting bands were stained using a Coomassie stain for visualization
of remaining α-synuclein.
        While these data showed that Aripiprazole was able to enhance the degradation of α-
synuclein by the 20S in vitro, this effect is not very potent when compared to other previously
identified activators, like TCH-165 (Fig. 3.4). The relatively poor potency and lack of translatable
activity of Aripiprazole when using α-synuclein as a substrate may cause difficulty in obtaining
clear 20S proteasome enhancement in more disease relevant systems. Despite this, analogue
development was underway to explore whether this activity could be improved upon, while also
making changes that may reduce the neuroleptic activity of Aripiprazole.
                                                   88


                                    20S proteasome                           10 μM TCH-165                             1 μM Epoxomicin
                                                                                                  Aripiprazole
                                                                                                      (μM)
                                                     α-synuclein
                           Ladder                                  Vehicle                                       100
                                                                                             3     10     30
                37 kDa
                25 kDa
                20 kDa
                15 kDa
                     Relative intensities:                         100         17            74    64     52     15
                        (% of vehicle)
Figure 3.4: Aripiprazole enhances 20S proteasome mediated degradation of the IDP α-
synuclein. Coomassie stain illustrating Aripiprazole’s enhancement of α-synuclein digestion by
the 20S at 3, 10, 30 and 100 μM. DMSO (vehicle), TCH-165 (10 μM) and proteasome inhibitor
(Epoxomicin, 1 μM) treatments were included as controls. Relative intensities, shown as percent
of vehicle, were obtained by densitometry of α-synuclein bands done using image J. Each lane was
normalized using densitometry of total 20S proteasome subunits to control for loading differences.
3.2.3 Synthesis and evaluation of Aripiprazole analogues did not yield any promising lead
molecules
        Despite the 20S activity seen for Aripiprazole in the fluorogenic peptide assays, analogue
development is necessary due to its dopamine D2 receptor activity.63 Additionally, Aripiprazole’s
potency for enhancing the rate of degradation of IDPs in vitro is notably worse than that of TCH-
165 (Fig. 3.4). Analogue development was undertaken to try to overcome these limitations and
grant a better understanding of Aripiprazole’s activity toward the 20S proteasome. This was done
by focusing on the two halves of the molecule, the quinolinone “head” and phenylpiperazine “tail”,
and functionalizing them with groups that were shown to be active with previously discovered
activators.51
                                                                             89


        The first series of analogues (compounds 3-1 through 3-6 (Scheme 3.1)) focused on the
phenylpiperazine-tail of Aripiprazole and incorporated functionalities that were explored during
analogue development of the phenothiazines.51 Compounds 3-1 through 3-6 retain the piperazine
and N-linked phenyl or dichlorophenyl functionalities of Aripiprazole and replaced the
quinolinone-head and linking alkyl chain with various benzyl-groups. This included, either an
unsubstituted benzyl, methyl 4-methylbenzoate, or p-tolyl acetate functionality. These analogues
were meant to explore whether the quinolinone functionality is necessary for 20S activation and
how substitution for a variety of other functionalities seen in previously synthesized
Chlorpromazine analogues would affect activity.51
        Compounds 3-1 through 3-6 were synthesized following a similar procedure to that of
Mohammadi et al.68 Briefly, the piperazines were dissolved with potassium carbonate in
dimethylformamide (DMF) and to this was added the corresponding benzyl bromide. The resulting
mixtures were stirred and refluxed in DMF for 3 hours to produce the desired products (compounds
3-1 through 3-6). The methyl ester analogue lacking the 2 chlorines on the phenyl piperazine was
originally targeted for synthesis through this method, but it was found that the phenol analogue
(compound 3-3) was isolated in its place. This was likely due to partial hydrolysis of the methyl
ester to the phenol, followed by isolation of the incorrect product. As will be discussed in the next
paragraph, evaluation of the 20S activities of these analogues showed no activation by any of the
analogues. So, considering compound 3-3 is further removed from the parent molecule than
compound 3-4, which showed no activity, the other methyl ester analogue was not pursued further.
                                                  90


Scheme 3.1: (A) Structures and (B) synthesis of Aripiprazole analogues 3-1 through 3-6.
  A
  B
       Following the synthesis of this first set of analogues (compounds 3-1 through 3-6), they
were evaluated for 20S activity using the fluorogenic peptide substrates, but none of these
analogues showed significant activity above the DMSO treated vehicle control (data not shown).
Despite their inability to activate the 20S, these results still provided information about the activity
of Aripiprazole and demonstrated that the functionalities explored here are not sufficient to
maintain that activity.
       Analogue development was continued by exploring the quinolinone-head group. Molecular
docking studies were performed using Autodock VinaTM, supported through computational
resources and services provided by the Institute for Cyber-Enabled Research at Michigan State
                                                   91


University. The entirety of the 20S proteasome was used to perform unbiased blind docking,
allowing for true conformational preference. From the resulting docking models, it was determined
that Aripiprazole is likely binding in the same α2-3 pocket as Chlorpromazine (Fig. 3.5A).
Additionally, both molecules seemed to orient similarly within the pocket. This resulted in the tail
portions of each molecule roughly overlapping in these overlaid images. Following these docking
studies, I hypothesized that analogues with Aripiprazole- and Chlorpromazine-like functionalities
could help to elucidate what functionalities of Aripiprazole are important for binding and
potentially yield active analogues. To this end, five new target molecules (Fig. 3.5B, compounds
3-7 through 3-11) were designed.
                                                 92


 A               α3           Chlorpromazine   B
                              and Aripiprazole
                                     α2
 C
Figure 3.5: Molecular docking led design of Aripiprazole and Chlorpromazine-like
analogues. (A) Preferred docking site of Chlorpromazine (blue) and Aripiprazole (red), utilizing
Autodock Vina, in the α2-3 inter-subunit binding pocket of the 20S proteasome’s α-ring. (B)
Proposed Aripiprazole and Chlorpromazine-like analogues (compounds 3-7 through 3-11).
        Compound 3-7 was designed to determine if the quinolinone functionality of Aripiprazole
mimics the phenothiazine portion of Chlorpromazine. The benzoate that was chosen as the tail-
portion of compound 3-7 was previously shown to enhance Chlorpromazine’s activity when
substituted in place of dimethylpropylamine.51 Compounds 3-8 and 3-9 represent hybrid molecules
with parts mimicking both the phenothiazine of Chlorpromazine, in the form of an Fmoc protecting
                                                 93


group, and Aripiprazole, in the phenyl piperazine-tail. Compounds 3-10 and 3-11 sought to expand
upon this and potentially provide further support for Fmoc as a potential mimic for phenothiazine
when binding to the 20S. These analogues sought to explore the possibility of a new functional
group, in Fmoc, that can be easily conjugated to a variety of tails and used in place of the
phenothiazine, seen with Chlorpromazine. Additionally, the substitution of the phenothiazine with
another similar functionality, like the Fmoc group seen here, would provide another route through
which to potentially eliminate the dopamine receptor activity seen with Chlorpromazine.51
Previous efforts to discover active substitutes for the phenothiazine functionality yielded few
promising results, so any progress that could be made to this end would be greatly beneficial for
development of future 20S activators.
        Compound 3-7 was synthesized by first adding sodium hydride to a solution of the
quinolinone-head group of Aripiprazole in DMF at 0°C for 15 minutes. This was followed by
addition of benzyl bromide, warming to room temperature, and stirring for an additional 3 hours
to form compound 3-7, in 61% yield (Scheme 3.2). Compound 3-8 through 3-11 were synthesized
by addition of Fmoc-Cl to a stirring solution of the corresponding amine and triethylamine (TEA)
at 0°C. The resulting mixtures were allowed to warm to room temperature and stirred overnight to
give compounds 3-8 through 3-11 (Scheme 3.2).
                                                 94


Scheme 3.2: Synthesis of (A) compound 3-7 and (B) compounds 3-8 through 3-11.
  A
  B
        Following the synthesis of compound 3-7 through 3-11 (Scheme 3.2), they were evaluated
for 20S proteasome acitivty using the fluorogenic peptide substrates. It was found that none of
these analogues showed any activation of the 20S proteasome in this assay (data not shown). The
lack of activity seen from compounds 3-8 through 3-11 suggest that the Fmoc functional group is
not a suitable sustitute for the phenothiazine of Chlorpromazine. Compound 3-7 being inactive
suggested that the quinolinone functionality of Aripiprazole does not directly mimic the
phenothiazine group of Chlorpromazine. This is clear, because the phenothiazine was shown to
have enhanced activity when conjugated to the same benzoate functionality, in place of the
dimethylamine.51 Additionally, it appears that the quinolinone group is not sufficient for
maintaining any of Aripiprazole’s prior activity on the 20S.
        All Aripiprazole analogues up until this point had undergone significant modification when
compared to Aripiprazole itself and it was thought that the truncated linker seen in compound 3-7
may not allow for enough flexibility in the molecule for it to properly bind to the 20S. To address
this possibility, compound 3-12 was designed and synthesized (Scheme 3.3) with the goal of
mimicking the overall structure and length of Aripiprazole more closely. Briefly, a solution of 1-
                                                 95


phenylpiprazeine and TEA in THF was slowly added to a solution of 4-bromobutanoyl chloride at
0 °C in THF with molecular sieves. The mixture was allowed to gradually warm to room
temperature overnight. Following workup, the crude material was carried forward due to an
unidentified contaminant that was not separated via column chromatography. The quinolinone-
head group of Aripiprazole was then dissolved in DMF and stirred in the presence of sodium
hydride for 10 minutes. To this was then added the crude intermediate, produced in the previous
reaction, and this mixture was stirred at room temperature overnight to yield compound 3-12, in
8% yield over 2 steps.
Scheme 3.3: Synthesis of compound 3-12.
        Following the synthesis of compound 3-12, it was tested in the fluorogenic peptide assay
and was found to be inactive (data not shown). This suggested that Aripiprazole may not be an
ideal scaffold from which to build novel 20S activators due to its activity apparently being highly
sensitive to structural modifications. Modifications, such as those undergone in compound 3-12,
would likely be required to effectively diminish its neuroleptic activity or to improve its potency.
This sensitivity to modification, coupled with Aripiprazole’s relatively poor potency when
translating activity to in vitro 20S proteasome mediated IDP digestions led me to explore other
                                                96


neuroleptic agent scaffolds for their potential as 20S proteasome activators. For this, I revisited the
HTPS run by our lab previously and further evaluated the activity of other 20S proteasome
activator hits.
3.2.4 The neuroleptic agent Fluspirilene activates all 3 catalytic sites of the 20S proteasome
but does not activate the 26S proteasome
         Upon returning to the HTPS run by our lab previously, I retested several other hit molecules
using fresh stocks of each compound. From these data (not shown), the small molecule
antipsychotic drug Fluspirilene64, 65 was identified as a promising new scaffold for the development
of 20S activators, due to its enhancement of 20S proteasome-mediated proteolysis of fluorogenic
peptide substrates. To further assess Fluspirilene’s 20S proteasome activity, a series of assays were
performed using the three previously mentioned fluorogenic peptide substrates. These substrates
were a CT-L, a Tryp-L and a Casp-L substrate, one for each of the catalytic sites of the proteasome.
It has also been shown that the proteasome’s active sites allosterically regulate each other in the
presence of their individual substrates.69 Therefore, a combination of the three fluorogenic
peptides, in equal amounts, was also used to represent the overall activity of a 20S activator more
accurately in a system in which all catalytic sites are interacting.69 This was the first time that this
combination of peptide substrates was utilized by our lab, and has since become a common practice
to obtain generalized levels of activity for a given compound, that can then be compared to other
identified activators. The continued testing of the individual catalytic sites remains important
because all of these catalytic sites will likely be required for the degradation of IDPs and other
native substrates of the 20S that have many cleavage sites. So, while the combination of the three
substrates provides a good overall view of activity, the individual substrate screenings each ensure
that all catalytic sites are contributing to these results.
                                                    97


        It was found that Fluspirilene activates all three catalytic sites of the 20S proteasome in a
concentration-dependent manner (Fig. 3.6A) and achieved an EC200 of 2.2 μM using the
combination of substrates, with a maximum fold enhancement of nearly 10-fold (i.e. 1000%)
relative to the DMSO treated vehicle control (Fig. 3.6B). The level of activity found for
Fluspirilene in these studies is on par with some of the most active and potent small molecule 20S
proteasome activators identified to date. This, coupled with Fluspirilene’s good drug-like
properties and BBB permeability,64, 65 suggested that it may represent an ideal scaffold from which
additional 20S proteasome activators can be developed.
                                                 98


                                          30
            A                                                                                       3 sites
                20S Proteasome Activity
                                                                                                    CT-L
                                          20                                                        Tryp-L
                                                                                                    Casp-L
                                                                                                    26S
                   (fold over control)
                                          10
                                          0
                                               -8        -7         -6          -5     -4          -3
                                                                log[M] Fluspirilene
      B
          Catalytic site                             3 sites             CT-L          Tryp-L            Casp-L
                                                                     Suc-LLVY         Boc-LRR              Z-LLE
           Substrate                                Combo
                                                                      -AMC             -AMC                -AMC
           EC200 (μM)                               2.2 ± 0.2        1.0 ± 0.3        10.9 ± 3.0        2.2 ± 0.3
            Max Fold
                                                    9.8 ± 0.5        12.9 ± 2.2       17.7 ± 5.3        16.3 ± 2.3
            Increase
Figure 3.6: The neuroleptic agent Fluspirilene activates all 3 catalytic sites of the 20S
proteasome but does not activate the 26S proteasome. (A) Concentration response (0–80 μM)
from fluorogenic peptide digestions with Fluspirilene. These data were collected in triplicate. Error
bars denote standard deviation. (B) Calculated EC200 and max fold increases in activity over the
vehicle control. Shown with calculated standard deviations.
3.2.5 Fluspirilene analogues designed using in silico docking models as a guide
       Fluspirilene is a potent dopamine D2 receptor antagonist that has been used for the
treatment of schizophrenia.64, 65 As such, it has good drug-like properties and penetrates the BBB
effectively, which makes it a promising scaffold for development of novel 20S activators and for
use in evaluating the potential of this therapeutic strategy. On the other hand, due to its potent D2
                                                                         99


receptor activity it cannot be repurposed therapeutically without modification. Therefore, we next
sought to explore whether Fluspirilene’s 20S proteasome activity is amenable to structural
modifications known to reduce the dopamine D2 receptor activity. For example, previous studies
by our group showed that eliminating or disrupting the interaction of the critical basic amine with
the D2 receptor abrogates this activity.51, 70
        Using molecular docking as a guide (this work was done collaboratively with Dr. Katarina
Keel), N-acylated Fluspirilene (Fig. 3.7A), was designed to eliminate Fluspirilene’s D2 receptor
activity. In this scaffold, the basicity of the piperidine’s amine has been reduced through conversion
to an amide. Additionally, Fluspirilene and N-acylated Fluspirilene dock similarly within the
proteasome. Molecular docking studies were performed using Autodock VinaTM, supported
through computational resources and services provided by the Institute for Cyber-Enabled
Research at Michigan State University. The entirety of the 20S proteasome was used to perform
unbiased blind docking, allowing for true conformational preference. Fluspirilene and N-acylated
Fluspirilene were found to preferentially bind to the α2-3 inter-subunit pocket (Fig. 3.7B and C).
This mode of binding is different from our previously reported 20S proteasome activator TCH-
165 which, when docked, preferentially binds to the α1-2 inter-subunit pocket.50 To test the
importance of the α2-3 inter-subunit pocket, two other analogues of Fluspirilene were devised as
negative controls. Compounds 3-13 and 3-14 (Fig. 3.7A) were designed to be less active towards
the proteasome, based on assumptions regarding the diphenyl tail’s optimal 3D conformation and
the importance of the difluoro substituents, respectively. Molecular docking of these analogues
showed less preference for the α2-3 inter-subunit pocket, supporting these assumptions. Through
these three analogues of Fluspirilene, we aimed to further understand the in-pocket binding
interactions of our newly discovered 20S proteasome activator scaffold.
                                                     100


 A
   B                                           C
Figure 3.7: Fluspirilene and N-acylated Fluspirilene preferentially dock in the α2-3 inter-
subunit binding pocket of the 20S proteasome. (A) Structures of N-acylated Fluspirilene,
compound 3-13 and compound 3-14. (B) Preferred docking site of N-acylated Fluspirilene,
utilizing Autodock VinaTM, in the α2-3 inter-subunit pocket of the 20S proteasome’s α-ring. (C)
Zoomed in image of N-acylated Fluspirilene docked in the α2-3 inter-subunit pocket.
                                              101


3.2.6 Activity differences between Fluspirilene analogues support what was predicted with
docking models
         Fluspirilene and its analogues were synthesized by Dr. Katarina Keel according to
literature.71 Each of the synthesized Fluspirilene analogues (Fluspirilene, N-acylated Fluspirilene,
compound 3-13 and compound 3-14) were tested for 20S proteasome activity (Fig. 3.8). To do
this, the previously described fluorogenic peptide assay was employed to assess their activity using
a combination of the three catalytic site substrates. The results obtained from the screening showed
that the Fluspirilene analogues (N-acylated Fluspirilene, compound 3-13 and compound 3-14) all
showed some degree of activity and clear concentration-responses (Fig. 3.8). Excitingly, N-
acylated Fluspirilene appeared to have comparable potency and greater maximum activity in this
assay (Fig. 3.8) when compared to that of Fluspirilene (Fig. 3.6B). N-acylated Fluspirilene
represents a promising scaffold from which to develop additional analogues due to its potent
activation of the 20S. Additionally, N-acylated Fluspirilene lacks a basic nitrogen, which is known
to be critical for D2 receptor activity.51, 70 While compounds 3-13 and 3-14 both showed some
activity towards the 20S proteasome in this assay, their activity relative to Fluspirilene was greatly
reduced, supporting what was predicted with the molecular docking models.
                                                  102


                                           25
                                                     Fluspirilene
                 20S proteasome activity
                                           20        N-acylated Fluspirilene
                                                     3-13
                                           15
                                                     3-14
                     (fold of control)
                                           10
                                           5
                                           0
                                                -8    -7       -6      -5      -4   -3
                                                             log[M] Agonist
Figure 3.8: Fluspirilene analogues activate the 20S proteasome with varying effectiveness.
Concentration response (0–80 μM) from fluorogenic peptide digestions using the combination of
three fluorogenic peptide substrates with Fluspirilene analogues. These data were collected in
triplicate. Error bars denote standard deviation.
3.2.7 Detailed analysis of molecular docking yields insights into potential key interactions for
Fluspirilene analogue activity
       To further analyze why Fluspirilene and N-acylated Fluspirilene show such promising 20S
proteasomal activity while compounds 3-13 and 3-14 display lessened activity, BIOVA Discovery
Studio 2020 was used to observe interactions of the analogues within the α2-3 inter-subunit pocket
(Fig. 3.9).72 In multiple binding modes obtained for both Fluspirilene and N-acylated Fluspirilene,
hydrogen bond interactions are observed between the imidazol-4-one’s amide N-H and a variety
of amino acid residues such as LYS77, ILE65, ASN84, TYR75, and GLN111 (Fig. 3.9A and B).
Comparatively, while compounds 3-13 and 3-14 show hydrogen bonding with the amide’s
carbonyl, they display no interactions between the amide’s N-H and any of the above-mentioned
amino acid residues (Fig. 3.9C and D). Furthermore, compounds 3-13 and 3-14 display less
                                                               103


preference for the α2-3 inter-subunit pocket, suggesting a strong N-H hydrogen bond interaction
is necessary to support preferential binding. Fluspirilene, N-acylated Fluspirilene, and compound
3-14 also display pi-pi interactions with the diphenyl tail, specifically interacting with PHE60,
PHE61, and TYR154, while compound 3-13 shows no pi-pi interactions between the amino acid
residues and diphenyl tail. This suggests that restricting the conformational flexibility of the
diphenyl tail with a double bond interferes with the binding of this scaffold. Further exploration is
necessary to explain the importance of the difluoro substituents for proteasomal activity.
Figure 3.9: Fluspirilene analogues binding models show varied interactions supporting
differences in activity. Binding models of Fluspirilene and the three analogues, viewed utilizing
BIOVIA Discovery Studio 2020 (A) Fluspirilene (B) N-acylated Fluspirilene (C) compound 3-13
(D) compound 3-14.
                                                104


3.2.8 N-acylated Fluspirilene activates all three catalytic sites of the 20S proteasome but not
the 26S proteasome
        The 20S activity of N-acylated Fluspirilene was further assessed in the fluorogenic peptide
assay, using each of the individual substrates (CT-L, Tryp-L and Casp-L) as well as the
combination of the three (Fig. 3.10). N-Acylated Fluspirilene displayed a clear concentration-
dependent enhancement of 20S proteasome-mediated degradation of each of the three fluorogenic
peptide substrates. When compared to Fluspirilene, N-acylated Fluspirilene performs similarly
using the combination of the three fluorogenic peptide substrates, with an EC200 of 1.9 M (Fig.
3.10B). Additionally, the N-acylated analogue achieved better activation of the Tryp-L site, but
reduced activation of the CT-L site relative to Fluspirilene itself, when tested with the individual
fluorogenic peptide substrates. The N-acylated analogue achieved higher max fold increases for
each substrate/combination (>1500% increase over vehicle) but required slightly higher
concentrations to reach doubling of activity at the CT-L (EC200 = 4.7 M) and Casp-L (EC200 = 4.1
M) sites (Fig. 3.10B). These results led to the decision to carry both Fluspirilene and N-acylated
Fluspirilene forward into more physiologically relevant assays.
                                                 105


                A                        50
                                                                                                 3 sites
               20S Proteasome Activity
                                         40                                                      CT-L
                                                                                                 Tryp-L
                                         30
                                                                                                 Casp-L
                                                                                                 26S
                   (fold of control)
                                         20
                                         10
                                         0
                                              -8         -7      -6           -5     -4         -3
                                                        log[M] N-acylated Fluspirlene
      B
          Catalytic site                            3 sites            CT-L         Tryp-L             Casp-L
                                                                  Suc-LLVY         Boc-LRR             Z-LLE
           Substrate                                Combo
                                                                   -AMC             -AMC               -AMC
           EC200 (μM)                              1.9 ± 0.5      4.7 ± 1.6        5.6 ± 0.8          4.1 ± 0.6
            Max Fold
                                                   20.8 ± 2.3     15.0 ± 6.9       30.6 ± 7.4        36.8 ± 12.2
            Increase
Figure 3.10: N-acylated Fluspirilene is comparable to Fluspirilene in its ability to activate all
3 sites of the 20S proteasome, but not the 26S proteasome. (A) Extended fluorogenic peptide
analysis of N-acylated Fluspirilene. These data were collected in triplicate. Error bars denote
standard deviation. (B) Calculated EC200 and max fold increases in activity over the vehicle
control. Shown with calculated standard deviations.
3.2.9 Fluspirilene and N-acylated Fluspirilene enhance the rate of 20S mediated proteolysis
of the Parkinson’s disease related IDP α-synuclein in vitro
       IDP accumulation and aggregation are commonly associated with the progression of
neurodegenerative diseases. It is believed that these IDPs represent a promising therapeutic target
that may allow for the development of the first disease altering treatment for many
                                                                      106


neurodegenerative diseases.17, 22, 73-77 Parkinson’s disease is currently the second most prevalent
neurodegenerative disease and is characterized by accumulation and aggregation of the IDP α-
synuclein.19, 78-83 Fluspirilene and N-acylated Fluspirilene were tested for their ability to enhance
degradation of α-synuclein, to demonstrate their potential to prevent the toxic IDP accumulation
and the translation of the activity towards the degradation of more relevant substrates, than the
fluorogenic peptides.
        Briefly, purified human 20S proteasome was incubated with DMSO (vehicle), Fluspirilene
or N-acylated Fluspirilene at various concentrations (1, 3 or 10 µM). Pure human α-synuclein
substrate was subsequently added to the mixtures and incubated for 4-hours at 37 °C. Following
this incubation, the remaining α-synuclein and 20S proteasome were subjected to denaturing
conditions and resolved using SDS-PAGE. The resulting bands were visualized using silver stain.
Enhanced 20S activity was measured as a reduction of remaining α-synuclein when compared to
the vehicle (DMSO) control. As shown in Fig. 3.11, both Fluspirilene and N-acylated Fluspirilene
were able to effectively enhance the degradation of α-synuclein by the 20S proteasome in vitro.
Both compounds displayed a significant (Fig. 3.11B-C, >50%, p<0.001) concentration-depended
decrease in α-synuclein at values near their EC200. These results grant confidence in this novel 20S
activator scaffold to induce the degradation of IDPs and prevent their accumulation. As such,
Fluspirilene and N-acylated Fluspirilene were selected as the ideal compounds with which to begin
developing novel methods to further evaluate the potential of 20S proteasome activation as a
therapeutic strategy for combating neurodegenerative diseases.
                                                 107


