IMIDAZOLINE SCAFFOLDS AS SMALL MOLECULE INHIBITORS OF THE HUMAN
PROTEASOME
By
Lauren Marie Azevedo

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Pharmacology and Toxicology – Master of Science
2014

ABSTRACT
IMIDAZOLINE SCAFFOLDS AS SMALL MOLECULE INHIBITORS OF THE HUMAN
PROTEASOME
By
Lauren Marie Azevedo
In the past decade, modulating NF-κB activation through inhibition of the
proteasome has emerged as a therapeutic target for the treatment of malignancies and
autoimmune diseases. In 2003, the proteasome inhibitor bortezomib was approved by
the FDA for the treatment of the plasma cell neoplasm, multiple myeloma. Although
bortezomib effectively treats multiple myeloma and has shown promising results for the
treatment of these other diseases, its therapeutic potential is limited by peripheral
neuropathy, reduced activity in solid tumors, and route of administration restrictions. In
2004, the Tepe laboratory first identified the imidazoline scaffold, as an inhibitor of the
NF-κB pathway and subsequently identified the proteasome as its target. The studies
presented in this thesis show that the noncompetitive imidazoline proteasome inhibitor,
TCH-013, is bioavailable and not hepatotoxic. This compound was also shown to have
anti-inflammatory properties and to reduce the broncheoalveolar lavage fluid cellularity
in an experimental model of asthma. In addition, we enhanced the efficacy of this
molecule by manipulating the functional groups. The result of these studies is the
discovery of compound 43, a noncompetitive proteasome inhibitor that is able to
overcome bortezomib resistance, is bioavailable, and is effective in an animal model of
systemic inflammation. The data presented herein suggest that a modified imidazoline
scaffold is an effective, noncompetitive and nontoxic proteasome inhibitor that has
therapeutic potential for the treatment of multiple myeloma and asthma.

Copyright by
LAUREN MARIE AZEVEDO
2014

iii

For my father, John Azevedo, who introduced me to the magic of science and the
wonders of medicine.

iv

ACKNOWLEDGEMENTS

I would like to first thank my mentor, Dr. Jetze Tepe. Over the years, he has
spent countless hours helping me develop into an independent and focused researcher.
I appreciate his understanding and willingness to take a chance on a multi-disciplinary
student. Without his support, inside and outside of the lab, this work would not have
been possible.
I would also like to thank my committee members, Dr. Norbert Kaminski, Dr.
Patricia Ganey, and Dr. Anne Dorrance. They have been a strong support network
since my first interview and I sincerely appreciate all of the time and effort they have put
into my education and professional development.
I would like to thank the College of Osteopathic Medicine, the DO/PhD program
and the Department of Pharmacology and Toxicology at Michigan State for the
unfaltering support.

Special thanks goes to Dr. Justin McCormick, Ms. Bethany

Heinlen, Dr. Keith Lookingland, Dr. Anne Dorrance, and Dr. Celia Guro for taking
special interest in my career and personal development. I would also like to thank the
support staff for their help with everything over the years.
I would like to acknowledge the collaborators that made these projects possible.
First, to Dr. Bill Henry, Dr. Jack Harkema, Dr. Jim Wagner and Dr. John Lapres who
spent countless hours laying the groundwork for this research. To the lab members,
especially Dr. Steve Proper and Dr. Devan Jackson-Humbles who taught me the
techniques and supported me throughout the studies.

v

I would like to thank my lab members and friends who have given me so
much emotional and intellectual support throughout this process. I owe a deep
debt of gratitude to the Tepe Lab and its members for their help with chemistry,
and to Teri