                               A
   B                  150
                                                            ***
                                                                                                 C                 150
                                                                                                                                              ****
                                                      ***                                                                                 ****
    α-syn remaining                                                                              α-syn remaining
                                           *                                                                                         **
                      100                                                                                          100
     (% of control)   50                                                                          (% of control)   50
                       0                                                                                            0
                                      e
                                   icl                µM                µM                µM                                          µM          µM         µM
                                                                                                                          cle
                               h                  1                 3                 0                                   hi
                            Ve                 u.                u.                .1                                    Ve          .1          .3      .1  0
                                          Fl                Fl               Flu
                                                                                                                                Flu         Flu        Flu
                                                                                                                              acyl        acyl    ac
                                                                                                                          N-          N-            yl
                                                                                                                                                 N-
Figure 3.11: Fluspirilene and N-acylated Fluspirilene enhance the rate of 20S mediated
proteolysis of the Parkinson’s disease related IDP α-synuclein in vitro. (A) Representative
silver stain illustrating Fluspirilene’s enhancement of α-synuclein digestion by the 20S at 1, 3 and
10 μM. (B) Densitometry of Fluspirilene α-synuclein digestions done using image J (n=3). (C)
Densitometry of N-acylated Fluspirilene α-synuclein digestions done using image J (n=5). Error
bars denote standard deviation. Ordinary one-way ANOVA statistical analysis was used to
determine statistical significance (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).
3.3 Conclusions
       In summary, these studies demonstrated that the previously FDA approved neuroleptic
agents, Aripiprazole and Fluspirilene both represent novel 20S proteasome activator scaffolds. It
                                                                                               108


was found that Aripiprazole, while being a 20S proteasome activator, does not show great
translation of activity to the enhancement of 20S-mediated degradation of IDP substrates, relative
to other activators discussed here. Additionally, the analogues of Aripiprazole that were
synthesized as part of these studies were all found to be inactive towards the 20S proteasome. For
these reasons, Aripiprazole is not an ideal scaffold from which to develop additional 20S
proteasome activators, nor for developing novel methods to further explore the potential of 20S
activation as a therapeutic method.
      However, the subsequent studies focused on Fluspirilene and analogues thereof, yielded
much more promising results. Fluspirilene and N-acylated Fluspirilene were both found to
effectively enhance 20S proteasome-mediated degradation of fluorogenic peptide substrates and
the IDP α-synuclein in vitro. Neither of these activators showed substantial activity towards the
26S proteasome, which is desirable for specific targeting of IDPs associated with
neurodegenerative diseases. Molecular docking analyses of the Fluspirilene analogues,
synthesized by Dr. Katarina Keel, provided insight into potential key binding interactions, and
suggest that these molecules act through a similar gate-opening mechanism as previously
identified 20S activators. The relatively good potency, translatable activity to disease relevant
substrates, BBB permeability and potential for docking-led analogue design seen with Fluspirilene
and N-acylated Fluspirilene led to the conclusion that it represents an ideal scaffold from which
novel activators can be developed. Additionally, Fluspirilene and N-acylated Fluspirilene were
selected as lead molecules for use in developing novel methods to further explore the potential of
20S activation as an innovative therapeutic strategy.
      1. The neuroleptic agents Aripiprazole and Fluspirilene were validated as 20S proteasome
          activators.
                                                 109


      2. Analogues of Aripiprazole and Fluspirilene were synthesized by me or Dr. Katarina Keel
          (Fluspirilenes) and evaluated for 20S activity.
      3. Fluspirilene and N-acylated Fluspirilene were determined to be ideal candidates for
          repurposing as 20S activators for further studies, based on a variety of advantages over
          other activators.
3.4 Experimental
General information
        Reactions were carried out under a nitrogen atmosphere in flame-dried glassware. Solvents
and reagents were purchased from commercial suppliers and used without further purification.
Anhydrous tetrahydrofuran (THF) was distilled over sodium (dryness was monitored by the color
of the solution, as indicated by benzophenone’s ketyl radical), triethylamine (TEA) was distilled
over calcium hydride, and dichloromethane (DCM) was dried over molecular sieves directly
before use. Magnetic stirring was used for all reactions. Yields refer to chromatographically and
spectroscopically pure compounds unless otherwise noted. Infrared spectra were recorded on a
Jasco Series 6600 FTIR spectrometer. 1H and 13C NMR spectra were recorded on a Varian Unity
Plus-500 spectrometer. Chemical shifts are reported relative to the residue peaks of the solvent
(CDCl3: 7.26 ppm for 1H and 77.0 ppm for 13C) (DMSO-d6: 2.50 ppm for 1H and 39.5 ppm for
13
  C). The following abbreviations are used to denote the multiplicities: s = singlet, d = doublet, dd
= doublet of doublets, t = triplet, and m = multiplet. The following abbreviation is used to denote
a broad signal: br. = broad. HRMS were obtained at the Mass Spectrometry Facility of Michigan
State University with a Micromass Q-ToF Ultima API LC-MS/MS mass spectrometer. Column
chromatography was performed using a Teledyne ISCO CombiFlash® NextGen system with
prepacked columns (RediSep® Normal-phase silica, 20-40 microns). TLCs were performed on
                                                110


pre-coated 0.25 mm thick silica gel 60 F254 plates and visualized using UV light and iodine
staining.
1-benzyl-4-phenylpiperazine (3-1). 1-phenylpiperazine (189 μL, 1.23 mmol) and K2CO3 (170
mg, 1.23 mmol) were dissolved in DMF (10 mL) at room temperature. To this mixture, benzyl
bromide (210.4 mg, 1.23 mmol) was added in DMF (5 mL). This solution was brought to reflux
and allowed to stir for 3 hours before being allowed to cool back to room temperature. The reaction
was then analyzed with TLC (3:1 hexanes/ethyl acetate) and it was determined that the reaction
had gone to completion. The reaction mixture was then cooled to 0°C in an ice bath. Once cooled,
the solution was acidified using 0.5 M HCl in water. This did not yield a precipitate that was
expected based on the literature reference for the procedure. The solution was then neutralized
with NaOH pellets. Then, ethyl acetate (40 mL) was added to the solution and the mixture was
transferred to a separatory funnel. The water layer was removed, and the leftover organic layer
was then washed three times with 10% LiBr solution to remove DMF and water-soluble
byproducts. The remaining ethyl acetate layer was dried with Na2SO4 and concentrated affording
1 (brown solid, 307.5 mg, 99% yield).
1
  H NMR (500 MHz, CDCl3) δ 7.54 – 7.45 (m, 4H), 7.42 – 7.37 (m, 3H), 7.07 – 7.03 (m, 2H), 6.99
(m, 1H), 3.69 (s, 2H), 3.32 (t, J = 5.0 Hz, 4H), 2.73 (t, J = 5.0 Hz, 4H).13C NMR (126 MHz,
CDCl3) δ 151.17, 137.86, 128.98, 128.88, 128.09, 126.94, 119.35, 115.78, 62.87, 52.91, 48.88. IR
(neat): 3052, 2967, 1599, 1503 cm-1. HRMS (ESI-TOF) m/z: [(M+H)+] calcd for (C17H21N2+):
253.1705. Found: 253.1750. m.p.: 39 – 40 °C.
                                                 111


1-benzyl-4-(2,3-dichlorophenyl)piperazine (3-2). 1-(2,3-dichlorophenyl)piperazine (200 mg,
0.747 mmol) and K2CO3 (103.2 mg, 0.747 mmol) were dissolved in DMF (10 mL) at room
temperature. To this solution, benzyl bromide (127.8 mg, 0.747 mmol) was added with more DMF
(5 mL). This mixture was then brought to reflux and allowed to stir for three hours. After three
hours, the reaction was allowed to cool to room temperature and TLC (3:1 hexanes/ethyl acetate)
was used to determine that the reaction had gone to completion. To the reaction mixture ethyl
acetate (40 mL) was added and the solution was transferred to a separatory funnel. The reaction
mixture was then washed four times with 10% LiBr solution to remove the DMF and water-soluble
byproducts. Next, the remaining ethyl acetate solution was dried with Na2SO4 and concentrated
under vacuum. The resulting crude product was then purified by column chromatography (silica,
2:1 ethyl acetate/hexanes) affording 2 (brown solid, 101.1 mg, 42% yield).
1
  H NMR (500 MHz, CDCl3) δ 7.41 – 7.27 (m, 5H), 7.17 – 7.12 (m, 2H), 6.96 (dd, J = 7.2, 2.4 Hz,
1H), 3.66 (s, 2H), 3.12 (s, 4H), 2.71 (s, 4H). 13C NMR (126 MHz, CDCl3) δ 151.01, 133.89, 129.41,
128.31, 127.40, 124.55, 118.58, 62.80, 53.00, 50.88. IR (neat): 3045, 2817, 1577 cm-1. HRMS
(ESI-TOF) m/z: [(M+H)+] calcd for (C17H19Cl2N2+): 321.0925. Found: 321.0929. m.p.: 59 – 60
°C.
                                                   112


4-((4-phenylpiperazin-1-yl)methyl)phenol (3-3). 1-phenylpiperazine (189 μL, 1.23 mmol) and
K2CO3 (170 mg, 1.23 mmol) were dissolved in DMF (10 mL) at room temperature. To this
solution, 4-(bromomethyl)phenyl acetate (281.76 mg, 1.23 mmol) was added with more DMF (5
mL). This mixture was then brought to reflux and allowed to stir for three hours. After three hours,
the reaction was allowed to cool to room temperature and TLC (3:1 hexanes/ethyl acetate) was
used to determine that the reaction had gone to completion. To the reaction mixture ethyl acetate
(40 mL) was added and the solution was transferred to a separatory funnel. The reaction mixture
was then washed four times with 10% LiBr solution to remove the DMF and water-soluble
byproducts. Next, the remaining ethyl acetate solution was dried with Na2SO4 and concentrated
under vacuum. The resulting crude product was then purified by flash column chromatography
(silica, ethyl acetate/hexanes) affording 3 (yellow powder, 50.4 mg, 15% yield).
1
  H NMR (500 MHz, DMSO-d6) δ 9.31 (s, 1H), 7.20 – 7.17 (m, 2H), 7.11 (d, J = 8.4 Hz, 2H), 6.91
(d, J = 8.2 Hz, 2H), 6.75 (t, J = 7.3 Hz, 1H), 6.72 (d, J = 8.4 Hz, 2H), 3.38 (s, 2H), 3.09 (t, J = 4.8
Hz, 4H), 2.46 (t, J = 4.8 Hz, 4H).     13
                                          C NMR (126 MHz, DMSO-d6) δ 156.36, 151.07, 130.18,
128.91, 128.01, 118.74, 115.35, 114.91, 61.69, 52.46, 48.20. IR (neat): 3098 (br.), 3006, 2814,
1598 cm-1. HRMS (ESI-TOF) m/z: [(M+H)+] calcd for (C17H21N2O+): 269.1654. Found: 269.1659.
m.p.: 177 – 178 °C.
                                                 113


4-((4-(2,3-dichlorophenyl)piperazin-1-yl)methyl)phenyl              acetate        (3-4).       1-(2,3-
dichlorophenyl)piperazine (200 mg, 0.747 mmol) and K2CO3 (103.2 mg, 0.747 mmol) were
dissolved in DMF (10 mL) at room temperature. To this solution, 4-(bromomethyl)phenyl acetate
(171.1 mg, 0.747 mmol) was added with more DMF (5 mL). This mixture was then brought to
reflux and allowed to stir for three hours. After three hours, the reaction was allowed to cool to
room temperature and TLC (3:1 hexanes/ethyl acetate) was used to determine that the reaction had
gone to completion. To the reaction mixture ethyl acetate (40 mL) was added and the solution was
transferred to a separatory funnel. The reaction mixture was then washed four times with 10%
LiBr solution to remove the DMF and water-soluble byproducts. Next, the remaining ethyl acetate
solution was dried with Na2SO4 and concentrated under vacuum. The resulting crude product was
then purified by flash column chromatography (silica, ethyl acetate/hexanes) affording 4 (yellow
powder, 163.1 mg, 58% yield).
1
  H NMR (500 MHz, CDCl3) δ 7.38 (d, J = 8.4 Hz, 2H), 7.16 – 7.12 (m, 2H), 7.06 (d, J = 8.5 Hz,
2H), 6.96 (dd, J = 6.8, 2.8 Hz, 1H), 3.57 (s, 2H), 3.06 (s, 4H), 2.64 (s, 4H), 2.30 (s, 3H). 13C NMR
(126 MHz, CDCl3) δ 169.57, 151.29, 149.71, 133.98, 130.12, 127.41, 124.49, 121.33, 118.58,
62.35, 53.18, 51.29, 21.14. IR (neat): 2942, 2830, 1747, 1577 cm-1. HRMS (ESI-TOF) m/z:
[(M+H)+] calcd for (C19H21Cl2N2O2+): 379.0980. Found: 379.0984. m.p.: 119 – 120 °C.
                                                114


methyl 4-((4-phenylpiperazin-1-yl)methyl)benzoate (3-5). 1-phenylpiperazine (189 μL, 1.23
mmol) and K2CO3 (170 mg, 1.23 mmol) were dissolved in DMF (10 mL) at room temperature. To
this solution, methyl-4-(bromomethyl)benzoate (281.76 mg, 1.23 mmol) was added with more
DMF (5 mL). This mixture was then brought to reflux and allowed to stir for three hours. After
three hours, the reaction was allowed to cool to room temperature and TLC (3:1 hexanes/ethyl
acetate) was used to determine that the reaction had gone to completion. To the reaction mixture
ethyl acetate (40 mL) was added and the solution was transferred to a separatory funnel. The
reaction mixture was then washed four times with 10% LiBr solution to remove the DMF and
water-soluble byproducts. Next, the remaining ethyl acetate solution was dried with Na2SO4 and
concentrated under vacuum. The resulting crude product was then purified by flash column
chromatography (silica, ethyl acetate/hexanes) affording 5 (pink/orange powder, 320.1 mg, 84%).
1
  H NMR (500 MHz, CDCl3) δ 8.02 (d, J = 8.2 Hz, 2H), 7.47 (d, J = 7.9 Hz, 2H), 7.27 – 7.24 (m,
2H), 6.93 (d, J = 8.0 Hz, 2H), 6.86 (t, J = 7.3 Hz, 1H), 3.91 (s, 3H), 3.65 (s, 2H), 3.23 (m, 4H),
2.65 (m, 4H).  13
                 C NMR (126 MHz, CDCl3) δ 166.93, 151.12, 129.64, 129.07, 119.78, 116.10,
62.45, 53.03, 52.05, 48.93. IR (neat): 3090, 2950, 1717, 1598, 1273 cm-1. HRMS (ESI-TOF) m/z:
[(M+H)+] calcd for (C19H23N2O2+): 311.1760. Found: 311.1779. m.p.: 86 – 87 °C.
                                                115


methyl        4-((4-(2,3-dichlorophenyl)piperazin-1-yl)methyl)benzoate           (3-6).      1-(2,3-
dichlorophenyl)piperazine (200 mg, 0.747 mmol) and K2CO3 (103.2 mg, 0.747 mmol) were
dissolved    in   DMF     (10   mL)    at  room    temperature.    To   this  solution,   methyl-4-
(bromomethyl)benzoate (171.1 mg, 0.747 mmol) was added with more DMF (5 mL). This mixture
was then brought to reflux and allowed to stir for three hours. After three hours, the reaction was
allowed to cool to room temperature and TLC (3:1 hexanes/ethyl acetate) was used to determine
that the reaction had gone to completion. To the reaction mixture ethyl acetate (40 mL) was added
and the solution was transferred to a separatory funnel. The reaction mixture was then washed four
times with 10% LiBr solution to remove the DMF and water-soluble byproducts. Next, the
remaining ethyl acetate solution was dried with Na2SO4 and concentrated under vacuum. The
resulting crude product was then purified by flash column chromatography (silica, ethyl
acetate/hexanes) affording 6 (pink/orange powder, 190 mg, 67% yield).
1
  H NMR (500 MHz, CDCl3) δ 8.03 (d, J = 7.1 Hz, 2H), 7.49 (m, 2H), 7.17 – 7.12 (m, 2H), 6.97
(m, 1H), 3.91 (s, 3H), 3.71 (s, 2H), 3.12 (m, 4H), 2.72 (m, 4H). 13C NMR (126 MHz, CDCl3) δ
166.88, 134.00, 129.73, 129.34, 127.47, 124.76, 118.66, 62.34, 53.10, 52.10, 50.87. IR (neat):
3095, 2967, 1712, 1577, 1270 cm-1. HRMS (ESI-TOF) m/z: [(M+H)+] calcd for (C19H21Cl2N2O2+):
379.0980. Found: 379.0985. m.p.: 92 – 93 °C.
                                                 116


methyl 4-(((2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)oxy)methyl)benzoate (3-7). To a solution of
7-hydroxy-3,4-dihydro-2(1H)-quinolinone (50 mg, 0.31 mmol) in DMF (10 mL) at 0°C NaH (8.9
mg, 0.372 mmol) was added. This mixture was stirred under nitrogen atmosphere for 15 minutes.
At this point, methyl-4-(bromomethyl)benzoate (71 mg, 0.31 mmol) was quickly added to avoid
H2O contamination. The resulting solution was then allowed to warm to room temperature and left
to stir for hours. Upon completion, the reaction was quenched with the addition of water (10 ml).
To this ethyl acetate (40 mL) was add and the resulting solution was transferred to a separatory
funnel and washed three times with 10% LiBr solution to remove DMF. The resulting organic
solution was then dried with Na2SO4 and concentrated under vacuum. The resulting crude product
was then purified by flash column chromatography (silica, ethyl acetate/hexanes) affording 7
(white powder, 59.1 mg, 61% yield).
1
  H NMR (500 MHz, CDCl3) δ 8.06 (d, J = 8.3 Hz, 2H), 7.67 (s, br., 1H), 7.49 (d, J = 8.3 Hz, 2H),
7.07 (d, J = 8.3 Hz, 1H), 6.59 (dd, J = 8.3, 2.5 Hz, 1H), 6.37 (d, J = 2.5 Hz, 1H), 5.10 (s, 2H), 4.12
(q, J = 7.2 Hz, 1H), 3.92 (s, 3H), 2.92 – 2.89 (m, 2H), 2.63 – 2.60 (m, 2H). 13C NMR (126 MHz,
CDCl3) δ 171.32, 141.89, 138.14, 129.91, 128.82, 126.91, 116.44, 108.81, 102.53, 69.49, 52.17,
31.00, 24.59. IR (neat): 3010, 2945, 1724, 1670, 1592 cm-1. HRMS (ESI-TOF) m/z: [(M+H)+]
calcd for (C18H18NO4+): 312.1236. Found: 312.1237. m.p.: 176 – 177 °C.
                                                 117


9H-fluoren-9-yl)methyl 4-phenylpiperazine-1-carboxylate (3-8). 1-phenylpiperazine (54 μL,
0.352 mmol) and triethylamine (54 μL, 0.387 mmol) were dissolved in anhydrous DCM (10 mL)
at 0°C. To this solution Fmoc-Cl (100 mg, 0.387 mmol) was added with a spatula. The resulting
solution was allowed to warm to room temperature and was stirred overnight. The resulting
solution was washed with water three times and extracted with ethyl acetate (40 mL). The
combined organic solution was then washed three time with brine solution and dried over Na2SO4.
The resulting crude product was purified by flash column chromatography (silica, ethyl
acetate/hexanes) affording 8 (white powder, 94.2 mg, 70% yield).
1
  H NMR (500 MHz, CDCl3) δ 7.79 (d, J = 7.5 Hz, 2H), 7.60 (d, J = 7.4 Hz, 2H), 7.43 (t, J = 7.5
Hz, 2H), 7.35 (m, 4H), 6.95 (m, 3H), 4.49 (d, J = 6.7 Hz, 2H), 4.29 (t, J = 6.7 Hz, 1H), 3.63 (m,
4H), 3.12 (m, 4H). 13C NMR (126 MHz, CDCl3) δ 155.11, 151.13, 143.95, 141.33, 129.21, 127.70,
127.05, 124.93, 120.46, 119.99, 116.72, 67.28, 49.38, 47.35. IR (neat): 3052, 2904, 1681, 1597,
1218 cm-1. HRMS (ESI-TOF) m/z: [(M+H)+] calcd for (C25H25N2O2+): 385.1916. Found:
385.1923. m.p.: 146 – 147 °C.
9H-fluoren-9-yl)methyl      4-(2,3-dichlorophenyl)piperazine-1-carboxylate       (3-9).    1-(2,3-
dichlorophenyl)piperazine (93.1 mg, 0.352 mmol) and triethylamine (54 μL, 0.387 mmol) were
dissolved in anhydrous DCM (10 mL) at 0°C. To this solution Fmoc-Cl (100 mg, 0.387 mmol)
                                              118


was added with a spatula. The resulting solution was allowed to warm to room temperature and
was stirred overnight. The resulting solution was washed with water three times and extracted with
ethyl acetate (40 mL). The combined organic solution was then washed three time with brine
solution and dried over Na2SO4. The resulting crude product was purified by flash column
chromatography (silica, ethyl acetate/hexanes) affording 9 (white semi-solid, 78.8 mg, 49% yield).
1
  H NMR (500 MHz, CDCl3) δ 7.78 (d, J = 7.5 Hz, 2H), 7.60 (d, J = 7.5 Hz, 2H), 7.41 (t, J = 7.4
Hz, 2H), 7.33 (t, J = 7.4 Hz, 2H), 7.21 – 7.15 (m, 2H), 6.92 (dd, J = 7.7, 1.8 Hz, 1H), 4.50 (d, J =
6.7 Hz, 2H), 4.27 (t, J = 6.7 Hz, 1H), 3.66 – 363 (m, 4H), 2.98 – 2.94 (m, 4H). 13C NMR (126
MHz, CDCl3) δ 155.20, 150.84, 143.95, 141.34, 134.15, 127.70, 127.51, 127.05, 125.09, 124.94,
120.00, 118.75, 67.27, 51.16, 47.37. IR (neat): 3065, 2918, 1698, 1577, 1204 cm-1. HRMS (ESI-
TOF) m/z: [(M+H)+] calcd for (C25H23Cl2N2O2+): 453.1136. Found: 453.1142.
9H-fluoren-9-yl)methyl dimethylcarbamate (3-10). Dimethylamine (176 μL, 0.352 mmol) and
triethylamine (54 μL, 0.387 mmol) were dissolved in anhydrous DCM (10 mL) at 0°C. To this
solution Fmoc-Cl (100 mg, 0.387 mmol) was added with a spatula. The resulting solution was
allowed to warm to room temperature and was stirred overnight. The resulting solution was washed
with water three times and extracted with ethyl acetate (40 mL). The combined organic solution
was then washed three time with brine solution and dried over Na2SO4. The resulting crude product
was purified by flash column chromatography (silica, ethyl acetate/hexanes) affording 10 (94 mg,
99%).
                                                119


1
  H NMR (500 MHz, CDCl3) δ 7.78 (d, J = 7.6 Hz, 2H), 7.62 (d, J = 7.6 Hz, 2H), 7.41 (t, J = 7.4
Hz, 2H), 7.32 (td, J = 7.5, 1.1 Hz, 2H), 4.40 (d, J = 7.0 Hz, 2H), 4.26 (t, J = 7.0 Hz, 1H), 2.95 (s,
6H).  13
         C NMR (126 MHz, CDCl3) δ 156.46, 144.12, 141.29, 127.61, 126.98, 125.00, 119.93,
67.35, 47.32, 36.48, 35.91. IR (neat): 3061, 2929, 2763, 1702, 1447 cm-1. m.p.: 146 – 147 °C.
Mass spectroscopy data was not possible to identify.
9H-fluoren-9-yl)methyl morpholine-4-carboxylate (3-11). Morpholine (30 μL, 0.352 mmol)
and triethylamine (54 μL, 0.387 mmol) were dissolved in anhydrous DCM (10 mL) at 0°C. To this
solution Fmoc-Cl (100 mg, 0.387 mmol) was added with a spatula. The resulting solution was
allowed to warm to room temperature and was stirred overnight. The resulting solution was washed
with water three times and extracted with ethyl acetate (40 mL). The combined organic solution
was then washed three time with brine solution and dried over Na2SO4. The resulting crude product
was purified by flash column chromatography (silica, ethyl acetate/hexanes) affording 11 (96.9
mg, 89%).
1
  H NMR (500 MHz, CDCl3) δ 7.78 (d, J = 7.5 Hz, 2H), 7.58 (d, J = 7.5 Hz, 2H), 7.42 (t, J = 7.5
Hz, 2H), 7.34 (td, J = 7.5, 1.0 Hz, 2H), 4.48 (d, J = 6.7 Hz, 2H), 4.26 (t, J = 6.7 Hz, 1H), 3.64 –
3.60 (m, 4H), 3.47 – 3.43 (m, 4H). 13C NMR (126 MHz, CDCl3) δ 143.90, 141.33, 127.71, 127.05,
124.89, 119.99, 67.29, 47.33. IR (neat): 3038, 2963, 1684, 1421 cm-1. m.p.: 110 – 111 °C. Mass
spectroscopy data was not possible to identify.
                                                120


4-bromo-1-(4-phenylpiperazin-1-yl)butan-1-one (intermediate to compound 12). To a round
bottom flask with a stir bar and molecular sieves was added 4-bromobutanoyl chloride (417 μL,
3.6 mmol) in dry THF (10 mL). This was then cooled to 0 °C and to it was slowly added a solution
of 1-phenylpiperazine (458 μL, 3.0 mmol) and triethylamine (416 μL, 3.0 mmol) in dry THF (10
mL). This mixture was stirred at 0 °C and gradually allowed to warm to room temperature
overnight. The reaction was then poured into DI H2O (40 mL) and extracted with DCM (40 mL).
The organic layers were then washed with brine solution, dried with Na2SO4 and concentrated
under vacuum. The crude material was then purified by flash column chromatography (silica, ethyl
acetate/hexanes) affording the intermediate to compound 12, but there remained significant
amounts of an unidentified contaminate. This prevented full characterization data and accurate
yields from being reported, but the crude material was carried forward for the synthesis of
compound 12.
HRMS (ESI-TOF) m/z: [(M+H)+] calcd for (C14H20BrN2O+): 311.0759. Found: 311.0766.
7-(4-oxo-4-(4-phenylpiperazin-1-yl)butoxy)-3,4-dihydroquinolin-2(1H)-one (3-12). To a
round bottom flask with a stir bar was added 7-hydroxy-3,4-dihydroquinolin-2(1H)-one (50 mg,
0.31 mmol) in DMF (3 mL) and NaH (14.9 mg, 0.372 mmol). This mixture was allowed to stir
under N2 atmosphere for about 10 minutes and then to it was added 4-bromo-1-(4-phenylpiperazin-
                                               121


1-yl)butan-1-one (115.8 mg, 0.372 mmol) in DMF (3 mL). The resulting mixture was stirred at
room temperature overnight. To the reaction was then added ethyl acetate (40 mL) and the organics
were washed with a 10% LiBr solution and then a brine solution to remove DMF. The organic
layer was collected, dried with Na2SO4 and concentrated under vacuum. The crude material was
then purified by flash column chromatography (silica, ethyl acetate/hexanes) affording 12 (9.5 mg,
8% yield).
1
  H NMR (500 MHz, CDCl3) δ 7.99 (s, br., 1H), 7.30 – 7.26 (m, 2H), 7.05 (d, J = 8.3 Hz, 1H),
6.93 – 6.89 (m, 3H), 6.54 (dd, J = 8.3, 2.4 Hz, 1H), 6.34 (m, 1H), 4.03 (t, J = 5.9 Hz, 2H), 3.79
(t, J = 5.1 Hz, 2H), 3.65 (t, J = 5.1 Hz, 2H), 3.15 (t, J = 5.2 Hz, 4H), 2.88 (t, J = 7.2 Hz, 2H),
2.59 (m, 4H), 2.19 – 2.10 (m, 2H). 13C NMR (126 MHz, CDCl3) δ 170.75, 158.45, 150.89,
129.23, 128.72, 120.57, 116.63, 115.83, 108.54, 102.10, 67.13, 49.69, 49.44, 45.38, 41.54, 31.05,
29.28, 24.77, 24.54. IR (neat): 3239, 2963, 1683, 1623, 1592 cm-1. HRMS (ESI-TOF) m/z:
[(M+H)+] calcd for (C23H28N3O3+): 394.2131. Found: 394.2136. m.p. data was not obtainable
due to insufficient amount of material remaining.
                                                  122


                                           REFERENCES
(1) Klaips, C. L.; Jayaraj, G.; Hartl, F. Pathways of cellular proteostasis in aging and disease. The
Journal of cell biology 2018, 217 (1), 51-63. DOI: 10.1083/jcb.201709072.
(2) Powers, E. T.; Morimoto, R. I.; Dillin, A.; Kelly, J. W.; Balch, W. E. Biological and
Chemical Approaches to Diseases of Proteostasis Deficiency. Annual Review of Biochemistry
2009, 78 (1), 959-991. DOI: 10.1146/annurev.biochem.052308.114844.
(3) Labbadia, J.; Morimoto, R. I. The biology of proteostasis in aging and disease. Annual review
of biochemistry 2015, 84, 435-464. DOI: 10.1146/annurev-biochem-060614-033955.
(4) Balch, W. E.; Morimoto, R. I.; Dillin, A.; Kelly, J. W. Adapting proteostasis for disease
intervention. 2008; Vol. 319, pp 916-919.
(5) Hartl, F. U. Cellular Homeostasis and Aging. Annual Review of Biochemistry 2016, 85 (1), 1-
4. DOI: 10.1146/annurev-biochem-011116-110806.
(6) Deshmukh, F. K.; Yaffe, D.; Olshina, M. A.; Ben-Nissan, G.; Sharon, M. The Contribution of
the 20S Proteasome to Proteostasis. Biomolecules 2019, 9 (5), 190, Review. DOI:
10.3390/biom9050190.
(7) Lehtonen, Š.; Sonninen, T. M.; Wojciechowski, S.; Goldsteins, G.; Koistinaho, J.
Dysfunction of Cellular Proteostasis in Parkinson's Disease. Frontiers in neuroscience 2019, 13
(10), 457. DOI: 10.3389/fnins.2019.00457.
(8) Kurtishi, A.; Rosen, B.; Patil, K. S.; Alves, G. W.; G., M. S. Cellular Proteostasis in
Neurodegeneration. Molecular neurobiology 2019, 56 (5), 3676-3689. DOI: 10.1007/s12035-
018-1334-z.
(9) Hipp, M. S.; Kasturi, P.; Hartl, F. U. The proteostasis network and its decline in ageing.
Nature reviews. Molecular cell biology 2019, 20 (7), 421-435. DOI: 10.1038/s41580-019-0101-
y.
(10) McNaught, K. S. P.; Olanow, C. W.; Halliwell, B.; Isacson, O.; Jenner, P. Failure of the
ubiquitin–proteasome system in Parkinson's disease. Nature Reviews Neuroscience 2001, 2 (8),
589-594. DOI: 10.1038/35086067.
(11) Saez, I.; Vilchez, D. The Mechanistic Links Between Proteasome Activity, Aging and Age-
related Diseases. Current genomics 2014, 15 (1), 38-51. DOI:
10.2174/138920291501140306113344.
(12) Lam, Y. A.; Pickart, C. M.; Alban, A.; Landon, M.; Jamieson, C.; Ramage, R.; Mayer, R. J.;
Layfield, R. Inhibition of the ubiquitin-proteasome system in Alzheimer's disease. Proceedings
of the National Academy of Sciences of the United States of America 2000, 97 (18), 9902-9906.
DOI: 10.1073/pnas.170173897.
                                                  123


(13) Ross, C. A.; Poirier, M. A. Protein aggregation and neurodegenerative disease. Nature
Medicine 2004, 10 (7), S10-S17. DOI: 10.1038/nm1066.
(14) Soto, C. Unfolding the role of protein misfolding in neurodegenerative diseases. Nature
Reviews Neuroscience 2003, 4 (1), 49-60. DOI: 10.1038/nrn1007.
(15) Keller, J. N.; Hanni, K. B.; Markesbery, W. R. Impaired Proteasome Function in
Alzheimer's Disease. Journal of Neurochemistry 2001, 75 (1), 436-439. DOI: 10.1046/j.1471-
4159.2000.0750436.x.
(16) Tofaris, G. K.; Layfield, R.; Spillantini, M. G. alpha-synuclein metabolism and aggregation
is linked to ubiquitin-independent degradation by the proteasome. FEBS letters 2001, 509 (1),
22-26. DOI: 10.1016/s0014-5793(01)03115-5.
(17) Alafuzoff, I.; Hartikainen, P. Alpha-synucleinopathies. Handbook of clinical neurology
2017, 145, 339-353. DOI: 10.1016/B978-0-12-802395-2.00024-9.
(18) Bengoa-Vergniory, N.; Roberts, R. F.; Wade-Martins, R.; Alegre-Abarrategui, J. Alpha-
synuclein oligomers: a new hope. Acta neuropathologica 2017, 134 (6), 819-838. DOI:
10.1007/s00401-017-1755-1.
(19) Berrocal, R.; Vasquez, V.; Krs, S. R.; Gadad, B. S.; Ks, R. α-Synuclein Misfolding Versus
Aggregation Relevance to Parkinson’s Disease: Critical Assessment and Modeling. Humana
Press Inc.: 2015; Vol. 51, pp 1417-1431.
(20) Uversky, V. N.; Oldfield, C. J.; Dunker, A. K. Intrinsically Disordered Proteins in Human
Diseases: Introducing the D <sup>2</sup> Concept. Annual Review of Biophysics 2008, 37 (1),
215-246. DOI: 10.1146/annurev.biophys.37.032807.125924.
(21) Uversky, V. N. Intrinsically disordered proteins and their (disordered) proteomes in
neurodegenerative disorders. Frontiers in aging neuroscience 2015, 7, 18-18. DOI:
10.3389/fnagi.2015.00018.
(22) Choi, M. L.; Gandhi, S. Crucial role of protein oligomerization in the pathogenesis of
Alzheimer's and Parkinson's diseases. The FEBS Journal 2018, 285 (19), 3631-3644. DOI:
10.1111/febs.14587.
(23) Levine, Z. A.; Larini, L.; LaPointe, N. E.; Feinstein, S. C.; Shea, J.-E. Regulation and
aggregation of intrinsically disordered peptides. Proceedings of the National Academy of
Sciences of the United States of America 2015, 112 (9), 2758-2763. DOI:
10.1073/pnas.1418155112.
(24) Korsak, M.; Kozyreva, T. Beta Amyloid Hallmarks: From Intrinsically Disordered Proteins
to Alzheimer’s Disease. Springer, Cham, 2015; pp 401-421.
(25) Cheng, B.; Gong, H.; Xiao, H.; Petersen, R. B.; Zheng, L.; Huang, K. Inhibiting toxic
aggregation of amyloidogenic proteins: a therapeutic strategy for protein misfolding diseases.
Biochimica et biophysica acta 2013, 1830 (10), 4860-4871. DOI: 10.1016/j.bbagen.2013.06.029.
                                                 124


(26) Castillo-Carranza, D. L.; Guerrero-Muñoz, M. J.; Sengupta, U.; Gerson, J. E.; Kayed, R. α-
Synuclein Oligomers Induce a Unique Toxic Tau Strain. Biological Psychiatry 2018, 84 (7),
499-508. DOI: 10.1016/j.biopsych.2017.12.018.
(27) Emmanouilidou, E.; Stefanis, L.; Vekrellis, K. Cell-produced alpha-synuclein oligomers are
targeted to, and impair, the 26S proteasome. Neurobiology of aging 2010, 31 (6), 953-968. DOI:
10.1016/j.neurobiolaging.2008.07.008.
(28) Ben-Nissan, G.; Sharon, M. Regulating the 20S proteasome ubiquitin-independent
degradation pathway. Biomolecules 2014, 4 (3), 862-884. DOI: 10.3390/biom4030862.
(29) Asher, G.; Reuven, N.; Shaul, Y. 20S proteasomes and protein degradation “by default”.
BioEssays 2006, 28 (8), 844-849. DOI: 10.1002/bies.20447.
(30) Njomen, E.; Tepe, J. J. Regulation of Autophagic Flux by the 20S Proteasome. Cell
Chemical Biology 2019, 26 (9), 1283-1294.e1285. DOI: 10.1016/j.chembiol.2019.07.002.
(31) Korovila, I.; Hugo, M.; Castro, J. P.; Weber, D.; Höhn, A.; Grune, T.; Jung, T. Proteostasis,
oxidative stress and aging. Redox biology 2017, 13, 550-567. DOI: 10.1016/j.redox.2017.07.008.
(32) Jung, T.; Grune, T. The proteasome and its role in the degradation of oxidized proteins.
IUBMB Life 2008, 60 (11), 743-752. DOI: 10.1002/iub.114.
(33) Jung, T.; Höhn, A.; Grune, T. The proteasome and the degradation of oxidized proteins: Part
II – protein oxidation and proteasomal degradation. Redox Biology 2014, 2, 99-104. DOI:
10.1016/J.REDOX.2013.12.008.
(34) Jung, T.; Höhn, A.; Grune, T. The proteasome and the degradation of oxidized proteins: Part
III—Redox regulation of the proteasomal system. Redox Biology 2014, 2, 388-394. DOI:
10.1016/J.REDOX.2013.12.029.
(35) Chondrogianni, N.; Petropoulos, I.; Grimm, S.; Georgila, K.; Catalgol, B.; Friguet, B.;
Grune, T.; Gonos, E. Protein damage, repair and proteolysis. Molecular aspects of medicine
2014, 35, 1-71. DOI: 10.1016/j.mam.2012.09.001.
(36) Myers, N.; Olender, T.; Savidor, A.; Levin, Y.; Reuven, N.; Shaul, Y. The Disordered
Landscape of the 20S Proteasome Substrates Reveals Tight Association with Phase Separated
Granules. PROTEOMICS 2018, 18 (21-22), 1800076. DOI: 10.1002/pmic.201800076.
(37) Zondler, L.; Kostka, M.; Garidel, P.; Heinzelmann, U.; Hengerer, B.; Mayer, B.; Weishaupt,
J. H.; Gillardon, F.; Danzer, K. M. Proteasome impairment by α-synuclein. PloS one 2017, 12
(9), e0184040-e0184040. DOI: 10.1371/journal.pone.0184040.
(38) Thibaudeau, T. A.; Anderson, R. T.; Smith, D. M. A common mechanism of proteasome
impairment by neurodegenerative disease-associated oligomers. Nature Communications 2018, 9
(1), 1097-1097. DOI: 10.1038/s41467-018-03509-0.
                                               125


(39) Smith, D. M. Could a Common Mechanism of Protein Degradation Impairment Underlie
Many Neurodegenerative Diseases? Journal of experimental neuroscience 2018, 12,
1179069518794675-1179069518794675. DOI: 10.1177/1179069518794675.
(40) Myeku, N.; Clelland, C. L.; Emrani, S.; Kukushkin, N. V.; Yu, W. H.; Goldberg, A. L.;
Duff, K. E. Tau-driven 26S proteasome impairment and cognitive dysfunction can be prevented
early in disease by activating cAMP-PKA signaling. Nature Medicine 2016, 22 (1), 46-53. DOI:
10.1038/nm.4011.
(41) Ruegsegger, C.; Saxena, S. Proteostasis impairment in ALS. Brain research 2016, 1648,
571-579. DOI: 10.1016/j.brainres.2016.03.032.
(42) Njomen, E.; Tepe, J. J. Proteasome Activation as a New Therapeutic Approach to Target
Proteotoxic Disorders. Journal of Medicinal Chemistry 2019, 62 (14), 6469-6481. DOI:
10.1021/acs.jmedchem.9b00101.
(43) Jones, C. L.; Tepe, J. J. Proteasome Activation to Combat Proteotoxicity. Molecules 2019,
24 (15), 2841, Review. DOI: 10.3390/molecules24152841.
(44) Coleman, R. A.; Trader, D. J. All About the Core: A Therapeutic Strategy to Prevent Protein
Accumulation with Proteasome Core Particle Stimulators. ACS Pharmacology & Translational
Science 2018, 1 (2), 140-142. DOI: 10.1021/acsptsci.8b00042.
(45) Opoku-Nsiah, K. A.; Gestwicki, J. E. Aim for the core: suitability of the ubiquitin-
independent 20S proteasome as a drug target in neurodegeneration. Mosby Inc.: 2018; Vol. 198,
pp 48-57.
(46) Maresh, M. E.; Salazar-Chaparro, A. F.; Trader, D. J. Methods for the discovery of small
molecules to monitor and perturb the activity of the human proteasome. Future Medicinal
Chemistry 2020, 13 (2), 99-116, review-article. DOI: 10.4155/fmc-2020-0288.
(47) Zerfas, B. L.; Coleman, R. A.; Salazar-Chaparro, A. F.; Macatangay, N. J.; Trader, D. J.
Fluorescent Probes with Unnatural Amino Acids to Monitor Proteasome Activity in Real-Time.
ACS Chemical Biology 2020, 15 (9), 2588– 2596, research-article. DOI:
10.1021/acschembio.0c00634.
(48) Coleman, R. A.; Trader, D. J. Methods to Discover and Evaluate Proteasome Small
Molecule Stimulators. Molecules 2019, 24 (12), 2341, Review. DOI:
10.3390/molecules24122341.
(49) Fiolek, T.; Magyar, C.; Wall, T.; Davies, S.; Campbell, M.; Savich, C.; Tepe, J.; Mosey, R.
Dihydroquinazolines enhance 20S proteasome activity and induce degradation of α-synuclein, an
intrinsically disordered protein associated with neurodegeneration. Bioorganic & Medicinal
Chemistry Letters 2021, 36, 127821. DOI: 10.1016/j.bmcl.2021.127821.
(50) Njomen, E.; Osmulski, P. A.; Jones, C. L.; Gaczynska, M.; Tepe, J. J. Small Molecule
Modulation of Proteasome Assembly. Biochemistry 2018, 57 (28), 4214-4224. DOI:
10.1021/acs.biochem.8b00579.
                                                126


(51) Jones, C. L.; Njomen, E.; Sjögren, B.; Dexheimer, T. S.; Tepe, J. J. Small Molecule
Enhancement of 20S Proteasome Activity Targets Intrinsically Disordered Proteins. ACS
Chemical Biology 2017, 12 (9), 2240-2247. DOI: 10.1021/acschembio.7b00489.
(52) Coleman, R. A.; Muli, C. S.; Zhao, Y.; Bhardwaj, A.; Newhouse, T. R.; Trader, D. J.
Analysis of chain length, substitution patterns, and unsaturation of AM-404 derivatives as 20S
proteasome stimulators. Bioorganic & Medicinal Chemistry Letters 2019, 29 (3), 420-423. DOI:
10.1016/J.BMCL.2018.12.030.
(53) Coleman, R. A.; Trader, D. J. Development and Application of a Sensitive Peptide Reporter
to Discover 20S Proteasome Stimulators. ACS Combinatorial Science 2018, 20 (5), 269-276.
DOI: 10.1021/acscombsci.7b00193.
(54) Trader, D. J.; Simanski, S.; Dickson, P.; Kodadek, T. Establishment of a suite of assays that
support the discovery of proteasome stimulators. Biochimica et Biophysica Acta (BBA) - General
Subjects 2017, 1861 (4), 892-899. DOI: 10.1016/J.BBAGEN.2017.01.003.
(55) Giżyńska, M.; Witkowska, J.; Karpowicz, P.; Rostankowski, R.; Chocron, E. S.; Pickering,
A. M.; Osmulski, P.; Gaczynska, M.; Jankowska, E. Proline- and Arginine-Rich Peptides as
Flexible Allosteric Modulators of Human Proteasome Activity. Journal of Medicinal Chemistry
2018, 62 (1), 359-370, research-article. DOI: 10.1021/acs.jmedchem.8b01025.
(56) Harder, B.; Blomquist, M.; Wang, J.; Kim, A.; Woodworth, G.; Winkles, J.; Loftus, J.; Tran,
N. Developments in Blood-Brain Barrier Penetrance and Drug Repurposing for Improved
Treatment of Glioblastoma. Frontiers in Oncology 2018, 8. DOI: doi:10.3389/fonc.2018.00462.
(57) Pushpakom, S.; Iorio, F.; Eyers, P. A.; Escott, K. J.; Hopper, S.; Wells, A.; Doig, A.;
Guilliams, T.; Latimer, J.; McNamee, C.; et al. Drug repurposing: progress, challenges and
recommendations. Nature Reviews Drug Discovery 2018, 18 (1), 41-58, ReviewPaper. DOI:
doi:10.1038/nrd.2018.168.
(58) Fletcher, E.; Kaminski, T.; Williams, G.; Duty, S. Drug repurposing strategies of relevance
for Parkinson’s disease. Pharmacology Research & Perspectives 2021, 9 (4), 841. DOI:
10.1002/prp2.841.
(59) Katsiki, M.; Chondrogianni, N.; Chinou, I.; Rivett, A. J.; Gonos, E. S. The Olive
Constituent Oleuropein Exhibits Proteasome Stimulatory Properties <i>In Vitro</i> and Confers
Life Span Extension of Human Embryonic Fibroblasts. Rejuvenation Research 2007, 10 (2),
157-172. DOI: 10.1089/rej.2006.0513.
(60) Huang, L.; Ho, P.; Chen, C.-H. Activation and inhibition of the proteasome by betulinic acid
and its derivatives. FEBS Letters 2007, 581 (25), 4955-4959. DOI:
10.1016/j.febslet.2007.09.031.
(61) Gaczynska, M.; Osmulski, P. A. Characterization of noncompetitive regulators of
proteasome activity. Academic Press Inc.: 2005; Vol. 398, pp 425-438.
                                                 127


(62) Boyd-Kimball, D.; Gonczy, K.; Lewis, B.; Mason, T.; Siliko, N.; Wolfe, J. Classics in
Chemical Neuroscience: Chlorpromazine. ACS chemical neuroscience 2019, 10 (1), 79-88. DOI:
10.1021/acschemneuro.8b00258.
(63) McGavin, J. K.; Goa, K. L. Aripiprazole. CNS Drugs 2012, 16 (11), 779-786,
OriginalPaper. DOI: doi:10.2165/00023210-200216110-00008.
(64) van Epen, J. H. Experience with fluspirilene (R 6218), a long-acting neuroleptic.
Psychiatria, neurologia, neurochirurgia 1970, 73 (4), 277-284.
(65) Janssen, P. A.; Niemegeers, C. J.; Schellekens, K. H.; Lenaerts, F. M.; Verbruggen, F. J.;
van Nueten, J. M.; Marsboom, R. H.; Hérin, V. V.; Schaper, W. K. The pharmacology of
fluspirilene (R 6218), a potent, long-acting and injectable neuroleptic drug. Arzneimittel-
Forschung 1970, 20 (11), 1689-1698.
(66) Bennett, M.; Bishop, J.; Leng, Y.; Chock, P.; Chase, T.; Mouradian, M. Degradation of α-
Synuclein by Proteasome. Journal of Biological Chemistry 1999, 274 (48), 33855-33858. DOI:
10.1074/jbc.274.48.33855.
(67) Meng, L.; Mohan, R.; Kwok, B. H. B.; Elofsson, M.; Sin, N.; Crews, C. M. Epoxomicin, a
potent and selective proteasome inhibitor, exhibits in vivo antiinflammatory activity. PNAS
1999, 96 (18), 10403-10408, research-article. DOI: 10.1073/pnas.96.18.10403.
(68) Mohammadi, A.; Khalili, B.; Tahavor, M. Novel push–pull heterocyclic azo disperse dyes
containing piperazine moiety: Synthesis, spectral properties, antioxidant activity and dyeing
performance on polyester fibers. Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy 2015, 150, 799-805. DOI: 10.1016/J.SAA.2015.06.024.
(69) Kisselev, A. F.; Akopian, T. N.; Castillo, V.; Goldberg, A. L. Proteasome active sites
allosterically regulate each other, suggesting a cyclical bite-chew mechanism for protein
breakdown. Mol. Cell 1999, 4 (3), 395-402.
(70) Homan, E. J.; Wikström, H. V.; Grol, C. J. Molecular modeling of the dopamine D2 and
serotonin 5-HT(1A) receptor binding modes of the enantiomers of 5-OMe-BPAT. Bioorganic
and Medicinal Chemistry 1999, 7 (9), 1805-1820. DOI: 10.1016/S0968-0896(99)00134-0.
(71) Chen, G.; Xia, H.; Cai, Y.; Ma, D.; Yuan, J.; Yuan, C. Synthesis and SAR study of
diphenylbutylpiperidines as cell autophagy inducers. Bioorg. Med. Chem. Lett. 2011, 21 (1),
234-239. DOI: 10.1016/j.bmcl.2010.11.029.
(72) BIOVIA, Dassault Systèmes, Discovery Studio. San Diego 2020.
(73) Longhena, F.; Spano, P.; Bellucci, A. Targeting of Disordered Proteins by Small Molecules
in Neurodegenerative Diseases. Handb Exp Pharmacol 2017. DOI: 10.1007/164_2017_60.
(74) Boland, B.; Yu, W. H.; Corti, O.; Mollereau, B.; Henriques, A.; Bezard, E.; Pastores, G. M.;
Rubinsztein, D. C.; Nixon, R. A.; Duchen, M. R.; et al. Promoting the clearance of neurotoxic
                                                 128


proteins in neurodegenerative disorders of ageing. Nature Reviews Drug Discovery 2018, 17 (9),
660-688. DOI: 10.1038/nrd.2018.109.
(75) Ciechanover, A.; Kwon, Y. T. Degradation of misfolded proteins in neurodegenerative
diseases: therapeutic targets and strategies. Experimental & Molecular Medicine 2015, 47 (3),
e147-e147. DOI: 10.1038/emm.2014.117.
(76) Deger, J. M.; Gerson, J. E.; Kayed, R. The interrelationship of proteasome impairment and
oligomeric intermediates in neurodegeneration. Aging cell 2015, 14 (5), 715-724. DOI:
10.1111/acel.12359.
(77) Gitler, A. D.; Dhillon, P.; Shorter, J. Neurodegenerative disease: Models, mechanisms, and
a new hope. Company of Biologists Ltd: 2017; Vol. 10, pp 499-502.
(78) Pinotsi, D.; Michel, C. H.; Buell, A. K.; Laine, R. F.; Mahou, P.; Dobson, C. M.; Kaminski,
C. F.; Kaminski Schierle, G. S. Nanoscopic insights into seeding mechanisms and toxicity of
alpha-synuclein species in neurons. Proc. Natl. Acad. Sci. U S A 2016, 113 (14), 3815-3819.
DOI: 10.1073/pnas.1516546113.
(79) Danzer, K. M.; Haasen, D.; Karow, A. R.; Moussaud, S.; Habeck, M.; Giese, A.;
Kretzschmar, H.; Hengerer, B.; Kostka, M. Different species of alpha-synuclein oligomers
induce calcium influx and seeding. J Neurosci 2007, 27 (34), 9220-9232. DOI:
10.1523/JNEUROSCI.2617-07.2007.
(80) Winner, B.; Jappelli, R.; Maji, S. K.; Desplats, P. A.; Boyer, L.; Aigner, S.; Hetzer, C.;
Loher, T.; Vilar, M.; Campioni, S.; et al. In vivo demonstration that alpha-synuclein oligomers
are toxic. Proc. Natl. Acad. Sci. U S A 2011, 108 (10), 4194-4199. DOI:
10.1073/pnas.1100976108.
(81) Wills, J.; Jones, J.; Haggerty, T.; Duka, V.; Joyce, J. N.; Sidhu, A. Elevated tauopathy and
alpha-synuclein pathology in postmortem Parkinson's disease brains with and without dementia.
Experimental Neurology 2010, 225 (1), 210-218. DOI: 10.1016/j.expneurol.2010.06.017.
(82) Ibáñez, P.; Bonnet, A. M.; Débarges, B.; Lohmann, E.; Tison, F.; Agid, Y.; Dürr, A.; Brice,
A.; Pollak, P. Causal relation between α-synuclein locus duplication as a cause of familial
Parkinson's disease. The Lancet 2004, 364 (9440), 1169-1171. DOI: 10.1016/S0140-
6736(04)17104-3.
(83) Ingelsson, M. Alpha-Synuclein Oligomers-Neurotoxic Molecules in Parkinson's Disease
and Other Lewy Body Disorders. Frontiers in neuroscience 2016, 10, 408-408. DOI:
10.3389/fnins.2016.00408.
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                                        Appendix:
Figure 3.12 1H and 13C NMR spectra of compound 3-1
                                          130


Figure 3.13 1H and 13C NMR spectra of compound 3-2
                                          131


Figure 3.14 1H and 13C NMR spectra of compound 3-3
                                          132


Figure 3.15 1H and 13C NMR spectra of compound 3-4
                                          133


Figure 3.16 1H and 13C NMR spectra of compound 3-5
                                          134


Figure 3.17 1H and 13C NMR spectra of compound 3-6
                                          135


Figure 3.18 1H and 13C NMR spectra of compound 3-7
                                          136


Figure 3.19 1H and 13C NMR spectra of compound 3-8
                                          137


Figure 3.20 1H and 13C NMR spectra of compound 3-9
                                          138


Figure 3.21 1H and 13C NMR spectra of compound 3-10
                                          139


Figure 3.22 1H and 13C NMR spectra of compound 3-11
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Figure 3.23 1H and 13C NMR spectra of compound 3-12
                                          141


                                                       CHAPTER FOUR
Development of Neurodegenerative Disease Model Systems for Evaluating 20S Proteasome
                               Activation as an Innovative Therapeutic Strategy
     Reproduced in part with permission from Fiolek J. Taylor, Keel L. Katarina and Tepe J. Jetze. Fluspirilene Analogs Activate the 20S
Proteasome and Overcome Proteasome Impairment by Intrinsically Disordered Protein Oligomers. ACS Chem. Neurosci. 2021. Copyright 2021
                                                        American Chemical Society
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4.1 Introduction
4.1.1 Background
         The utility of small molecule activators of the 20S proteasome as biochemical tools for
studying the proteasome is unquestionable, given the wide use of SDS for studying proteasome
inhibition1-3 and the proteasomes involvement in numerous essential biological pathways.4-11
However, regarding their potential for use as therapeutics targeting neurodegenerative diseases,
there remains much to be explored. Several recent studies have suggested this potential application
of small molecule 20S proteasome activators and have begun to explore it in purified protein and
cell-based systems.11-18 These studies have made use of a variety of substrates, including IDPs that
are associated with neurodegenerative disease pathogenesis, like α-synuclein and tau.11-13, 17 As a
result, interest in this potential method for combating neurodegenerative diseases has grown, but
several key questions remain unanswered regarding their potential in more disease relevant
systems.
         In neurodegenerative diseases, like Parkinson’s disease and Alzheimer’s disease, IDPs are
known to exist in a variety of states of oligomerization and fibrilization. In fact, these various
aggregate forms of IDPs are often considered hallmarks of the development of these diseases.19-30
The mixture of IDP monomers, oligomers and large aggregates or fibrils seen in diseased systems
represents a very different potential substrate and environment for the proteasome, when compared
to purely monomeric IDPs. While the precise role that each form of these IDPs play within
neurodegenerative disease pathogenesis is not fully understood, mounting evidence suggests that
smaller oligomeric forms may represent the most toxic species, as opposed to the monomers, large
aggregates, and fibrils.31-37 Additionally, some forms of these IDP oligomers have been shown to
directly inhibit the proteasome, which may contribute to their further accumulation, aggregation,
                                                  143


and disease progression.38-40 The complex nature of IDPs seen within neurodegenerative diseases
necessitates that we address a couple of important questions regarding small molecule activation
of the 20S proteasome. First, can small molecule activators of the 20S proteasome assist in
maintaining its activity in the presence of inhibitory IDP oligomers? Secondly, can they effect the
levels of accumulated IDP oligomers via 20S proteasome activation, or only the monomeric IDPs?
Answering these questions, regarding the interplay of small molecule activators of the 20S
proteasome and the various oligomeric forms of IDPs, is critical to better understand the potential
of this therapeutic strategy.
         In addition to the various forms of IDPs seen in neurodegenerative diseases, there are yet
other compounding factors. For example, multiple cell types are affected during the progression
of these diseases. In addition to neurons, there exist immune cells that are critical to healthy brain
function and are sensitive to disruptions in brain homeostasis. The perturbation of this delicate
system, as seen in neurodegenerative diseases, leads to the development of neuroinflammation.41-
43
   Neuroinflammation is deeply involved in the progression of neurodegenerative diseases, and
while the extent to which it contributes to disease progression is not fully understood, it is widely
thought to have a detrimental role.41, 44-47 Microglia, the primary native immune cells of the brain,
are thought to be one of the key drivers of neuroinflammation in neurodegenerative disease
pathogenesis.43, 48 IDPs that are released by degenerating neurons into the extracellular space can
induce activation of microglia, leading to the secretion of pro-inflammatory signaling molecules,
like cytokines, chemokines, and reactive oxygen species. The inflammation resulting from their
release contributes to further neuron degeneration and death. This is thought to contribute to the
initiation of a deleterious cycle of neuron degeneration, IDP release, and neuroinflammation,
where each aspect further aggravates the others.41-43, 46, 49-52 Whether 20S proteasome activators
                                                  144


can break this cycle of increasing neuron degeneration and neuroinflammation is not yet known.
Exploration of the effects of 20S activators on neurons, microglia and neuroinflammation will be
necessary to fully understand their effects in diseased systems.
        To address the questions posed above and push forward the development of small molecule
activators of the 20S proteasome as therapeutics targeting neurodegenerative diseases, novel
assays and disease models will need to be developed and implemented in their evaluation. Prior to
the studies outlined here, there had been no published attempts at exploring the interplay between
small molecule activators of the 20S proteasome and IDP oligomers that inhibit the proteasome.38,
39
   Additionally, while some cell-based assays have been explored for validation of the activity of
small molecule 20S proteasome activators, there remains a need for more neurodegenerative
disease relevant models in which they can be evaluated.11-18 Furthermore, what effect these 20S
proteasome activators might have on neuroinflammation, seen in the development of these
diseases, has yet to be explored.41, 44-47 Considering the complex nature of these diseases, involving
numerous forms of IDPs and cell types, much work remains to be done to validate this proposed
therapeutic strategy.
        To this end, I sought to begin to address some of these questions surrounding the potential
of small molecule activators of the 20S proteasome as a therapeutic strategy for treating
neurodegenerative diseases, by developing novel assays and models to expand our experimental
program. To begin this effort, I followed work done by the Smith group, that demonstrated IDP
oligomer-mediated inhibition of the proteasome38 and worked to explore the effects of 20S
activators in similar systems. My hypothesis was that small molecule activators of the 20S
proteasome can maintain 20S proteasome activity in the presence of inhibitory IDP oligomers,
thus helping to reestablish proteostasis (Fig. 4.1). Additionally, I explored the effects of 20S
                                                   145


activators in a cell-based model of familial Parkinson’s disease, making use of the A53T mutant
form of α-synuclein.53-55 Finally, I wanted to ensure that the 20S activators are not going to have
a detrimental effect on disease relevant cell types and begin to explore their effects on
neuroinflammation. For this, I evaluated their effect on the viability of immortalized microglia and
on IDP-induced release of the inflammatory cytokine TNF-α, by the same cells. I will also briefly
outline efforts made towards the development of other tools and model systems for use in future
studies by the Tepe lab, focused on furthering our ability to evaluate the potential of this
therapeutic strategy.
Figure 4.1: Cartoon of proposed inhibition of 20S activity by IDP oligomers38 and activation
via small molecule activators being developed by the Tepe lab.12, 13, 17, 56
                                                146


4.1.2 Objective
        The goals of these studies were: (1) investigate the interplay between small molecule 20S
proteasome activators and inhibitory IDP oligomers, (2) develop novel methods, tools, and cell-
based models to further demonstrate the potential of small molecule 20S proteasome activation as
an innovative therapeutic strategy targeting neurodegenerative diseases, (3) explore the effects of
20S proteasome activators on microglia and neuroinflammation induced by α-synuclein.
4.2 Results and Discussion
4.2.1 20S activators maintain activity in the presence of inhibitory α-synuclein oligomers
        In neurodegenerative diseases, like Alzheimer’s disease and Parkinson’s disease,
proteasome impairment is a major contributor to the accumulation of neurotoxic IDP oligomers.57-
68
   In fact, it was recently shown that IDP oligomers associated with neurodegenerative diseases,
such as α-synuclein, amyloid β, and Huntingtin protein, can directly inhibit the 20S proteasome.38,
40
   This IDP oligomer-induced 20S proteasome impairment has the potential to contribute to further
accumulation of the IDPs, thus promoting disease progression. I hypothesized that small molecule
20S proteasome activators can protect against IDP-mediated impairment of the 20S proteasome,
and as a result may assist in reestablishing the clearing of IDPs. If IDP clearance by the proteasome
can be reestablished and free IDP levels can be reduce, then proteostasis can begin to be
reestablished as well.
        To monitor the effects of small molecule 20S proteasome activators, like TCH-165,
Fluspirilene and N-acylated Fluspirilene, on an IDP oligomer-impaired 20S proteasome I followed
work published by the Smith group.38 Briefly, purified 20S proteasome was incubated with the
compounds and an aggregate mixture of α-synuclein (purchased from Novus Biologicals) was
introduced prior to addition of a fluorogenic peptide substrate (CT-L). Consistent with their
                                                  147


findings,38 it was found that the α-synuclein aggregate mixture significantly reduced 20S-mediated
proteolysis of the fluorogenic peptide substrate (Fig. 4.2). Excitingly, it was also found that the
20S proteasome activators TCH-165, Fluspirilene and N-acylated Fluspirilene, were each able to
maintain 20S proteasome activity in the presence of the mixed α-synuclein aggregates, in a
concentration dependent manner (Fig. 4.2).
        These findings were very exciting, because prior to this experiment no one had explored
the interplay between inhibitory IDP oligomers and small molecule activators of the 20S
proteasome. It was previously unknown whether small molecule activators of the 20S proteasome
would have any effect on its activity in the presence of an inhibitory IDP species, such as these
mixed α-synuclein aggregates. This represents a promising step towards validating the potential of
this method, considering that in neurodegenerative disease afflicted systems there will likely be a
variety of IDP species, including inhibitory oligomeric forms.19-30, 38-40 So, for 20S proteasome
activators to have substantial effects on neurodegenerative disease progression, they must still
affect 20S proteasome activity in such a system.
                                                 148


                                                                                                   Mixed α-synuclein aggregates
                                                       A
                                                                                                             TCH-165
                                                                   400
                                                                                                                           ****
                                                                   300
                                                   20S activity
                                                                                                                      ****
                                                                   200                                        ****
                                                  (% of control)                          ***
                                                                                                          ns
                                                                   100
                                                                    0
                                                                                i   cle            sy
                                                                                                     n
                                                                                                                 μM            μM        μM         μM
                                                                             eh               α-             1             3         5         10
                                                                         V                +
                                                                                i   cle
                                                                         V   eh
                                    Mixed α-synuclein aggregates                                                                           Mixed α-synuclein aggregates
         B                                                                                                           C
                                           Fluspirilene                                                                                             N-acylated Fluspirilene
                     200                                                                                                  200
                                                                                                                                                               ****
                     150                             ****                                                                 150                                ****
     20S activity                ****
                                                 ****                                                     20S activity                    ****
                                                                                                                                                          ****
                     100                       **                                                                         100                         *
                                          ns
    (% of control)                                                                                       (% of control)
                     50                                                                                                   50
                      0                                                                                                        0
                           cle      syn   μM      μM           μM        μM                                                         cle       syn     μM      μM      μM    μM
                           hi     α-      1      3          5        10                                                            hi     α-        1        3      5      10
                      Ve        +                                                                                              Ve        +
                            cle                                                                                                     cle
                           hi                                                                                                      hi
                       Ve                                                                                                      Ve
Figure 4.2: 20S proteasome activators maintain 20S activity in the presence of inhibitory α-
synuclein oligomers. 20S proteasome-mediated degradation of a fluorogenic peptide substrate
(CT-L) impaired by α-synuclein mixed aggregates in the presence of a concentration-gradient of
(A) TCH-165, (B) Fluspirilene, or (C) N-acylated Fluspirilene. These data were collected in
triplicate (n=3). Error bars denote standard deviation. One-way ANOVA statistical analysis was
used to determine statistical significance. (ns=not significant, *p<0.05, **p<0.01, ***p<0.001,
****p<0.0001).
                                                                                              149


        It should be noted that differences in level of activity were seen between TCH-165 and the
Fluspirilene analogues tested here, especially at higher concentrations (Fig. 4.2). Several factors
could contribute to these differences, including which inter-subunit pocket they interact with (α1/2
for TCH-16513 and α2/3 for the Fluspirilenes (Chapter 3)), interactions between the small
molecules and the oligomers, their normal differences in activity levels, or some combination
thereof. Studies being undertaken by other members of the Tepe lab may shed light on these
differences in activity going forward, but that is beyond the scope of this work.
4.2.2 20S activators maintain 20S activity in the presence of inhibitory Abeta oligomers
        Similar to what is seen in Parkinson’s disease with α-synuclein accumulation and
oligomerization, in Alzheimer’s disease the IDP amyloid β is known to accumulate and
aggregate.23, 29, 38, 69-72 This effect is thought to similarly contribute to disease progression, although
the exact mechanisms are not fully understood, as with Parkinson’s disease. As was mentioned
above, Smith and co-workers were able to demonstrate that mixed amyloid β oligomers were also
able to directly inhibit the activity of the proteasome in vitro.38 While this inhibition across the
different IDPs in their study seemed to be similar, it is possible that the exact interactions taking
place could be varied. As a result, it is possible that the interplay between different inhibitory IDP
oligomers and 20S proteasome activators could be varied. I hypothesized that the 20S proteasome
activators TCH-165, Fluspirilene and N-acylated Fluspirilene could also maintain 20S proteasome
activity in the presence of amyloid β mixed aggregates, like what was seen with α-synuclein. This
would further support the hypothesis that small molecule 20S proteasome activation is a promising
method by which to address the problem of IDP accumulation and aggregation in multiple
neurodegenerative diseases that share similarities in terms of IDP accumulation and aggregation
but differ in the IDP that is associated with their pathogenesis.
                                                       150


        For this study, amyloid β mixed aggregates were generated following literature
precedence.38, 73 Briefly, synthetic amyloid β (1–42) (purchased from Eurogentec) was dissolved
in 100% hexafluoroisopropanol (HFIP) and incubated at 37 °C for 2 hours to remove any pre-
existing aggregates. The HFIP was removed via lyophilization, and the resulting peptide films
were stored at −80 °C until use. Aggregate mixtures were prepared by resuspending amyloid β
films in DMSO, followed by addition of ultrapure H2O and rapid addition of 2 M Tris-base at pH
7.6. The solution was then briefly vortexed and allowed to incubate at room temperature for 5 min.
The amyloid β aggregate mixture was then diluted to the desired concentration and used
immediately. Following generation of the mixed amyloid β aggregates, the same procedure that
was used for generating the data seen in Fig. 4.3 was followed, except the addition of α-synuclein
mixed aggregates was replaced with the amyloid β aggregate mixture.
        Consistent with the findings of Smith and co-workers38 and my own findings with α-
synuclein mixed aggregates (Fig. 4.2), the amyloid β aggregate mixture also inhibited the 20S
proteasome’s ability to degrade the fluorogenic peptide substrate (Fig 4.3). Additionally, as was
found with α-synuclein, the 20S proteasome activators (TCH-165, Fluspirilene or N-acylated
Fluspirilene) maintained 20S proteasome activity in the presence of the amyloid β aggregates, in
a concentration-dependent manner. At the higher concentrations (3–10 µM depending on the
activator) each 20S activator was able to achieve levels of activity on par with or above that of the
untreated 20S proteasome control, for both the α-synuclein and amyloid β treated 20S proteasomes
(Fig. 4.2 and Fig. 4.3).
                                                151


                                                                                                                                Mixed Abeta aggregates
                                                                          A                                                            TCH-165
                                                                                           300                                              ****
                                                                                                                                                  ****
                                                                           20S activity    200
                                                                                                                                           **
                                                                                                                         *
                                                                          (% of control)
                                                                                                                                     ns
                                                                                           100
                                                                                                0
                                                                                                           icl
                                                                                                              e
                                                                                                                            e   ta        μM            μM        μM        μM
                                                                                                    Ve
                                                                                                       h                 Ab           1             3         5        10
                                                                                                                     +
                                                                                                           i   cle
                                                                                                    V   eh
                                                      Mixed Abeta aggregates                                                                                              Mixed Abeta aggregates
    B                                                       Fluspirilene                                                             C                                      N-acylated Fluspirilene
                     200                                                                                                                          200
                                                                                                                                                                                        ****
                     150                                              ****                                                                        150                                 **
     20S activity                                                                                                                 20S activity
                                            ***                                                                                                                                   *
                                                                     **                                                                                             **
                                                                ns
                     100                                   ns                                                                                     100                        ns
    (% of control)                                                                                                               (% of control)
                     50                                                                                                                            50
                      0                                                                                                                             0
                                       e               a
                                    icl              et         μM        μM               μM           μM
                                                                                                                                                             cle       ta    μM       μM       μM    μM
                                                Ab
                                h                          1          3               5                                                                            Ab
                                                                                                10
                                                                                                                                                         hi
                           Ve                                                                                                                       Ve                e     1         3    5        10
                                            +                                                                                                                  +
                                        e
                                    icl
                                                                                                                                                             cle
                                h                                                                                                                        hi
                           Ve                                                                                                                       Ve
Figure 4.3: 20S proteasome activators maintain 20S activity in the presence of inhibitory
amyloid β oligomers. 20S proteasome-mediated degradation of fluorogenic peptide substrate
(CT-L) impaired by amyloid β mixed aggregates in the presence of a concentration-gradient of (A)
TCH-165, (B) Fluspirilene, or (C) N-acylated Fluspirilene. These data were collected in triplicate
(n=3). Error bars denote standard deviation. One-way ANOVA statistical analysis was used to
determine statistical significance. (ns=not significant, *p<0.05, **p<0.01, ***p<0.001,
****p<0.0001).
                                                                                                                         152


        The results obtained for both experiments were very encouraging and suggested that 20S
proteasome activators have the potential to preserve 20S proteasome activity at or above normal
levels in diseased systems where inhibitory IDP oligomers are present. These data represent the
first examples of 20S proteasome small molecule activators being used in conjunction with IDP
oligomers. To further investigate the interplay between 20S activators and oligomeric IDPs that
can inhibit the 20S proteasome, I wanted to examine what effects the activated 20S proteasome
was having on the IDPs themselves.
4.2.3 Fluspirilene analogues reduce monomeric and oligomeric α-synuclein in vitro
        IDP oligomers, like those that inhibit the 20S proteasome,38-40 are thought to exist in a
dynamic equilibrium with the monomeric form.25, 71, 74, 75 According to the studies by Smith and
co-workers, the medium size oligomers (~50 kDa) were the species responsible for the proteasome
inhibition, not the monomeric forms or larger aggregates.38 I hypothesized that small molecule 20S
activators can maintain its activity in the presence of these inhibitory species, enhance 20S-
mediated degradation of monomeric α-synuclein, and shift the equilibrium towards formation of
monomers, thus indirectly reducing the inhibitory medium sized oligomers.38
        This was explored by examining α-synuclein oligomer levels after an incubation of 24
hours with 20S proteasome, pretreated with either Fluspirilene, N-acylated Fluspirilene, or vehicle
control (DMSO). The remaining α-synuclein monomers and oligomers were visualized using
western blot, and the medium sized oligomers (as indicated within the green box, Fig. 4.4A and
B) were quantified (Fig. 4.4C and D). It was found that the addition of Fluspirilene or N-acylated
Fluspirilene led to a significant concentration-dependent reduction in remaining inhibitory α-
synuclein oligomers, relative to the vehicle control (Fig. 4.4C and D). This is the first evidence
that small molecule 20S proteasome activation can influence levels of oligomeric IDP.
                                               153


 A                                                                               C                           Fluspirilene
                                                remaining inhibitory oligomers
                                                                                 150
                                                                                                             ns         ns
                                                                                 100
                                                        (% of control)
                                                                                 50
                                                                                                                                  **
                                                                                     0
                                                                                                    l
                                                                                                 ro             M        M           M
                                                                                            nt             1µ       3µ        10
                                                                                                                                 µ
                                                                                          Co
 B                                                                               D
                                                                                                        N-acylated Fluspirilene
                                                remaining inhibitory oligomers
                                                                                 150                         ns
                                                                                                                        ns
                                                                                 100
                                                        (% of control)
                                                                                  50                                                 *
                                                                                     0
                                                                                           ro l            µM       µM        µM
                                                                                         Cont            1          3        10
Figure 4.4: Fluspirilene and N-acylated Fluspirilene reduce monomeric and oligomeric α-
synuclein in vitro. (A) Western blot analysis of 20S proteasome-mediated α-synuclein oligomer
digestion with Fluspirilene (1, 3 or 10 µM) and (C) quantification of remaining oligomeric α-
synuclein. Densitometry of Fluspirilene α-synuclein oligomer digestions done using image J (n=3).
(B) Western blot analysis of 20S proteasome-mediated α-synuclein oligomer digestion with N-
acylated Fluspirilene (1, 3 or 10 µM) and (D) quantification of remaining oligomeric α-synuclein.
Densitometry of N-acylated Fluspirilene α-synuclein oligomer digestions done using image J
(n=4). Error bars denote standard deviation. One-way ANOVA statistical analysis was used to
determine statistical significance (ns=not significant, (*p<0.05, **p<0.01).
                                               154


        These studies (Figs. 4.2 – 4.4) demonstrate the potential of 20S activators to assist in
reestablishing proteostasis in systems where inhibitory IDP oligomers are present, as seen in
many neurodegenerative diseases, by maintaining 20S proteasome activity and reducing
accumulated IDPs.38-40
4.2.4 20S activators reduce the accumulation of A53T-mutant α-synuclein in transfected
HEK-293T cells
        Next, I sought to develop a cellular model that could be employed to begin to evaluate 20S
proteasome activators within a cell-based system and on a neurodegenerative disease relevant
substrate. The A53T mutation of α-synuclein has been linked to some cases of early onset familial
Parkinson’s disease. This point mutation on α-synuclein leads to faster oligomerization than the
wild-type protein and as a result more readily disrupts proteostasis in neurons.53-55 Because of its
involvement with some cases of familial Parkinson’s disease and the disordered nature of α-
synuclein, cells expressing the A53T mutant form of α-synuclein could serve as a promising model
for evaluating small molecule 20S proteasome activators.53-55
        To this end, I transiently transfected a plasmid containing A53T α-synuclein into HEK-
293T cells. Following transfection, the cells were incubated for 24 hours to allow for accumulation
of the A53T α-synuclein to begin. Then, the cells were treated with DMSO (vehicle) or 20S
proteasome activators (Fluspirilene or N-acylated Fluspirilene) for 8 hours. The cells were then
lysed, and total protein concentration was normalized for each sample. The resulting cell lysates
were subjected to SDS-PAGE, western blot and probed for A53T α-synuclein protein that had
accumulated. To ensure that the observed effects were due to changes at the protein level,
cycloheximide was added prior to treatment with 20S activators to block protein synthesis.
                                                155


        HEK-293T cells do not normally express easily detectable levels of α-synuclein, as can be
seen when comparing the no plasmid control to the transfected vehicle control (Fig. 4.5).
Excitingly, it was found that both Fluspirilene and N-acylated Fluspirilene effectively reduced the
accumulation of A53T α-synuclein protein in these transfected HEK-293T cells in a concentration-
dependent manner (Fig. 4.5). Importantly, the effect of the 20S proteasome activator N-acylated
Fluspirilene was abrogated by blocking proteasome activity, with the proteasome inhibitor
bortezomib (BTZ),76 thereby implicating proteasome-mediated degradation as responsible for this
reduced accumulation of A53T α-synuclein. These results demonstrated that Fluspirilene
analogues can enhance the activity of cellular 20S proteasomes and reduce the accumulation of
disease-causing IDPs, like A53T α-synuclein. Moreover, this cellular model of familial
Parkinson’s disease53-55 can be used to evaluate novel 20S proteasome activators and assist in their
optimization for cell-based systems.
                                                156


Figure 4.5: Fluspirilene and N-acylated Fluspirilene reduce the accumulation of A53T α-
synuclein in transiently transfected HEK-293T cells in a concentration-dependent manner.
Western blot analysis of remaining A53T α-synuclein in HEK-293T cell lysates following 8 h
treatment with DMSO (vehicle), Fluspirilene (10 or 30 µM), N-acylated Fluspirilene (10 or 30
µM), or a combination of N-acylated Fluspirilene (30 µM) and Bortezomib (100 nM).
Densitometry of resulting bands was performed using image J. Lanes were normalized for loading
differences using the housekeeping protein GAPDH. Quantifications shown as percent of vehicle.
        This cell-based model system of familial Parkinson’s disease continues to serve as a
valuable tool for evaluating the effectiveness of various 20S proteasome activators in cells.
However, improvements in terms of throughput, workflow and consistency could still be made to
this model. For example, the development of a cell line that is stably transfected with an expression
system that allows for switching or tuning of expression, such as a tetracycline or doxycycline-
controlled system, could offer some of these improvements. Such a system would eliminate the
                                               157


need to perform transient transfections prior to every experiment and allow for easier tuning of
A53T α-synuclein levels prior to treatments, providing enhanced reproducibility and ease of
optimization. The development of such a system is currently being explored by other members of
the Tepe lab.
4.2.5 Treatment of primary mouse hippocampal neurons with pre-formed α-synuclein fibrils
seeds accumulation of phosphorylated α-synuclein aggregates
         To continue the investigation of 20S proteasome activation as a potential therapeutic
strategy for neurodegenerative diseases, their efficacy will need to be explored in more relevant
cell types, prior to movement into in vivo models. In an attempt to address this need, and to further
explore the effects of 20S activators on accumulation of oligomeric IDPs, I sought to develop a
cellular model using primary mouse hippocampal neurons. In this proposed model, I aimed to
induce aggregation of endogenous α-synuclein by treating primary mouse hippocampal neurons
with pre-formed α-synuclein aggregate seeds, following protocols similar to those reported in the
literature.77, 78 These cells could then serve as a disease relevant model system to evaluate 20S
proteasome activators for their ability to reduce α-synuclein aggregation caused by exogenous
seeding, as is believed to occur in neurodegenerative disease pathogenesis.24, 27, 47, 77-79 This would
also represent one of the most relevant models of neurodegenerative disease, in terms of cell type,
that has been explored with small molecule 20S proteasome activators to date.
         These studies were done in collaboration with Dr. Caryl Sortwell and her lab at MSU’s
Grand Rapids campus. The Sortwell lab obtained pre-formed α-synuclein fibrils from a
collaborator and then assisted me through training on primary cell culture and processing of the
pre-formed α-synuclein fibrils, via sonication, to generate the desired α-synuclein pre-formed fibril
seeds. I was then able to employ these techniques myself using our own equipment and facilities.
                                                 158


Following this training, our collaboration continued, where I cultured the primary neurons and
performed pre-formed fibril and 20S activator treatments. The Sortwell lab then confirmed proper
pre-formed fibril seed formation, via AFM imaging, and imaged the final fixed neurons with a
Lionheart fluorescent microscope. Unfortunately, after several rounds of optimization the progress
of these studies was halted, due to the prohibitive costs associated with purchasing the neurons
and other required supplies for their culturing and treatment. Prior to the cessation of these
experiments, however, we were able to achieve seeding of endogenous α-synuclein that could be
visualized via immunostaining for phosphorylated α-synuclein (Fig. 4.6). Additional optimization
was deemed necessary to achieve better seeding efficiency and to reduce background signal. This
need for further optimization at this stage, along with the likelihood of needing further optimization
upon incorporation of 20S proteasome activator treatments, would have been quite costly, despite
this successful preliminary experiment. It was for this reason that these studies were put on hold.
                                                  159


Figure 4.6: Seeding of primary mouse hippocampal neurons with pre-formed α-synuclein
fibrils induced aggregation of endogenous α-synuclein. Fluorescence microscopy images of
primary mouse hippocampal neurons following 14-day treatment with pre-formed α-synuclein
fibril seeds. Cell bodies (green) were probed for tubulin using a primary IgM mouse antibody for
5H1 and a GFP-conjugated (488) secondary goat anti-mouse IgM antibody. Phosphorylated α-
synuclein (red) was probed with a primary IgG2a mouse antibody, followed by an RFP-conjugated
(594 nm) secondary goat anti-mouse IgG2a antibody. The bottom imaged is zoomed in portion of
the top image displaying an area with high levels of phosphorylated α-synuclein aggregation.
                                               160


        I later planned an alternative method, but its development has not yet begun. This
alternative method would make use of an immortalized mouse hippocampal neuron cell line (HT-
22 cells), in place of the primary neurons, but retain the same α-synuclein seeding strategy. These
neurons can be grown and passaged, unlike primary neurons, under normal mammalian cell culture
conditions and are much less sensitive to perturbations. These cells would allow for much more
cost efficient and rapid optimization of a similar model, if this cell line is amendable to seeding
with α-synuclein pre-formed fibril seeds, like the primary neurons. In the next section, I will
discuss the synthesis and testing of a fluorescent probe for IDP aggregation that could be used in
conjunction with this proposed HT-22 cellular model to further simplify the workflow and allow
for live-cell labeling.
4.2.6 Synthesis and testing of a fluorescent probe for monitoring IDP aggregation
        Cell-based models of IDP aggregation and neurodegeneration can be challenging and
expensive to develop (see discussion above). As such, any tools that can simplify the workflow
and provide greater experimental flexibility can provide substantial benefits. While working on
the development of the primary neuron α-synuclein aggregation model above, I undertook the
synthesis (Scheme 4.1) and testing (Fig. 4.7) of a fluorescent probe (compound 4.3) that had been
reported in the literature.80 This probe was developed to provide a simple method to monitor IDP
aggregation in living cellular models of neurodegenerative disease. Compound 4-3 functions as a
molecular rotor-like dye, which following excitation can dissipate energy through nonradiative
pathways, because of its free intramolecular rotation around the vinyl linkage in non-rigid
environments. Upon binding to IDP aggregates however, this rotation is restricted and relaxation
back to the ground-state occurs through fluorescence (λem = 605 nm), much like what is seen with
thioflavin T and other amyloid fibril dyes.80-84 This probe was designed to improve on the cell
                                                 161


permeability of thioflavin T and to reduce background fluorescence, which makes this probe more
ideal than the commercially available thioflavin T for our purposes.80 Such a probe would provide
a simpler method to probe for IDP aggregation, relative to immunostaining, and allow for
monitoring this aggregation in living cells without the need for fixation. This could allow for live-
cell imaging, time-lapse studies, and a simplified and less expensive workflow. Since this probe is
not commercially available, the synthesis was undertaken to obtain the probe following the
published route (Scheme 4.1).80 Our lab had the 2-methylbenzothiazole intermediate on hand, so
3 steps were needed to form the final product.
        Briefly, compound 4-1 was synthesized by refluxing 2-methylbenzothiazole and methyl
iodide in acetonitrile (ACN) overnight. Subsequent cooling of the reaction mixture, filtering and
washing with ethyl acetate yielded 90% of pure compound 4-1. Compound 4-2 was synthesized
by dissolving piperidine and 4-fluorobenzaldehyde in DMF with potassium carbonate and heating
to 90 °C overnight. The resulting mixture was then cooled to room temperature and added dropwise
to ice water. The product was then extracted using DCM and dried to yield pure compound 4-2 in
65% yield. Compound 4-3 was synthesized by dissolving compounds 4-1 and 4-2 in EtOH and
refluxing while stirring overnight. The reaction was then cooled to room temperature, filtered and
the solid was rinsed 3 times with ethyl acetate. Pure compound 4-3 was obtained directly as a dark
red powder in 93% yield.
                                                 162


Scheme 4.1: Synthesis of a fluorescent probe for monitoring IDP oligomerization and fibrilization,
following work by Shvadchak et al.80
        Following the synthesis of compound 4-3, it was tested for its ability to preferentially detect
α-synuclein oligomers over their monomeric form, seen as an increase in fluorescence intensity
(λexc = 570 nm, λem = 605 nm). This was done by briefly incubating pure monomeric α-synuclein
or the α-synuclein oligomeric mixture that was used previously (Fig. 4.2 and Fig. 4.4) with
compound 4-3. Fluorescence was then measured using our SpectraMax plate reader (λexc = 570
nm, λem = 605 nm), following a similar protocol to what was used in the initial report of this
probe.80 It was found that incubation of the probe with the α-synuclein oligomeric mixtures
resulted in a large (~300-fold) increase in fluorescence intensity, relative to that of the probe alone
or with monomeric α-synuclein (Fig. 4.7).
                                                 163


                                                                                      ✱✱✱✱
                                                                  3000                     ✱✱✱✱
                                   Fluorescence intensity (RFU)
                                                                  2000
                                                                                     ns
                                                                  1000
                                                                       0
                                                                                   in     s           s
                                                                                cle    er          er
                                                                                      m           m
                                                                             n u on o       lig
                                                                                                o
                                                                        - sy        M    O
                                                                       α
                                                                  No
Figure 4.7: Compound 4-3 shows increased fluorescence in the presence of oligomeric α-
synuclein. Brief incubation (15 mins) of compound 4-3 (4 µM) in PBS buffer with either
monomeric α-synuclein (10 µM) or the mixed aggregates of α-synuclein (10 µM), followed by
reading on our SpectraMax plate reader (λexc = 570 nm, λem = 605 nm). These data were collected
in triplicate (n=3). Error bars denote standard deviations. One-way ANOVA statistical analysis
was used to determine statistical significance (ns=not significant, ****p<0.001).
       Compound 4-3 will be used to help develop cell-based models of IDP aggregation for the
testing of 20S proteasome activators going forward. It should provide a more simplified workflow
and lower cost option, relative to immunostaining, and will allow for live-cell imaging using
confocal microscopy, further diversifying our toolset for evaluating this potential therapeutic
strategy. It is planned to be used in conjunction with the immortalized mouse hippocampal
neurons, discussed above, to allow for live-cell imaging and quantitative evaluation of pre-formed
fibril seed-induced aggregation of endogenous α-synuclein. Additionally, it is serving as a valuable
                                                                                164


tool for use in other cell-based models currently being worked on by other members of the Tepe
lab.
4.2.7 Fluspirilene analogues and TCH-165 are well tolerated by microglia
        The involvement of neuroinflammation in the pathogenesis of neurodegenerative diseases
has been an area of increasing interest, as more ties between the two are identified. Numerous
studies have shown that neuroinflammation appears to play a critical role in disease progression.
However, it is still unclear at what stage in disease development it begins to contribute or to what
degree it is responsible for the continued neuron degeneration.41,            44-47
                                                                                     The initiation of
neuroinflammation seen in neurodegenerative diseases appears to be due, at least in part, to the
release of accumulated IDPs, like α-synuclein in Parkinson’s disease, by degenerating neurons into
the extracellular space. These IDPs released by degenerating neurons are not normally present in
the extracellular space within the brain. As such, the primary native immune cells of the brain,
microglia, detect these abnormal proteins, through their binding to multiple potential receptors on
                                                                                    41-43, 46, 49-52
the cell surface (TLR2, TLR4, FCγR, and CD36), and become activated.                                 These
activated microglia undergo a phenotype switch to their activated form, much like what is seen
with macrophages when they detect pathogens and switch to their M1 (pro-inflammatory)
phenotype. As a result, the activated microglia begin to secrete inflammatory signaling molecules,
including cytokines, chemokines, and reactive oxygen species.41, 43, 48, 85 These pro-inflammatory
signaling molecules have been shown to contribute to further neuron degeneration. These events
are believed to culminate in the initiation of a deleterious cycle of increasing neuron degeneration,
IDP release, microglia activation, and neuroinflammation.41-43, 46, 49-52
        I hypothesized that 20S proteasome activators can help to disrupt this cycle of degeneration
by enhancing the degradation of the IDPs that are activating the microglia. This could take place
                                                  165


at the level of the neurons that are producing and releasing the IDPs, but I also wanted to explore
whether they could have an effect at the level of the microglia and their activation. To explore this,
I set out to develop a cell-based model of neuroinflammation caused by IDPs. For this, I cultured
immortalized mouse microglial (IMG) cells and then treated them with 20S proteasome activators,
TCH-165 or N-acylated Fluspirilene, followed by addition of A53T α-synuclein directly into the
media. Following a 24-hour incubation together with the A53T α-synuclein, an MTS assay was
performed on the microglial cells to monitor viability and a TNF-α ELISA was performed on media
taken from the cells. TNF-α is a cytokine that is commonly associated with inflammatory responses
triggered by immune cells and has been implicated in neuroinflammation development.41, 42, 49
TNF-α is secreted by microglial cells following their activation, so by monitoring the levels of
TNF-α in the media with an ELISA, I could monitor the degree of microglia activation seen in
each treatment.41, 42, 49
         Following treatment of the IMG cells with 20S activators and A53T α-synuclein, it was
found that both TCH-165 and N-acylated Fluspirilene were well tolerated by the microglia,
showing no significant decrease in viability up to 5 µM for TCH-165 and upwards of 10 µM for
N-acylated Fluspirilene (Fig. 4.8). This result was very important, because if 20S proteasome
activators show significant deleterious effects on microglial viability it could lead to more
inflammatory signaling and further disruption of brain homeostasis.
                                                  166


 A                   150                                                                                    B                  150
    cell viability   100                                                                                      cell viability   100
   (% of control)    50                                                                                      (% of control)    50
                      0                                                                                                         0
                                       d                 d                                                                                        d             d
                                a   te             a   te              M              M              M                                a   te           a   te         M        M        M        M
                                                                   1               3               5                                                                .1        .3        .5        10
                            tre                tre            65              65              65                                     tre           lu tre lu    lu    .
                       Un              3   T             -1              -1              -1                                     Un         3T yl F yl F yl F l Flu
                                    A5            T    CH          T   CH          T   CH                                               A5
                                                                                                                                                ac     ac    ac    cy
                                                                                                                                             N-     N-     N- N- a
Figure 4.8: TCH-165 and N-acylated Fluspirilene are well tolerated by microglia. MTS assay
monitoring cell viability of immortalized mouse microglia treated with A53T α-synuclein and
either DMSO (A53T treated), (A) TCH-165 at various concentrations or (B) N-acylated
Fluspirilene at various concentrations. These data were collected in triplicate (n=3). Error bars
denote standard deviations. One-way ANOVA statistical analysis was used to determine statistical
significance. No statistically significant differences between treatments were found.
4.2.8 20S activators reduce the release of the pro-inflammatory cytokine TNF-α by
immortalized microglia activated with A53T α-synuclein
               Alongside the MTS assay being performed on the treated microglia cells, a TNF-α ELISA
was performed on the media removed from the cultures. This was done to monitor TNF-α release
associated with microglia activation.41, 42, 49 As anticipated, the addition of A53T α-synuclein to
the microglia cultures induced increased release of TNF-α into the media, as detected by the
ELISA (Fig. 4.9), corresponding to an increase in activated microglia. This was consistent with
previous literature precedence and the consensus in the field surrounding IDP-induced
neuroinflammation as a contributing factor to neurodegenerative disease pathogenesis.42, 43, 49, 51
Excitingly, it was also found that treatment with TCH-165 or N-acylated Fluspirilene resulted in
                                                                                                            167


concentration-dependent reductions in the A53T α-synuclein-induced TNF-α release by these
microglia (Fig. 4.9). Both, TCH-165 and N-acylated Fluspirilene were able to reduce the levels of
TNF-α release back down to near what was seen in the media of untreated microglia, despite the
addition of the activating IDP. These results suggest that 20S proteasome activators have the
potential to help to not only reduce accumulating IDPs associated with neurodegenerative disease
(Fig 4.4 and Fig. 4.5), but also to help combat their pro-inflammatory effects on microglial cells
that are believed to contribute to the progression of these diseases (Fig. 4.9).
                                                                                 ✱✱                                                                                     ✱✱
 A                                                                        ✱
                                                                                                               B
                                                                                                                                                                    ✱
                                               ✱✱                ns                                                                                            ns
                         400
                                                                                                                                         400
                                                                                                                                                  ✱✱
   TNF- concentration
                                                                                                                                                          ns
                                                                                                                   TNF- concentration
                         300
                                                                                                                                         300
                         200
                                                                                                                                         200
      (% of control)
                                                                                                                      (% of control)
                         100
                                                                                                                                         100
                          0
                                                                                                                                          0
                                           d                 d
                                        te                 te             M              M              M                                          d        ed M M M M
                                    a                  a              1               3               5                                            te       at
                                tre                tre           65              65              65                                         rt e
                                                                                                                                                 a
                                                                                                                                                        rt e lu. 1 lu. 3 lu. 5 . 10
                           Un              3   T
                                                          H-
                                                             1
                                                                          H-
                                                                             1
                                                                                          H-
                                                                                             1
                                        A5              C               C               C                                                 Un 53T yl F yl F yl F l Flu
                                                      T               T               T                                                         A          ac       ac   ac    cy
                                                                                                                                                        N-       N-    N- N- a
Figure 4.9: TCH-165 and N-acylated Fluspirilene reduce the release of TNF-α by microglia
activated with A53T α-synuclein. Sandwich ELISA of TNF-α secreted into the media by IMG
cell cultures treated with A53T α-synuclein and either DMSO (A53T treated), (A) TCH-165 at
various concentrations (1, 3 or 5 µM) or (B) N-acylated Fluspirilene at various concentrations (1,
3, 5 or 10 µM). These data were collected in triplicate (n=3). Error bars denote standard deviations.
One-way ANOVA statistical analysis was used to determine statistical significance (ns=not
significant, *p<0.05, **p<0.01).
                                                                                                               168


        While these results were very exciting and suggested that 20S activators have the potential
to address multiple factors related to neurodegenerative disease pathogenesis (i.e., IDP
accumulation (Fig. 4.5), oligomer-induced proteasome inhibition (Fig. 4.2–4.4), and
neuroinflammation (Fig. 4.9)), the exact mechanism by which this reduction of TNF-α release is
occurring is not made clear by the results of this experiment. While the hypothesis was that 20S
activators could enhance the degradation of the activating IDPs to lead to a reduction in
inflammatory signaling, this experiment cannot directly show that IDP reduction is what led to
TNF-α release being reduced. Several methods could be used to explore the mechanism. For
example, the addition of a proteasome inhibitor could block this effect if it were indeed occurring,
but proteasome inhibitors are generally toxic to cells.6 Using western blotting or an ELISA to look
at the amounts of remaining α-synuclein is a strategy that could be employed to explore whether
it is being degraded at an enhanced rate, but whether this should be done on extracellular α-
synuclein, α-synuclein that has entered the microglia, or both is not clear. The exact mechanism
for α-synuclein induced microglia activation is not fully understood, so which fractions should be
analyzed is also unclear. Additionally, there is mounting evidence for the presence of extracellular
proteasomes,86, 87 so it is possible that degradation may take place outside, as well as inside of
these cells, further complicating the matter.
        To begin to shed light on the mechanism by which these 20S activators were able to reduce
TNF-α release, I chose to explore whether this effect of 20S activators was dependent on activation
via an IDP, like A53T α-synuclein. To do this, lipopolysaccharide (LPS) was used to activate the
microglia in place of the A53T α-synuclein. LPS is commonly used to induce inflammatory
responses in cell culture because it is readily identified by immune cells as foreign, due to its
presence being indicative of bacterial infection when present in the body.48, 49, 85, 88 As expected,
                                                 169


LPS induces a large increase in TNF-α release by the microglia, making use of the same ELISA
for TNF-α detection and quantification (Fig. 4.10). Interestingly, it was found that the 20S
proteasome activators TCH-165 and N-acylated Fluspirilene were also able to reduce the amount
LPS-induced TNF-α release in this assay (Fig. 4.10), like what was seen with A53T α-synuclein
(Fig. 4.9). This suggests that the initial hypothesis, that 20S activators could reduce the release of
TNF-α caused by A53T α-synuclein by enhancing the degradation of the A53T α-synuclein, should
be rejected. In fact, these results suggest that the reduction in TNF-α release seen in these assays
may represent a broader anti-inflammatory effect caused by these 20S proteasome activators,
independent of activation via extracellular IDPs.
                                                   600
                             TNF- concentration   400
                                (% of control)
                                                   200
                                                     0
                                                         ated     ated            lu .
                                                                        ac  TCH-
                                                    Un        St          yl F   16
                                                       tre      re   N-             5
                                                             LP
Figure 4.10: TCH-165 and N-acylated Fluspirilene reduce the release of TNF-α by microglia
activated with LPS. TNF-α ELISA of the media from immortalized mouse microglia cultures
treated with LPS and either DMSO (LPS treated), TCH-165 (5 µM) or N-acylated Fluspirilene (10
µM).
       While these results complicate the mechanistic study, this potential anti-inflammatory
effect could still prove to be beneficial in the case of neurodegenerative disease treatment, where
                                                                  170


both IDP accumulation and neuroinflammation are thought to contribute to disease progression.41-
43, 46, 49-52
              Further studies are required to elucidate the mechanism through which 20S proteasome
activators, such as TCH-165 and N-acylated Fluspirilene, can reduce TNF-α release by microglia
following their activation with pro-inflammatory stimuli.
4.3 Conclusions
         In summary, several novel methods were developed herein to further evaluate 20S
proteasome activators as a therapeutic method for the treatment of neurodegenerative diseases.
These studies focused on providing disease relevant models to evaluate 20S activators and
answering crucial questions regarding the implications of 20S activation in neurodegenerative
disease model systems. It was found that TCH-165 and the Fluspirilene analogues could maintain
20S proteasome activity in the presence of inhibitory IDP oligomers and can begin to reduce
monomeric and oligomeric forms of α-synuclein in vitro. These findings support the hypothesis
that 20S proteasome activators can reestablish proteostasis in neurodegenerative disease systems,
where IDP accumulation and inhibitory IDP oligomers contribute to the disruption of proteostasis.
19-30, 38-40
         Additional cell-based models of Parkinson’s disease were explored, focusing on differing
aspects of the disease. First, HEK-293T cells transfected with A53T α-synuclein served as a model
of early onset familial Parkinson’s disease.53-55 This model has allowed for evaluation of the ability
of 20S proteasome activators to reduce accumulation of a disease relevant substrate in live cells.
         To further the studies focused on IDP oligomers, I began to develop a cellular model of IDP
aggregation, using primary mouse hippocampal neurons treated with pre-formed α-synuclein fibril
seeds.77, 78 While the development of this method did not reach completion, due to cost constraints,
the preliminary results suggest that this seeding was successful in inducing aggregation of
                                                    171


endogenous α-synuclein. The development of a closely related model, using immortalized mouse
hippocampal neurons, was planned to achieve the same goal of exploring the effects of 20S
activators on cellular IDP aggregation. In conjunction with the fluorescent probe (compound 4-3)
synthesized herein, this model could be developed in a more cost-effective way, with a simplified
workflow and allow for analysis in living neurons.80
      To explore the effects of small molecule 20S activators on the development of
neuroinflammation associated with Parkinson’s disease,42, 43, 49, 51 immortalized mouse microglial
cells were treated with 20S activators and A53T α-synuclein. Treatment with A53T α-synuclein
induced increased secretion of the inflammatory cytokine TNF-α. This effect was prevented by
addition of small molecule 20S proteasome activators, in a concentration-dependent manner. A
similar effect was seen when LPS was used in place of A53T α-synuclein to activate the microglia,
which suggests that this anti-inflammatory effect is not specific to activation by IDPs. As a result,
the hypothesis that small molecule 20S proteasome activators could reduce the activation of
microglia by reducing the activating IDPs was rejected. However, despite this effect not being IDP
specific, it could still prove to be beneficial to the treatment of neurodegenerative diseases where
neuroinflammation is believed to contribute to disease pathogenesis.41, 44-47 Further studies are
required to elucidate the exact mechanism by which 20S activators reduce TNF-α release in this
model system.
    1. 20S activators were found to be able to preserve 20S proteasome activity in the presence
        of inhibitory IDP oligomers and reduce monomeric and oligomeric forms of α-synuclein
        in those systems.
    2. Novel methods and tools were developed to enable evaluation of 20S activators effects on
        inhibition of the 20S by IDP oligomers, direct effects on IDP oligomers, cellular
                                                   172


        accumulation of A53T α-synuclein and neuroinflammation caused by A53T α-synuclein.
        The tools, general protocol and initial attempts at development of a model to allow for
        monitoring of α-synuclein aggregation in live neurons was also explored herein.
    3. 20S proteasome activators can reduce A53T α-synuclein-induced release of TNF-α by
        microglia, however this effect is not specific to activation of microglia by IDPs. Additional
        studies are needed to elucidate the mechanism of reduced TNF-α release.
4.4 Experimental
General information
        Reactions were carried out under a nitrogen atmosphere in flame-dried glassware. Solvents
and reagents were purchased from commercial suppliers and used without further purification.
Magnetic stirring was used for all reactions. Yields refer to chromatographically and
spectroscopically pure compounds unless otherwise noted. Infrared spectra were recorded on a
Jasco Series 6600 FTIR spectrometer. 1H and 13C NMR spectra were recorded on a Varian Unity
Plus-500 spectrometer. Chemical shifts are reported relative to the residue peaks of the solvent
(CDCl3: 7.26 ppm for 1H and 77.0 ppm for 13C) (CD3OD: 3.31 ppm for 1H and 47.6 ppm for 13C)
(DMSO-d6: 2.50 ppm for 1H and 39.5 ppm for 13C). The following abbreviations are used to denote
the multiplicities: s = singlet, d = doublet, and m = multiplet. HRMS were obtained at the Mass
Spectrometry Facility of Michigan State University with a Micromass Q-ToF Ultima API LC-
MS/MS mass spectrometer. Column chromatography was performed using a Teledyne ISCO
CombiFlash® NextGen system with prepacked columns (RediSep® Normal-phase silica, 20-40
microns). TLCs were performed on pre-coated 0.25 mm thick silica gel 60 F254 plates and
visualized using UV light and iodine staining.
                                                 173


Compound 4-1: 2-Methylbenzothiazole (1.0 eq) and methyl iodide (2.0 eq) were dissolved in dry
acetonitrile (3.6 mL) and refluxed overnight. The reaction mixture was then cooled to room
temperature and the product was collected by filtration. The product was then washed with ethyl
acetate three times to yield pure product. Purple solid (2.70 g, 90%). 1H NMR (500 MHz, CD3OD)
δ 8.32 (d, J = 8.2 Hz, 1H), 8.24 (d, J = 8.5 Hz, 1H), 7.94 – 7.90 (m, 1H), 7.84 – 7.80 (m, 1H), 4.30
(s, 3H), 3.22 (s, 3H).  13
                           C NMR (126 MHz, CD3OD) δ 176.85, 142.01, 129.51, 128.90, 128.30,
123.77, 116.38, 35.67, 16.20. IR: 3056, 2965 cm-1. HRMS (ESI-TOF) m/z: [(M-I)+] calcd for
(C9H10NS+) 164.0534; Found 164.0538. Melting point: >200 °C. Reported 258°C.80
Compound 4-2: A mixture of piperidine (1.0 eq.), 4-fluorobenzaldehyde (1.2 eq.) and potassium
carbonate (2 eq.) in 4 mL of DMF was heated to 90°C overnight. The mixture was then cooled to
room temperature, added dropwise to ice water, and extracted with DCM to yield pure product.
Brown solid (40.3 mg, 21%). 1H NMR (500 MHz, CDCl3) δ 9.71 (s, 1H), 7.70 (d, J = 8.9 Hz, 2H),
6.86 (d, J = 8.5 Hz, 2H), 3.37 (m, 4H), 1.63 (m, 6H).       13
                                                               C NMR (126 MHz, CDCl3) δ 190.22,
154.94, 131.92, 113.36, 48.46, 25.21, 24.23. IR: 3002, 2947, 1662 cm-1. HRMS (ESI-TOF) m/z:
[(M+H)+] calcd for (C12H16NO+) 190.1232; Found 190.1233. Melting point: 56-58°C.
Compound 4-3: Compounds 4-1 (0.91 eq.) and 4-2 (1.0 eq.) were dissolved in 20mL (5mL/mmol)
of EtOH and refluxed while stirring overnight. The reaction was then cooled to room temperature,
                                                 174


filtered and the solid product was rinsed 3 times with ethyl acetate. Pure compound 3 was obtained
directly as a violet powder (1.63 g, 93%). 1H NMR (500 MHz, DMSO-d6) δ 8.32 (d, J = 7.4 Hz,
1H), 8.11 (d, J = 7.5 Hz, 1H), 8.07 (d, J = 15.0 Hz, 1H), 7.90 (d, J = 7.2 Hz, 2H), 7.79 (m, 1H),
7.70 – 7.64 (m, 2H), 7.06 (d, J = 7.0 Hz, 2H), 4.24 (s, 3H), 3.50 (m, 4H), 1.60 (m, 6H). 13C NMR
(126 MHz, DMSO-d6) δ 171.42, 153.57, 149.66, 141.96, 132.88, 128.90, 127.52, 126.88, 123.84,
122.25, 116.02, 113.45, 106.92, 47.54, 35.64, 25.10, 23.94. IR (neat): 3043, 2926, 1563, 1241 cm-
1
  . HRMS (ESI-TOF) m/z: [(M-I)+] calcd for (C21H23N2S+) 335.1576; Found 335.1621. Melting
point: >200 °C. Reported 252 °C.80
                                                 175


                                            REFERENCES
(1) Dahlmann, B.; Rutschmann, M.; Kuehn, L.; Reinauer, H. Activation of the multicatalytic
proteinase from rat skeletal muscle by fatty acids or sodium dodecyl sulphate. Biochemical
Journal 1985, 228 (1), 171-177. DOI: 10.1042/bj2280171.
(2) Tanaka, K.; Yoshimura, T.; Ichihara, A. Role of substrate in reversible activation of
proteasomes (multi-protease complexes) by sodium dodecyl sulfate. Journal of biochemistry
1989, 106 (3), 495-500.
(3) Azevedo, L. M.; Lansdell, T. A.; Ludwig, J. R.; Mosey, R. A.; Woloch, D. K.; Cogan, D. P.;
Patten, G. P.; Kuszpit, M. R.; Fisk, J. S.; Tepe, J. J. Inhibition of the Human Proteasome by
Imidazoline Scaffolds. Journal of Medicinal Chemistry 2013, 56 (14), 5974-5978. DOI:
10.1021/jm400235r.
(4) Voges, D.; Zwickl, P.; Baumeister, W. The 26S Proteasome: A Molecular Machine Designed
for Controlled Proteolysis. Annual Review of Biochemistry 1999, 68, 1015-1068, review-article.
DOI: 10.1146/annurev.biochem.68.1.1015.
(5) McNaught, K. S. P.; Olanow, C. W.; Halliwell, B.; Isacson, O.; Jenner, P. Failure of the
ubiquitin–proteasome system in Parkinson's disease. Nature Reviews Neuroscience 2001, 2 (8),
589-594. DOI: 10.1038/35086067.
(6) Voorhees, P.; Dees, E.; O'Neil, B.; Orlowski, R. The proteasome as a target for cancer
therapy. Clinical cancer research 2003, 9 (17), 6316-6325.
(7) Ostrowska, H. The ubiquitin-proteasome system: a novel target for anticancer and anti-
inflammatory drug research. Cellular & Molecular Biology Letters 2008, 13 (3), 353-365. DOI:
10.2478/s11658-008-0008-7.
(8) Calamini, B.; Morimoto, R. Protein homeostasis as a therapeutic target for diseases of protein
conformation. Current Topics in Medicinal Chemistry 2012, 12 (22), 2623-2640. DOI:
10.2174/1568026611212220014.
(9) Huang, X.; Dixit, V. M. Drugging the undruggables: exploring the ubiquitin system for drug
development. Cell Research 2016, 26 (4), 484-498, ReviewPaper. DOI: doi:10.1038/cr.2016.31.
(10) Tundo, G.; Sbardella, D.; Santoro, A.; Coletta, A.; Oddone, F.; Grasso, G.; Milardi, D.;
Lacal, P.; Marini, S.; Purrello, R.; et al. The proteasome as a druggable target with multiple
therapeutic potentialities: Cutting and non-cutting edges. Pharmacology & therapeutics 2020,
213, 107579. DOI: 10.1016/j.pharmthera.2020.107579.
(11) Njomen, E.; Tepe, J. J. Proteasome Activation as a New Therapeutic Approach to Target
Proteotoxic Disorders. Journal of Medicinal Chemistry 2019, 62 (14), 6469-6481. DOI:
10.1021/acs.jmedchem.9b00101.
                                                  176


(12) Jones, C. L.; Njomen, E.; Sjögren, B.; Dexheimer, T. S.; Tepe, J. J. Small Molecule
Enhancement of 20S Proteasome Activity Targets Intrinsically Disordered Proteins. ACS
Chemical Biology 2017, 12 (9), 2240-2247. DOI: 10.1021/acschembio.7b00489.
(13) Njomen, E.; Osmulski, P. A.; Jones, C. L.; Gaczynska, M.; Tepe, J. J. Small Molecule
Modulation of Proteasome Assembly. Biochemistry 2018, 57 (28), 4214-4224. DOI:
10.1021/acs.biochem.8b00579.
(14) Njomen, E.; Tepe, J. J. Regulation of Autophagic Flux by the 20S Proteasome. Cell
Chemical Biology 2019, 26 (9), 1283-1294.e1285. DOI: 10.1016/j.chembiol.2019.07.002.
(15) Trader, D. J.; Simanski, S.; Dickson, P.; Kodadek, T. Establishment of a suite of assays that
support the discovery of proteasome stimulators. Biochimica et Biophysica Acta (BBA) - General
Subjects 2017, 1861 (4), 892-899. DOI: 10.1016/J.BBAGEN.2017.01.003.
(16) Coleman, R. A.; Trader, D. J. Methods to Discover and Evaluate Proteasome Small
Molecule Stimulators. Molecules 2019, 24 (12), 2341, Review. DOI:
10.3390/molecules24122341.
(17) Fiolek, T.; Magyar, C.; Wall, T.; Davies, S.; Campbell, M.; Savich, C.; Tepe, J.; Mosey, R.
Dihydroquinazolines enhance 20S proteasome activity and induce degradation of α-synuclein, an
intrinsically disordered protein associated with neurodegeneration. Bioorganic & Medicinal
Chemistry Letters 2021, 36, 127821. DOI: 10.1016/j.bmcl.2021.127821.
(18) Jones, C. L.; Tepe, J. J. Proteasome Activation to Combat Proteotoxicity. Molecules 2019,
24 (15), 2841, Review. DOI: 10.3390/molecules24152841.
(19) Alam, P.; Bousset, L.; Melki, R.; Otzen, D. E. α-synuclein oligomers and fibrils: a spectrum
of species, a spectrum of toxicities. Journal of neurochemistry 2019, 150 (5). DOI:
10.1111/jnc.14808.
(20) Bengoa-Vergniory, N.; Roberts, R. F.; Wade-Martins, R.; Alegre-Abarrategui, J. Alpha-
synuclein oligomers: a new hope. Acta neuropathologica 2017, 134 (6), 819-838. DOI:
10.1007/s00401-017-1755-1.
(21) Cárdenas-Aguayo, M. d. C.; Gómez-Virgilio, L.; DeRosa, S.; Meraz-Ríos, M. A. The Role
of Tau Oligomers in the Onset of Alzheimer's Disease Neuropathology. ACS Chemical
Neuroscience 2014, 5 (12), 1178-1191. DOI: 10.1021/cn500148z.
(22) Castillo-Carranza, D. L.; Guerrero-Muñoz, M. J.; Sengupta, U.; Gerson, J. E.; Kayed, R. α-
Synuclein Oligomers Induce a Unique Toxic Tau Strain. Biological Psychiatry 2018, 84 (7),
499-508. DOI: 10.1016/j.biopsych.2017.12.018.
(23) Cheng, B.; Gong, H.; Xiao, H.; Petersen, R. B.; Zheng, L.; Huang, K. Inhibiting toxic
aggregation of amyloidogenic proteins: a therapeutic strategy for protein misfolding diseases.
Biochimica et biophysica acta 2013, 1830 (10), 4860-4871. DOI: 10.1016/j.bbagen.2013.06.029.
                                                 177


(24) Choi, M. L.; Gandhi, S. Crucial role of protein oligomerization in the pathogenesis of
Alzheimer's and Parkinson's diseases. The FEBS Journal 2018, 285 (19), 3631-3644. DOI:
10.1111/febs.14587.
(25) Cremades, N.; Cohen, S. I. A.; Deas, E.; Abramov, A. Y.; Chen, A. Y.; Orte, A.; Sandal,
M.; Clarke, R. W.; Dunne, P.; Aprile, F. A.; et al. Direct observation of the interconversion of
normal and toxic forms of α-synuclein. Cell 2012, 149 (5), 1048-1059. DOI:
10.1016/j.cell.2012.03.037.
(26) Deger, J. M.; Gerson, J. E.; Kayed, R. The interrelationship of proteasome impairment and
oligomeric intermediates in neurodegeneration. Aging cell 2015, 14 (5), 715-724. DOI:
10.1111/acel.12359.
(27) Danzer, K. M.; Haasen, D.; Karow, A. R.; Moussaud, S.; Habeck, M.; Giese, A.;
Kretzschmar, H.; Hengerer, B.; Kostka, M. Different Species of -Synuclein Oligomers Induce
Calcium Influx and Seeding. Journal of Neuroscience 2007, 27 (34), 9220-9232. DOI:
10.1523/JNEUROSCI.2617-07.2007.
(28) Ingelsson, M. Alpha-Synuclein Oligomers-Neurotoxic Molecules in Parkinson's Disease
and Other Lewy Body Disorders. Frontiers in neuroscience 2016, 10, 408-408. DOI:
10.3389/fnins.2016.00408.
(29) Sengupta, U.; Nilson, A. N.; Kayed, R. The Role of Amyloid-β Oligomers in Toxicity,
Propagation, and Immunotherapy. Elsevier B.V.: 2016; Vol. 6, pp 42-49.
(30) Levine, Z. A.; Larini, L.; LaPointe, N. E.; Feinstein, S. C.; Shea, J.-E. Regulation and
aggregation of intrinsically disordered peptides. Proceedings of the National Academy of
Sciences of the United States of America 2015, 112 (9), 2758-2763. DOI:
10.1073/pnas.1418155112.
(31) Kayed, R.; Dettmer, U.; Lesné, S. E. Soluble endogenous oligomeric α-synuclein species in
neurodegenerative diseases: Expression, spreading, and cross-talk. Journal of Parkinson's
disease 2020, 10 (3), 791-818. DOI: 10.3233/JPD-201965.
(32) Haass, C.; Selkoe, D. J. Soluble protein oligomers in neurodegeneration: lessons from the
Alzheimer's amyloid beta-peptide. Nature reviews. Molecular cell biology 2007, 8 (2), 101-112.
DOI: 10.1038/nrm2101.
(33) Brettschneider, J.; Del Tredici, K.; Lee, V. M.; Trojanowski, J. Q. Spreading of pathology in
neurodegenerative diseases: a focus on human studies. Nature reviews. Neuroscience 2015, 16
(2), 109-120. DOI: 10.1038/nrn3887.
(34) Rubinsztein, D. C. The roles of intracellular protein-degradation pathways in
neurodegeneration. Nature 2006, 443 (7113), 780-786. DOI: 10.1038/nature05291.
(35) Selkoe, D. J. Folding proteins in fatal ways. Nature 2023, 426 (6968), 900-904,
ReviewPaper. DOI: doi:10.1038/nature02264.
                                                 178


(36) Selkoe, D. J.; Hardy, J. The amyloid hypothesis of Alzheimer's disease at 25 years. EMBO
molecular medicine 2016, 8 (6), 595-608. DOI: 10.15252/emmm.201606210.
(37) Chabrier, M. A.; Blurton-Jones, M.; Agazaryan, A. A.; Nerhus, J. L.; Martinez-Coria, H.;
LaFerla, F. M. Soluble aβ promotes wild-type tau pathology in vivo. The Journal of
neuroscience : the official journal of the Society for Neuroscience 2012, 32 (48), 17345-17350.
DOI: 10.1523/JNEUROSCI.0172-12.2012.
(38) Thibaudeau, T. A.; Anderson, R. T.; Smith, D. M. A common mechanism of proteasome
impairment by neurodegenerative disease-associated oligomers. Nature Communications 2018, 9
(1), 1097-1097. DOI: 10.1038/s41467-018-03509-0.
(39) Zondler, L.; Kostka, M.; Garidel, P.; Heinzelmann, U.; Hengerer, B.; Mayer, B.; Weishaupt,
J. H.; Gillardon, F.; Danzer, K. M. Proteasome impairment by α-synuclein. PLOS ONE 2017, 12
(9), e0184040-e0184040. DOI: 10.1371/journal.pone.0184040.
(40) Smith, D. M. Could a Common Mechanism of Protein Degradation Impairment Underlie
Many Neurodegenerative Diseases? Journal of experimental neuroscience 2018, 12,
1179069518794675-1179069518794675. DOI: 10.1177/1179069518794675.
(41) Yang, Q.; Zhou, J. Neuroinflammation in the central nervous system: Symphony of glial
cells. Glia 2019, 67 (6), 1017-1035. DOI: 10.1002/glia.23571.
(42) Su, X.; Maguire-Zeiss, K.; Giuliano, R.; Prifti, L.; Venkatesh, K.; Federoff, H. Synuclein
activates microglia in a model of Parkinson's disease. Neurobiology of Aging 2007, 29 (11),
1690-1701. DOI: 10.1016/j.neurobiolaging.2007.04.006.
(43) Song, N.; Chen, L.; Xie, J. Alpha-Synuclein Handling by Microglia: Activating, Combating,
and Worsening. Neuroscience Bulletin 2021, 37 (5), 751-753, OriginalPaper. DOI:
doi:10.1007/s12264-021-00651-6.
(44) Cao, S.; Standaert, D.; Harms, A. The gamma chain subunit of Fc receptors is required for
alpha-synuclein-induced pro-inflammatory signaling in microglia. Journal of neuroinflammation
2012, 9, 259. DOI: 10.1186/1742-2094-9-259.
(45) Nilson, A. N.; English, K. C.; Gerson, J. E.; Barton Whittle, T.; Nicolas Crain, C.; Xue, J.;
Sengupta, U.; Castillo-Carranza, D. L.; Zhang, W.; Gupta, P.; et al. Tau Oligomers Associate
with Inflammation in the Brain and Retina of Tauopathy Mice and in Neurodegenerative
Diseases. Journal of Alzheimer's disease : JAD 2017, 55 (3), 1083-1099. DOI: 10.3233/JAD-
160912.
(46) Tu, H.; Yuan, B.; Hou, X.; Zhang, X.; Pei, C.; Ma, Y.; Yang, Y.; Fan, Y.; Qin, Z.; Liu, C.;
et al. α-synuclein suppresses microglial autophagy and promotes neurodegeneration in a mouse
model of Parkinson's disease. Aging cell 2021, 20 (12), e13522. DOI: 10.1111/acel.13522.
(47) Garcia, P.; Jürgens-Wemheuer, W.; Huarte, O.; Michelucci, A.; Masuch, A.; Brioschi, S.;
Weihofen, A.; Koncina, E.; Coowar, D.; Heurtaux, T.; et al. Neurodegeneration and
neuroinflammation are linked, but independent of alpha-synuclein inclusions, in a
                                                 179


seeding/spreading mouse model of Parkinson's disease. Glia 2022, 70 (5), 935-960. DOI:
10.1002/glia.24149.
(48) Aloisi, F. Immune function of microglia. Glia 2001, 36 (2), 165-179. DOI:
10.1002/glia.1106.
(49) Hoenen, C.; Gustin, A.; Birck, C.; Kirchmeyer, M.; Beaume, N.; Felten, P.; Grandbarbe, L.;
Heuschling, P.; Heurtaux, T. Alpha-Synuclein Proteins Promote Pro-Inflammatory Cascades in
Microglia: Stronger Effects of the A53T Mutant. PloS one 2016, 11 (9), e0162717. DOI:
10.1371/journal.pone.0162717.
(50) Raffaele, S.; Lombardi, M.; Verderio, C.; Fumagalli, M. TNF Production and Release from
Microglia via Extracellular Vesicles: Impact on Brain Functions. Cells 2020, 9 (10), 2145. DOI:
10.3390/cells9102145.
(51) Wood, H. α-Synuclein-activated microglia are implicated in PD pathogenesis. Nature
Reviews Neurology 2022, 18 (4), 188-188, BriefCommunication. DOI: doi:10.1038/s41582-022-
00631-y.
(52) Xia, Y.; Zhang, G.; Kou, L.; Yin, S.; Han, C.; Hu, J.; Wan, F.; Sun, Y.; Wu, J.; Li, Y.; et al.
Reactive microglia enhance the transmission of exosomal α-synuclein via toll-like receptor 2.
Brain : a journal of neurology 2021, 144 (7), 2024-2037. DOI: 10.1093/brain/awab122.
(53) Conway, K. A.; Harper, J. D.; Lansbury, P. T. Accelerated in vitro fibril formation by a
mutant α-synuclein linked to early-onset Parkinson disease. Nature Medicine 1998, 4 (11), 1318-
1320. DOI: 10.1038/3311.
(54) Wills, J.; Credle, J.; Haggerty, T.; Lee, J.-H.; Oaks, A. W.; Sidhu, A. Tauopathic changes in
the striatum of A53T α-synuclein mutant mouse model of Parkinson's disease. PloS one 2011, 6
(3), e17953-e17953. DOI: 10.1371/journal.pone.0017953.
(55) Smith, W. W.; Jiang, H.; Pei, Z.; Tanaka, Y.; Morita, H.; Sawa, A.; Dawson, V. L.; Dawson,
T. M.; Ross, C. A. Endoplasmic reticulum stress and mitochondrial cell death pathways mediate
A53T mutant alpha-synuclein-induced toxicity. Human Molecular Genetics 2005, 14 (24), 3801-
3811. DOI: 10.1093/hmg/ddi396.
(56) Fiolek, T. J.; Keel, K. L.; Tepe, J. J. Fluspirilene Analogs Activate the 20S Proteasome and
Overcome Proteasome Impairment by Intrinsically Disordered Protein Oligomers. ACS Chemical
Neuroscience 2021, 12 (8), 1438-1448. DOI: 10.1021/acschemneuro.1c00099.
(57) Cecarini, V.; Bonfili, L.; Amici, M.; Angeletti, M.; Keller, J.; Eleuteri, A. Amyloid peptides
in different assembly states and related effects on isolated and cellular proteasomes. Brain
research 2008, 1209, 8-18. DOI: 10.1016/j.brainres.2008.03.003.
(58) Díaz-Hernández, M.; Valera, A.; Morán, M.; Gómez-Ramos, P.; Alvarez-Castelao, B.;
Castaño, J.; Hernández, F.; Lucas, J. Inhibition of 26S proteasome activity by huntingtin
filaments but not inclusion bodies isolated from mouse and human brain. Journal of
neurochemistry 2006, 98 (5), 1585-1596. DOI: 10.1111/j.1471-4159.2006.03968.x.
                                                  180


(59) Gregori, L.; Fuchs, C.; Figueiredo-Pereira, M.; Van Nostrand, W.; Goldgaber, D. Amyloid
beta-protein inhibits ubiquitin-dependent protein degradation in vitro. The Journal of Biological
Chemistry 1995, 270 (34), 19702-19708. DOI: 10.1074/jbc.270.34.19702.
(60) Lindersson, E.; Beedholm, R.; Højrup, P.; Moos, T.; Gai, W.; Hendil, K. B.; Jensen, P. H.
Proteasomal inhibition by alpha-synuclein filaments and oligomers. The Journal of biological
chemistry 2004, 279 (13), 12924-12934. DOI: 10.1074/jbc.M306390200.
(61) Bence, N.; Sampat, R.; Kopito, R. Impairment of the ubiquitin-proteasome system by
protein aggregation. Science (New York, N.Y.) 2001, 292 (5521). DOI:
10.1126/science.292.5521.1552.
(62) Oh, S.; Hong, H.; Hwang, E.; Sim, H.; Lee, W.; Shin, S.; Mook-Jung, I. Amyloid peptide
attenuates the proteasome activity in neuronal cells. Mechanisms of ageing and development
2005, 126 (12), 1292-1299. DOI: 10.1016/j.mad.2005.07.006.
(63) Tanaka, K.; Matsuda, N. Proteostasis and neurodegeneration: the roles of proteasomal
degradation and autophagy. Biochimica et biophysica acta 2014, 1843 (1), 197-204. DOI:
10.1016/j.bbamcr.2013.03.012.
(64) Tanaka, Y.; Engelender, S.; Igarashi, S.; Rao, R. K.; Wanner, T.; Tanzi, R. E.; Sawa, A.; L.
Dawson, V.; Dawson, T. M.; Ross, C. A. Inducible expression of mutant alpha-synuclein
decreases proteasome activity and increases sensitivity to mitochondria-dependent apoptosis.
Human Molecular Genetics 2001, 10 (9), 919-926. DOI: 10.1093/hmg/10.9.919.
(65) Tseng, B. P.; Green, K. N.; Chan, J. L.; Blurton-Jones, M.; LaFerla, F. M. Abeta inhibits the
proteasome and enhances amyloid and tau accumulation. Neurobiology of aging 2008, 29 (11),
1607-1618. DOI: 10.1016/j.neurobiolaging.2007.04.014.
(66) Emmanouilidou, E.; Stefanis, L.; Vekrellis, K. Cell-produced alpha-synuclein oligomers are
targeted to, and impair, the 26S proteasome. Neurobiology of aging 2010, 31 (6), 953-968. DOI:
10.1016/j.neurobiolaging.2008.07.008.
(67) Kristiansen, M.; Deriziotis, P.; Dimcheff, D. E.; Jackson, G. S.; Ovaa, H.; Naumann, H.;
Clarke, A. R.; van Leeuwen, F. W. B.; Menéndez-Benito, V.; Dantuma, N. P.; et al. Disease-
associated prion protein oligomers inhibit the 26S proteasome. Molecular cell 2007, 26 (2), 175-
188. DOI: 10.1016/j.molcel.2007.04.001.
(68) Deriziotis, P.; Tabrizi, S. Prions and the proteasome. Biochimica et biophysica acta 2008,
1782 (12), 713-722. DOI: 10.1016/j.bbadis.2008.06.011.
(69) Hampel, H.; Hardy, J.; Blennow, K.; Chen, C.; Perry, G.; Kim, S. H.; Villemagne, V. L.;
Aisen, P.; Vendruscolo, M.; Iwatsubo, T.; et al. The Amyloid-β Pathway in Alzheimer’s Disease.
Molecular Psychiatry 2021, 26 (10), 5481-5503, ReviewPaper. DOI: doi:10.1038/s41380-021-
01249-0.
(70) Ono, K. Alzheimer's disease as oligomeropathy. Neurochemistry international 2018, 119,
57-70. DOI: 10.1016/j.neuint.2017.08.010.
                                                 181


(71) Michaels, T. C. T.; Šarić, A.; Curk, S.; Bernfur, K.; Arosio, P.; Meisl, G.; Dear, A. J.;
Cohen, S. I. A.; Dobson, C. M.; Vendruscolo, M.; et al. Dynamics of oligomer populations
formed during the aggregation of Alzheimer’s Aβ42 peptide. Nature Chemistry 2020, 12 (5),
445-451, OriginalPaper. DOI: doi:10.1038/s41557-020-0452-1.
(72) Korsak, M.; Kozyreva, T. Beta Amyloid Hallmarks: From Intrinsically Disordered Proteins
to Alzheimer’s Disease. Springer, Cham, 2015; pp 401-421.
(73) Jan, A.; Hartley, D. M.; Lashuel, H. A. Preparation and characterization of toxic Abeta
aggregates for structural and functional studies in Alzheimer's disease research. Nature protocols
2010, 5 (6), 1186-1209. DOI: 10.1038/nprot.2010.72.
(74) Carulla, N.; Caddy, G. L.; Hall, D. R.; Zurdo, J.; Gairí, M.; Feliz, M.; Giralt, E.; Robinson,
C. V.; Dobson, C. M. Molecular recycling within amyloid fibrils. Nature 2023, 436 (7050), 554-
558, OriginalPaper. DOI: doi:10.1038/nature03986.
(75) Gruning, C.; Klinker, S.; Wolff, M.; Schneider, M.; Toksoz, K.; Klein, A.; Nagel-Steger, L.;
Willbold, D.; Hoyer, W. The Off-rate of Monomers Dissociating from Amyloid-β Protofibrils.
Journal of Biological Chemistry 2013, 288 (52), 37104-37111. DOI: 10.1074/jbc.M113.513432.
(76) Kane, R.; Bross, P.; Farrell, A.; Pazdur, R. Velcade®: U.S. FDA Approval for the
Treatment of Multiple Myeloma Progressing on Prior Therapy. The Oncologist 2003, 8 (6), 508-
513. DOI: 10.1634/theoncologist.8-6-508.
(77) Volpicelli-Daley, L. A.; Luk, K. C.; Patel, T. P.; Tanik, S. A.; Riddle, D. M.; Stieber, A.;
Meaney, D. F.; Trojanowski, J. Q.; Lee, V. M. Y. Exogenous α-synuclein fibrils induce Lewy
body pathology leading to synaptic dysfunction and neuron death. Neuron 2011, 72 (1), 57-71.
DOI: 10.1016/j.neuron.2011.08.033.
(78) Tanik, S.; Schultheiss, C.; Volpicelli-Daley, L.; Brunden, K.; Lee, V. Lewy body-like α-
synuclein aggregates resist degradation and impair macroautophagy. The Journal of biological
chemistry 2013, 288 (21), 15194-15210. DOI: 10.1074/jbc.M113.457408.
(79) Pinotsi, D.; Michel, C. H.; Buell, A. K.; Laine, R. F.; Mahou, P.; Dobson, C. M.; Kaminski,
C. F.; Kaminski Schierle, G. S. Nanoscopic insights into seeding mechanisms and toxicity of α-
synuclein species in neurons. Proceedings of the National Academy of Sciences 2016, 113 (14),
3815-3819. DOI: 10.1073/pnas.1516546113.
(80) Pankaj Gaur, M. G., Andrii Kurochka, Subrata Ghosh, Dmytro A. Yushchenko, and
Volodymyr V. Shvadchak. Fluorescent Probe for Selective Imaging of α-Synuclein Fibrils in
Living Cells. 2021, 12, 1293-1298.
(81) Haidekker, M. A.; Theodorakis, E. A. Environment-sensitive behavior of fluorescent
molecular rotors. Journal of Biological Engineering 2010, 4 (1), 1-14, ReviewPaper. DOI:
doi:10.1186/1754-1611-4-11.
(82) Kitts, C.; Beke-Somfai, T.; Nordén, B. Michler's hydrol blue: a sensitive probe for amyloid
fibril detection. Biochemistry 2011, 50 (17), 3451-3461. DOI: 10.1021/bi102016p.
                                                182


(83) Wördehoff, M. M.; Hoyer, W. α-Synuclein Aggregation Monitored by Thioflavin T
Fluorescence Assay. Bio-protocol 2018, 8 (14). DOI: 10.21769/BioProtoc.2941.
(84) Sulatskaya, A. I.; Rodina, N. P.; Sulatsky, M. I.; Povarova, O. I.; Antifeeva, I. A.;
Kuznetsova, I. M.; Turoverov, K. K. Investigation of α-Synuclein Amyloid Fibrils Using the
Fluorescent Probe Thioflavin T. International journal of molecular sciences 2018, 19 (9). DOI:
10.3390/ijms19092486.
(85) Orihuela, R.; McPherson, C.; Harry, G. Microglial M1/M2 polarization and metabolic
states. British Journal of Pharmacology 2015, 173 (4), 649-665. DOI: 10.1111/bph.13139.
(86) Choi, W. H.; Kim, S.; Park, S.; Lee, M. J. Concept and application of circulating
proteasomes. Experimental & Molecular Medicine 2021, 53 (10), 1539-1546, ReviewPaper.
DOI: doi:10.1038/s12276-021-00692-x.
(87) Ben-Nissan, G.; Katzir, N.; Füzesi-Levi, M. G.; Sharon, M. Biology of the Extracellular
Proteasome. Biomolecules 2022, 12 (5), 619, Review. DOI: 10.3390/biom12050619.
(88) Ye, X.; Zhu, M.; Che, X.; Wang, H.; Liang, X.; Wu, C.; Xue, X.; Yang, J.
Lipopolysaccharide induces neuroinflammation in microglia by activating the MTOR pathway
and downregulating Vps34 to inhibit autophagosome formation. Journal of neuroinflammation
2020, 17 (1), 18. DOI: 10.1186/s12974-019-1644-8.
                                                183


                                       APPENDIX
Figure 4.10 1H and 13C NMR spectra of compound 4-1
                                          184


Figure 4.11 1H and 13C NMR spectra of compound 4-2
                                          185


Figure 4.12 1H and 13C NMR spectra of compound 4-3
                                          186


        CHAPTER FIVE
Conclusions and Future Directions
              187


5.1 Conclusions and Future Directions
         The UPS has long been envisioned as a promising therapeutic target due to its involvement
in numerous critical biological pathways.1-10 The success of inhibitors of the proteasome in the
treatment of some cancers validated it as a therapeutic target.11 In recent years, the importance of
the lone 20S form of the proteasome as a player in the maintenance of proteostasis has come to
light.12 As a result, there has been a surge in interest in targeting the 20S form of the proteasome
for treatment of a variety of diseases.7,        13-26
                                                       One area of particular interest is that of
neurodegenerative disease treatment, considering the lack of disease modifying treatments and the
devastation that is wrought on the lives of patients and their loved ones by these diseases.
         My PhD studies have focused on furthering the development of small molecules 20S
proteasome activators as an innovative therapeutic strategy aimed at treating neurodegenerative
diseases. This was done by first identifying novel small molecule scaffolds that enhance 20S
proteasome-mediated proteolysis, evaluating their activities, ensuring that this activity is
translatable to disease relevant IDP substrates and initiating SAR studies focused on developing
potent analogues.22, 23 With these novel activators in hand, I sought to further explore the potential
of this proposed therapeutic strategy on more disease relevant substrates and in more disease
relevant systems.23 The result is an expanded toolbox of activators and methods with which they
can be evaluated for their potential to effect neurodegenerative disease pathogenesis.
         I was the first to explore the interplay between IDP oligomers seen in neurodegenerative
diseases and small molecule 20S proteasome activators. Through these studies I found that small
molecule 20S proteasome activators can maintain 20S proteasome activity in the presence of
inhibitory IDP oligomers and reduce them in vitro.23 These studies were the foundation for several
ongoing studies in the Tepe lab aimed at further exploring this interplay, expanding to other disease
                                                  188


related IDPs and developing more disease relevant model systems for furthering small molecule
20S proteasome activation as a therapeutic strategy. Of particular interest is the development of a
neuronal cell-based model for IDP accumulation and aggregation, as was initiated with the primary
mouse hippocampal neuron studies outlined above. Substitution of this cell line with the
immortalized hippocampal cells (HT-22 cell line) mentioned above could allow for a much more
efficient, in terms of time and cost, means to develop this model system to explore the effects of
small molecule 20S proteasome activators in a neuron model of IDP aggregation. These cells could
also serve in the development of a model for IDP accumulation through stable transfection with
an IDP, such as A53T α-synuclein. If toxicity proves to be a limiting factor here, an inducible
expression system, like a Tet-on/off system, may permit for more controlled expression and
accumulation.
        I also demonstrated that small molecule 20S proteasome activators can reduce the release
of the pro-inflammatory cytokine TNF-α by microglia that have been activated by the familial
Parkinson’s disease related IDP A53T α-synuclein. Chronic neuroinflammation is one of the
hallmarks of neurodegenerative diseases, like Parkinson’s disease, and is believed to be partially
a result of activation of microglia by IDPs, as seen in these studies.27-32 So, reduction of this pro-
inflammatory signaling could show promise for development of therapeutics for these diseases.
The reduction of TNF-α release seen in these studies was not specific to microglial activation with
IDPs, but also those activated with LPS,33 suggesting the possibility of a more general anti-
inflammatory response caused by small molecule 20S proteasome activators in this system. While
this lacks the specificity that was hypothesized, it may still serve as a promising secondary function
during development of neurodegenerative disease therapeutics, where both inflammation and IDP
accumulation, aggregation and toxicity play a role.34-40 Further studies are needed to elucidate the
                                                  189


mechanism associated with this reduction in TNF-α release by activated microglia. Some potential
avenues to explore this mechanism include monitoring intracellular levels and release of other
inflammatory signaling molecules as well as changes in vesicle trafficking. Proteomic and
genomic analyses in these cells may also provide insight into how small molecule 20S proteasome
activation might be affecting TNF-α release in this system. Going forward with these studies on
neuroinflammation induced by IDP release, co-culture experiments with microglia and neuronal
cells could serve as promising cell-based models of neurodegenerative disease for further
evaluation of the effects of small molecule 20S proteasome activation. If a stably transfected
neuronal model for IDP accumulation or aggregation can be developed, a cellular model
demonstrating how IDPs can lead to the development of neuroinflammation could be conceived.
Alternatively, activation of microglia through direct addition of IDPs could be used to develop a
model in which the deleterious effects of neuroinflammation on neurons can be explored and
potentially prevented with the addition of small molecule 20S proteasome activators.
                                               190


                                           REFERENCES
(1) Voges, D.; Zwickl, P.; Baumeister, W. The 26S Proteasome: A Molecular Machine Designed
for Controlled Proteolysis. Annual Review of Biochemistry 1999, 68, 1015-1068, review-article.
DOI: 10.1146/annurev.biochem.68.1.1015.
(2) McNaught, K. S. P.; Olanow, C. W.; Halliwell, B.; Isacson, O.; Jenner, P. Failure of the
ubiquitin–proteasome system in Parkinson's disease. Nature Reviews Neuroscience 2001, 2 (8),
589-594. DOI: 10.1038/35086067.
(3) Voorhees, P.; Dees, E.; O'Neil, B.; Orlowski, R. The proteasome as a target for cancer therapy.
Clinical cancer research 2003, 9 (17), 6316-6325.
(4) Ostrowska, H. The ubiquitin-proteasome system: a novel target for anticancer and anti-
inflammatory drug research. Cellular & Molecular Biology Letters 2008, 13 (3), 353-365. DOI:
10.2478/s11658-008-0008-7.
(5) Calamini, B.; Morimoto, R. Protein homeostasis as a therapeutic target for diseases of protein
conformation. Current Topics in Medicinal Chemistry 2012, 12 (22), 2623-2640. DOI:
10.2174/1568026611212220014.
(6) Huang, X.; Dixit, V. M. Drugging the undruggables: exploring the ubiquitin system for drug
development. Cell Research 2016, 26 (4), 484-498, ReviewPaper. DOI: doi:10.1038/cr.2016.31.
(7) Njomen, E.; Tepe, J. J. Proteasome Activation as a New Therapeutic Approach to Target
Proteotoxic Disorders. Journal of Medicinal Chemistry 2019, 62 (14), 6469-6481. DOI:
10.1021/acs.jmedchem.9b00101.
(8) Tundo, G.; Sbardella, D.; Santoro, A.; Coletta, A.; Oddone, F.; Grasso, G.; Milardi, D.; Lacal,
P.; Marini, S.; Purrello, R.; et al. The proteasome as a druggable target with multiple therapeutic
potentialities: Cutting and non-cutting edges. Pharmacology & therapeutics 2020, 213, 107579.
DOI: 10.1016/j.pharmthera.2020.107579.
(9) Thibaudeau, T. A.; Smith, D. M. A Practical Review of Proteasome Pharmacology.
Pharmacological reviews 2019, 71 (2), 170-197. DOI: 10.1124/pr.117.015370.
(10) Shang, F.; Taylor, A. Ubiquitin-proteasome pathway and cellular responses to oxidative
stress.    Free     radical     biology     &    medicine    2011,     51    (1),    5-16.    DOI:
10.1016/j.freeradbiomed.2011.03.031.
(11) Kane, R.; Bross, P.; Farrell, A.; Pazdur, R. Velcade®: U.S. FDA Approval for the Treatment
of Multiple Myeloma Progressing on Prior Therapy. The Oncologist 2003, 8 (6), 508-513. DOI:
10.1634/theoncologist.8-6-508.
(12) Deshmukh, F. K.; Yaffe, D.; Olshina, M. A.; Ben-Nissan, G.; Sharon, M. The Contribution
of the 20S Proteasome to Proteostasis. Biomolecules 2019, 9 (5), 190, Review. DOI:
10.3390/biom9050190.
                                                 191


(13) Jones, C. L.; Njomen, E.; Sjögren, B.; Dexheimer, T. S.; Tepe, J. J. Small Molecule
Enhancement of 20S Proteasome Activity Targets Intrinsically Disordered Proteins. ACS
Chemical Biology 2017, 12 (9), 2240-2247. DOI: 10.1021/acschembio.7b00489.
(14) Trader, D. J.; Simanski, S.; Dickson, P.; Kodadek, T. Establishment of a suite of assays that
support the discovery of proteasome stimulators. Biochimica et Biophysica Acta (BBA) - General
Subjects 2017, 1861 (4), 892-899. DOI: 10.1016/J.BBAGEN.2017.01.003.
(15) Coleman, R. A.; Trader, D. J. Development and Application of a Sensitive Peptide Reporter
to Discover 20S Proteasome Stimulators. ACS Combinatorial Science 2018, 20 (5), 269-276. DOI:
10.1021/acscombsci.7b00193.
(16) Njomen, E.; Osmulski, P. A.; Jones, C. L.; Gaczynska, M.; Tepe, J. J. Small Molecule
Modulation of Proteasome Assembly. Biochemistry 2018, 57 (28), 4214-4224. DOI:
10.1021/acs.biochem.8b00579.
(17) Giżyńska, M.; Witkowska, J.; Karpowicz, P.; Rostankowski, R.; Chocron, E. S.; Pickering,
A. M.; Osmulski, P.; Gaczynska, M.; Jankowska, E. Proline- and Arginine-Rich Peptides as
Flexible Allosteric Modulators of Human Proteasome Activity. Journal of Medicinal Chemistry
2018, 62 (1), 359-370, research-article. DOI: 10.1021/acs.jmedchem.8b01025.
(18) Opoku-Nsiah, K. A.; Gestwicki, J. E. Aim for the core: suitability of the ubiquitin-
independent 20S proteasome as a drug target in neurodegeneration. Mosby Inc.: 2018; Vol. 198,
pp 48-57.
(19) Coleman, R. A.; Muli, C. S.; Zhao, Y.; Bhardwaj, A.; Newhouse, T. R.; Trader, D. J. Analysis
of chain length, substitution patterns, and unsaturation of AM-404 derivatives as 20S proteasome
stimulators. Bioorganic & Medicinal Chemistry Letters 2019, 29 (3), 420-423. DOI:
10.1016/J.BMCL.2018.12.030.
(20) Njomen, E.; Tepe, J. J. Regulation of Autophagic Flux by the 20S Proteasome. Cell Chemical
Biology 2019, 26 (9), 1283-1294.e1285. DOI: 10.1016/j.chembiol.2019.07.002.
(21) Coleman, R. A.; Trader, D. J. Methods to Discover and Evaluate Proteasome Small Molecule
Stimulators. Molecules 2019, 24 (12), 2341, Review. DOI: 10.3390/molecules24122341.
(22) Fiolek, T.; Magyar, C.; Wall, T.; Davies, S.; Campbell, M.; Savich, C.; Tepe, J.; Mosey, R.
Dihydroquinazolines enhance 20S proteasome activity and induce degradation of α-synuclein, an
intrinsically disordered protein associated with neurodegeneration. Bioorganic & Medicinal
Chemistry Letters 2021, 36, 127821. DOI: 10.1016/j.bmcl.2021.127821.
(23) Fiolek, T. J.; Keel, K. L.; Tepe, J. J. Fluspirilene Analogs Activate the 20S Proteasome and
Overcome Proteasome Impairment by Intrinsically Disordered Protein Oligomers. ACS Chemical
Neuroscience 2021, 12 (8), 1438-1448. DOI: 10.1021/acschemneuro.1c00099.
(24) Ben-Nissan, G.; Katzir, N.; Füzesi-Levi, M. G.; Sharon, M. Biology of the Extracellular
Proteasome. Biomolecules 2022, 12 (5), 619, Review. DOI: 10.3390/biom12050619.
                                                 192


(25) Jones, C. L.; Tepe, J. J. Proteasome Activation to Combat Proteotoxicity. Molecules 2019, 24
(15), 2841, Review. DOI: 10.3390/molecules24152841.
(26) George, D.; Tepe, J. Advances in Proteasome Enhancement by Small Molecules.
Biomolecules 2021, 11 (12), 1789. DOI: 10.3390/biom11121789.
(27) Heneka, M.; Carson, M.; El Khoury, J.; Landreth, G.; Brosseron, F.; Feinstein, D.; Jacobs,
A.; Wyss-Coray, T.; Vitorica, J.; Ransohoff, R.; et al. Neuroinflammation in Alzheimer's disease.
The Lancet. Neurology 2015, 14 (4), 388-405. DOI: 10.1016/S1474-4422(15)70016-5.
(28) Calsolaro, V.; Edison, P. Neuroinflammation in Alzheimer's disease: Current evidence and
future directions. Alzheimer's & dementia : the journal of the Alzheimer's Association 2016, 12
(6), 719-732. DOI: 10.1016/j.jalz.2016.02.010.
(29) Liu, J.; Wang, F. Role of Neuroinflammation in Amyotrophic Lateral Sclerosis: Cellular
Mechanisms and Therapeutic Implications. Frontiers in immunology 2017, 8, 1005. DOI:
10.3389/fimmu.2017.01005.
(30) Yang, Q.; Zhou, J. Neuroinflammation in the central nervous system: Symphony of glial cells.
Glia 2019, 67 (6), 1017-1035. DOI: 10.1002/glia.23571.
(31) Tu, H.; Yuan, B.; Hou, X.; Zhang, X.; Pei, C.; Ma, Y.; Yang, Y.; Fan, Y.; Qin, Z.; Liu, C.; et
al. α-synuclein suppresses microglial autophagy and promotes neurodegeneration in a mouse
model of Parkinson's disease. Aging cell 2021, 20 (12), e13522. DOI: 10.1111/acel.13522.
(32) Hoenen, C.; Gustin, A.; Birck, C.; Kirchmeyer, M.; Beaume, N.; Felten, P.; Grandbarbe, L.;
Heuschling, P.; Heurtaux, T. Alpha-Synuclein Proteins Promote Pro-Inflammatory Cascades in
Microglia: Stronger Effects of the A53T Mutant. PloS one 2016, 11 (9), e0162717. DOI:
10.1371/journal.pone.0162717.
(33) Ye, X.; Zhu, M.; Che, X.; Wang, H.; Liang, X.; Wu, C.; Xue, X.; Yang, J. Lipopolysaccharide
induces neuroinflammation in microglia by activating the MTOR pathway and downregulating
Vps34 to inhibit autophagosome formation. Journal of neuroinflammation 2020, 17 (1), 18. DOI:
10.1186/s12974-019-1644-8.
(34) Uversky, V. N.; Oldfield, C. J.; Midic, U.; Xie, H.; Xue, B.; Vucetic, S.; Iakoucheva, L. M.;
Obradovic, Z.; Dunker, A. K. Unfoldomics of human diseases: linking protein intrinsic disorder
with diseases. BMC genomics 2009, 10 Suppl 1 (Suppl 1), S7-S7. DOI: 10.1186/1471-2164-10-
S1-S7.
(35) Uversky, V. Intrinsically disordered proteins and their (disordered) proteomes in
neurodegenerative disorders. Frontiers in Aging Neuroscience 2015, 7. DOI:
10.3389/fnagi.2015.00018.
(36) Smith, D. M. Could a Common Mechanism of Protein Degradation Impairment Underlie
Many Neurodegenerative Diseases? Journal of experimental neuroscience 2018, 12,
1179069518794675-1179069518794675. DOI: 10.1177/1179069518794675.
                                                193


(37) Thibaudeau, T. A.; Anderson, R. T.; Smith, D. M. A common mechanism of proteasome
impairment by neurodegenerative disease-associated oligomers. Nature Communications 2018, 9
(1), 1097-1097. DOI: 10.1038/s41467-018-03509-0.
(38) Ross, C. A.; Poirier, M. A. Protein aggregation and neurodegenerative disease. Nature
Medicine 2004, 10 (7), S10-S17. DOI: 10.1038/nm1066.
(39) Gammon, K. Neurodegenerative disease: brain windfall. Nature 2014, 515 (7526), 299-300.
DOI: 10.1038/nj7526-299a.
(40) Onyango, I.; Jauregui, G.; Čarná, M.; Bennett, J.; Stokin, G. Neuroinflammation in
Alzheimer's Disease. Biomedicines 2021, 9 (5), 524. DOI: 10.3390/biomedicines9050524.
                                             194


  CHAPTER SIX
Materials and Methods
         195


6.1 Materials
6.1.1 Key resource tables
Table 6.1: Antibodies
                                                                              Catalogue
              Reagent or Resources                          Source
                                                                               number
  GAPDH (14C10) Rabbit mAb HRP-linked           Cell Signaling Technology    3683S
  α-synuclein (Syn204) mouse monoclonal
                                                Cell Signaling Technology    2647S
  antibody
  Recombinant anti-alpha-synuclein antibody
                                                Abcam                        ab138501
  [MJFR1] Rabbit
  Goat anti-Rabbit IgG HRP-linked               Cell Signaling Technology    7074S
  Horse anti-Mouse IgG HRP-linked               Cell Signaling Technology    7076S
  Goat anti-Mouse IgG2a Alexa FluorTM 594       Thermofisher Scientific      A-21135
  Goat anti-Mouse IgM Alexa FluorTM 488         Thermofisher Scientific      A-21042
Table 6.2: Peptides and recombinant proteins
                                                                            Catalogue
             Reagent or Resources                      Source
                                                                             number
  Human 20S proteasome                        BostonBiochem             E-360
                                                                        BML-PW8720-
  Human 20S proteasome                        Enzo Life Sciences
                                                                        0050
  19S Proteasome                              BostonBiochem             E-366
  Recombinant WT human a-synuclein            Abcam                     ab51189
  Recombinant human GAPDH protein             Abcam                     ab77109
  N-Succinyl-Leu-Leu-Val-Tyr-7-amido-4-
                                              BostonBiochem             S-280
  methylcoumarin (Suc-LLVY-AMC)
  Z-Leu-Leu-Glu-7-amido-4-methylcoumarin
                                              BostonBiochem             S-230
  (Z-LLE-AMC)
  Boc-Leu-Arg-Arg-7-amido-4-
                                              BostonBiochem             S-300
  methylcoumarin (Boc-LRR-AMC)
  Recombinant Human α-synuclein Protein       Novus Biologicals         NBC1-18331
  Recombinant Human GAPDH Protein             Novus Biologicals         NBC1-18528
  Recombinant Human α-synuclein Active                                  NBP2-54789-
                                              Novus Biologicals
  Pre-formed Fibrils (type 1) Protein                                   100ug
  Human α-synuclein, A53T                     Kerafast Inc.             EGP013
  Beta-Amyloid (1-42), Human                  Eurogentec                AS-20276
                                            196


Table 1.3: Cell lines, cell culture and transfection reagents
                                                                           Catalogue
            Reagent or Resources                          Source
                                                                            number
 Immortalized mouse microglia (IMG) cells       Sigma Aldrich           SCC134
 HEK-293T                                       ATCC                    CRL-3216
 Primary Mouse Hippocampal Neurons              Gibco                   A15587
 Neurobasal Plus Medium                         Gibco                   A3582901
 DMEM medium                                    Gibco                   11995-065
 0.25% Trypsin-EDTA (1x)                        Gibco                   25200-056
 Fetal Bovine Serum                             Gibco                   16000044
 Penicillin-Streptomycin                        Gibco                   15140-122
 GlutaMAX (100x)                                Gibco                   35050-061
 B-27 Supplement (50x)                          Gibco                   17504-044
 Amphotericin B                                 Gibco                   15290018
 Gentamicin                                     Gibco                   15750-060
 X-tremeGENE HP DNA Transfection
                                                Sigma Aldrich           06366236001
 Reagent
 Dulbecco’s Phosphate Buffered Saline           Sigma Aldrich           D8537-500ML
Table 6.4: Oligonucleotides and recombinant DNA
                                                                            Catalogue
            Reagent or Resources                           Source
                                                                             number
 pHM6-alphasynuclein-A53T                       Addgene                  40825
Table 6.5: Bacterial strains
                                                                            Catalogue
            Reagent or Resources                           Source
                                                                             number
 DH5α Chemically Competent E. coli              ThermoFisher Scientific   18265017
Table 6.6: Commercial assay kits
                                                                            Catalogue
            Reagent or Resources                           Source
                                                                             number
 Pierce BCA Protein Assay Kit                   Thermofisher Scientific   23225
 Pierce Silver Stain Kit                        Thermofisher Scientific   24612
 CellTiter 96 Aqueous One Solution Cell
                                                Promega                   G3580
 Proliferation Assay
 Mouse TNF-α ELISA kit                          Invitrogen                BMS607-3
 Plasmid Maxi kit                               QIAGEN                    12163
                                               197


Table 6.7: Other chemicals and reagents
                                                                             Catalogue
             Reagent or Resources                        Source
                                                                              number
  TCH-165                                    Tepe lab                      NA
  Fluspirilene                               Tepe lab                      NA
  N-acylated Fluspirilene                    Tepe lab                      NA
  Other Fluspirilene analogues               Tepe lab                      NA
  Aripiprazole                               Cayman Chemical               19989
  Dihydroquinazoline analogues               Mosey lab                     NA
  Bortezomib                                 Cayman Chemical               10008822
  Epoxomicin                                 Cayman Chemical               10007806
  Cycloheximide                              Cell Signaling Technology     2112
  LB Broth                                   Sigma-Aldrich                 L7275
  cOmplete Mini, EDTA-free protease
                                             Sigma Aldrich                 11836170001
  inhibitor cocktail tablets
  Adenosine 5′-triphosphate magnesium salt   Sigma Aldrich                 A9187
  Pierce RIPA buffer                         Thermofisher Scientific       89901
  Mini-PROTEAN® TGX™ Precast Gels            Bio-Rad                       4561094
  Immun-Blot® PVDF Membrane                  Bio-Rad                       1620177
  Clarity™ Western ECL Substrate             Bio-Rad                       1705060
  Radiance Plus HRP Substrate                Azure Biosystems              AC2103
  InstantBlueTM Coomassie stain              Expedeon                      ISB1L
  Blocking-grade Blocker (non-fat dry milk)  Bio-Rad                       1706404
6.2 Methods
6.2.1 Cell culture
        General cell culture: Human embryonic kidney cells (HEK-293T) or immortalized mouse
microglial cells (IMG) were maintained in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum and 100 units/mL penicillin/streptomycin, at 37 °C
with 5% CO2.
6.2.2 Primary neuron cell culture
        Plating media: Neurobasal Plus media supplemented with 10% FBS, 2% B-27
supplement, 1% GlutaMAX supplement, 1% Amphotericin B and 50 μg/mL of Gentamicin was
                                            198


used for initial plating at the manufacturers recommended density and for the first 4 days of
culturing the cells.
        Culturing media: Beginning on day 4 after plating, plating media was exchanged for
Neurobasal Plus media supplemented with 2% B-27 supplement, 1% GlutaMAX supplement, 1%
Amphotericin B and 50 μg/mL of Gentamicin, without FBS, and was used to maintain the cells
for the remainder of the culture time.
        Plating and culturing procedures: From liquid nitrogen, the cells were rapidly thawed in
a 37 °C water bath until a small ice crystal remained. The vial was then transferred to the cell
culture hood and disinfected with 70% ethanol. Prior to every liquid transfer step with cells, all
pipette tips and tubes must be pre-rinsed with media to prevent cells from adhering to the surfaces
and to maximize viable cell count. The cells were then gently transferred with a pipette to a pre-
rinsed 50 mL conical tube. Next, the cryo-preservation tube that the cells came from was washed
using 1 mL of the plating media and this media was added dropwise while gently swirling the cells.
To this was then added dropwise 2 mL of additional plating media. The cells were gently mixed
with a pipette without creating any air bubbles. The cells were counted using 0.4% trypan blue and
a hemocytometer. 1.7x104 cells were plated per well in a poly-D-lysine-coated 96-well plate. The
cells were then diluted to 200 μL with the plating media and transferred to an incubator at 37 °C
with humidified atmosphere of 5% CO2 in air. After 24 h, half of the media was aspirated and
replaced with fresh plating media. After 3 more days (72 h), 3/4 of the media was aspirated off and
replaced with culture media (no FBS). This was done every 3 days for general culture or every 7
days for experiments with two treatments over 14 days. All media exchanges on primary mouse
hippocampal neurons were done one well at a time to minimize any time exposed to air.
                                                 199


        Pre-formed α-synuclein fibril seed preparation: Pre-formed α-synuclein fibrils,
obtained from our collaborated Dr. Sortwell, (2 mg/mL) were sonicated with a micro probe tip
sonicator with 60 pulses at 10% power (total of 30 s, 0.5 s on, 0.5 s off). Following sonication,
pre-formed fibril seeds were diluted in culturing media to the desired final concentration (0.001
mg/mL) and immediately used for treatment of primary mouse hippocampal neurons.
        Seeding of primary mouse hippocampal neurons with α-synuclein pre-formed fibrils:
Following the initial plating procedure outlined above, on day 4 ~80% of the plating media was
removed from primary mouse hippocampal cultures and was replaced with fresh culturing media
containing desired final concentration of pre-formed α-synuclein fibril seeds. The cells were
returned to the incubator for 7 days, after which 50% of the media was exchanged for fresh
culturing media, without any pre-formed α-synuclein fibril seeds. When compound treatments
were attempted (data not shown), treatments were done shortly following initial treatment with
pre-formed α-synuclein fibril seeds and again 7 days later, for a total of 2 treatments. However,
these experiments did not yield conclusive results due to suboptimal cell growth and seeding
efficiencies. Following the 14-day incubation with pre-formed α-synuclein fibril seeds, all media
was gently removed from neuron cultures and the cells were washed with warm TBS buffer 1x,
fixed with 4% paraformaldehyde in TBS for 30 min at RT, and washed 2x with TBS buffer again.
Cells could then be stored at 4 °C. Immunostaining and confocal immunofluorescence of resulting
fixed primary mouse hippocampal neurons was performed by Dr. Caryl Sortwell and her lab.
6.2.3 Transient transfection of A53T α-synuclein plasmid into HEK-293T cells
        HEK-293T cells were grown using the methods stated above to ~50-70% confluency in 60
mm plates. DNA (5 μg of A53T α-synuclein plasmid) was mixed with 0.5 mL of serum free-
DMEM medium. X-tremeGENE transfection reagent (10 μL) was briefly vortexed, added to the
                                               200


plasmid solution and then this mixture was incubated for 20 min at RT. The mixture was then
added dropwise with swirling to the HEK-293T cells and allowed to incubate for 4 h at 37ºC, 5%
CO2, in a tissue culture incubator. The transfection medium was replaced with fresh culture
medium and cultured for a further 24 h.
6.2.4 Proteasome-mediated degradation of A53T α-synuclein in HEK-293T cells
        HEK-293T cells were transfected as described above. The cells were then treated with 50
μg/mL of cycloheximide, in combination with either vehicle (DMSO), Fluspirilene (10 or 30 μM),
N-acylated Fluspirilene (10 or 30 μM), Bortezomib (100 nM), or a combination thereof for 8 h.
The cells were then lysed, and the resulting cellular extracts were immunoblotted to monitor A53T
α-synuclein levels.
6.2.5 Immunoblot
        Transfected HEK-293T cells at 70-80% confluency were treated with test compounds at
the reported concentrations and time as indicated in figure legends. Samples meant for immunoblot
only were washed 2x with warm DPBS buffer and scrapped with chilled RIPA buffer
supplemented with protease inhibitor cocktail. Samples were incubated at 0 °C for 15 min,
centrifuged at ~14,000 g and the supernatant assayed for total protein content with bicinchoninic
acid (BCA) assay. Normalized and boiled samples were resolved on 4-20% Tris-glycine gels,
resolved via SDS-PAGE and blotted onto PVDF membranes. Membranes were blocked in 5%
non-fat milk in TBST buffer for 60 min at RT. Membranes were then incubated with primary
antibody in 5% non-fat milk TBST buffer, overnight at 4ºC. Membranes were then washed 5x for
5 min each with TBST buffer and incubated in secondary antibody at RT for 60 min. Membranes
were washed again as above and developed with ECL clarity or Azure Biosystems Radiance Plus
                                                 201


reagent. Images were captured with an Azure Biosystems 300Q Imager. All primary antibodies
were used at a dilution of 1:1000.
6.2.6 Immortalized mouse microglia TNF-α and viability experiments
        Culture and treatment procedure: IMG cells were plated in 96-well cell culture plates
at a density of 10,000 cells per well. The cells were cultured under normal culture conditions for
24 h and then media was exchanged for fresh media (90 µL). Compounds were first dissolved in
DMSO at 1,000x the final desired concentration, and then further diluted to 10x the final desired
concentration in media. To each well was then added DMSO, TCH-165 or N-acylated Fluspirilene
(10 µL) at various concentrations (see Figure legends for details). Each treatment was done on six
different wells, so that three wells could be utilized for cell viability assessments and the media of
the other three wells could be used to measure TNF-α release. This prevented loss of any cells
when removing media influencing cell viability measurements. The cells were further incubated
for 1 h under normal culture conditions, and then A53T α-synuclein was added to a final
concentration of 10 µM. The cells were then returned to the incubator for 24 h.
        Cell viability assay: At the end of the treatments described above, to the cells was added
100 µL of fresh media (totaling 200 µL of media) and CellTiter 96 Aqueous One Solution Cell
Proliferation Assay solution (20 μL). The cells were then incubated for 1–4 h at 37 °C with 5%
CO2, while monitoring for color change. The assay plate was then allowed to equilibrate for 15
min at RT, gently agitated to ensure even distribution of color and absorbance readings were taken
on a SpectraMax M5e Spectrometer. Data are presented as a percentage of the vehicle control for
each experimental condition, after background subtraction.
        TNF-α ELISA: At the end of the treatments described above, the media from the cells and
from blank wells was removed and subjected to a TNF-α ELISA. This was done using a Mouse
                                                  202


TNF-α ELISA kit purchased from Invitrogen, following their recommended protocol and using
their provided standards. Absorbance readings were taken on a SpectraMax M5e Spectrometer. A
standard curve was generated from the provided standards and resulting absorbance readings using
the GraphPad Prism 7 software. Using this standard curve, concentrations for the various
treatments were determined from their absorbance readings. Data is represented as a percentage of
the TNF-α concentration relative to the untreated IMG cell control.
6.2.7 Molecular docking studies.
        Docking was performed using PyRx’s Vina Wizard program, supported through
computational resources and services provided by the Institute for Cyber-Enabled Research at
Michigan State University. The macromolecule for these docking studies was defined using a
crystal structure of the closed gate human 20S proteasome, obtained from the PDB database (PDB
ID: 4R3O). Small molecules ligands were generated in Perkin Elmer’s Chem3D. These small
molecule ligands were minimized using the MM2 force field, converted into PDB files and
uploaded to PyRx. Vina was run on a maximized grid of the h20S proteasome (grid box 153.2 ×
138.0 × 189.4 Å) to allow for unbiased docking on the entire human 20S proteasome. Docking
was performed on each ligand three times with exhaustiveness set to 1000. The top nine reported
docking states were analyzed using Schrödinger’s PyMOL Molecular Graphics System. In the case
of the Fluspirilene analogues, BIOVIA Discovery Studio 2020 was used to further analyze the
docking models using the receptor-ligand interactions function. This allowed for exploration of
how the human 20S proteasome (receptor) was predicted to interact with the Fluspirilene
analogues at each of the 9 top docking states.
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6.2.8 Fluorogenic peptide degradation 20S proteasome activity assay.
        Activity assays were carried out in a 100 μL reaction volume. Different concentrations (1–
80 μM) of test compounds in DMSO were added (1 µL) to a black flat/clear bottom 96-well plate
containing 1 nM of human constitutive 20S proteasome, in assay buffer (50 mM Tris-HCl at pH
7.8, 100 mM NaCl) and allowed to incubate for 15 min at 37 °C. Fluorogenic substrates were then
added and the enzymatic activity measured at 37 °C on a SpectraMax M5e spectrometer by
measuring the change in fluorescence unit per min for 1 h at 380/460 nm. Medium-throughput
screening (MTPS) was carried out in 384-well plates as described above, with the following
exceptions. Three concentrations of compounds (3, 10 and 30 μM; 150 nL of 200x stocks in
DMSO) were dispensed into a black flat bottom 384-well plate containing 25 μL 20S proteasome
in assay buffer, followed by 5 μL of 6x substrate working solution (Suc-LLVY-AMC diluted in
assay buffer), using automated liquid dispensers. Enzymatic activity was measured at 37 ºC on a
BioTek plate reader by measuring change in fluorescence unit per min for 1 h at 380/460 nm. The
fluorescence units for the vehicle control were set as 100%, and the ratio of drug-treated sample
relative to that of vehicle control was used to calculate the fold change in enzymatic activity. The
fluorogenic substrates used were one of the following: Suc-LLVY-AMC (CT-L activity, 20 μM),
Z-LLE-AMC (Casp-L activity, 20 μM), Boc-LRR-AMC (T-L activity, 40 μM) or a combination of
the three substrates (each at 6.67 μM). Magnesium chloride (5 mM) and ATP (2.5 mM) were
included in assays containing 26S proteasome.
6.2.9 In vitro purified α-synuclein degradation assay
        Digestion of α-synuclein was carried out in a 50 μL reaction volume made of 50 mM Tris
at pH 7.8; 0.33 μM purified α-synuclein and 6.7 nM purified human 20S proteasome. Briefly, 20S
proteasome was diluted to 7.58 nM in the reaction buffer. Test compounds or vehicle (1 μL of 50×
                                                 204


stock or DMSO, see Figures for treatment details) were added to 44 μL of 7.58 nM 20S and
incubated at 37 °C for 20 min. 5 μL of 3.3 μM α-synuclein substrate was then added to the reaction
mixture and incubated at 37 °C for 4 h. The reactions were quenched with concentrated sodium
dodecyl sulfate (SDS) loading buffer. After boiling for 10 min, samples were resolved on a 4–20%
Tris-glycine SDS-PAGE gel. The gels were then stained using InstantBlue Coomassie based
staining solution or Pierce Silver Stain Kit and the manufacturers recommended procedures.
6.2.10 Amyloid beta aggregate preparation.
         Synthetic amyloid beta was purchased from Eurogentec. To remove preexisting aggregates,
synthetic amyloid beta peptide was dissolved in 100% HFIP and incubated at ° °C for 2 h. The
HFIP was removed, and the remaining peptide films were stored at −80 °C until use. Aggregates
were prepared by resuspending amyloid beta films with DMSO (50 μL per 1 mg of peptide),
followed by addition of ultrapure H2O (800 μL) and rapid addition of 2 M Tris-base (10 μL) at pH
7.6. The solution was then vortexed for 5 seconds and allowed to incubate at room temperature for
5 min. The Amyloid beta mixture was then diluted to the desired concentration and used
immediately.
6.2.11 IDP oligomer inhibition in fluorogenic peptide degradation assay
         Assays were carried out in a 100 μL reaction volume. Different concentrations (1–10 μM)
of test compounds were added to a black flat/clear bottom 96-well plate containing 1 nM of human
constitutive 20S proteasome, in 50 mM Tris-HCl at pH 7.8 and allowed to incubate for 15 min at
37 °C. Then, 1 μL of α-synuclein or amyloid beta oligomer mixture was added to each sample to
a final concentration of 500 nM for α-synuclein and 2.5 μM for amyloid beta. This mixture was
then allowed to incubate again for 15 min at 37 °C. Next, 10 μL of CT-L fluorogenic substrate was
added to a final concentration of 20 μM. The enzymatic activity was measured at 37 °C on a
                                                205


SpectraMax M5e spectrometer by measuring the change in fluorescence unit per min for 1 h at
380−460 nm. The fluorescence units for the vehicle control were set to 100%, and the ratio of
drug-treated or just oligomer-treated samples to the vehicle control was used to calculate the
relative enzymatic activity.
6.2.12 Proteasome-mediated degradation of α-synuclein oligomeric mixture
        Digestion of α-synuclein oligomer mixture was carried out in a 50 μL reaction volume
made of 50 mM Tris at pH 7.8; 0.33 μM α-synuclein oligomer mixture and 6.7 nM purified human
20S proteasome. Briefly, 20S proteasome was diluted to 7.58 nM in the reaction buffer.
Fluspirilene, N-acylated Fluspirilene or vehicle (1 μL of 50x stock or DMSO) were added to 44
μL of 7.58 nM 20S and incubated at 37 °C for 20 min. The substrate (5 μL of 3.3 μM synuclein
oligomer mixture) was then added to the reaction mixture and incubated at 37 °C for 24 h. The
reactions were then quenched with concentrated SDS loading buffer. Samples were resolved on a
4–20% Tris-glycine SDS-PAGE and immunoblotted with mouse monoclonal anti α-synuclein IgG
(1:2000) and anti-mouse HRP-linked IgG (1:2000). Blots were developed with ECL western
reagent and imaged with an Azure Biosystems 300Q imager.
6.2.13 Plasmid preparation
        Bacterial culture: E. coli were grown at 37 ºC in LB supplemented ampicillin (25 μg/mL).
        Plasmid purification: A53T α-synuclein plasmid was purified and prepared from E. coli
that had been transformed with the plasmid previously. Purification was done using a plasmid
maxi prep kit obtained from Qiagen and was performed following their provided protocol.
Resulting plasmid was diluted to ~1 µg/µL in the provided TE buffer for use in transfections.
                                               206


6.2.14 Quantification and statistical analysis
        Data are presented as mean ± standard deviation (SD). For each figure, the number of
replicates is indicated in the figure legends. Statistical analysis was only performed on experiments
with three or more n (biological replicates for cellular experiments or individual experiments for
biochemical assays). Western blot quantifications were performed with ImageJ software.
Statistical analysis was performed with GraphPad Prism 7 software. One-way ANOVA analysis
with post Šidák correction test was used for multiple comparisons of means. Effect was considered
significant for *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Confocal immunofluorescence (By Dr. Caryl Sortwell)
                                                   207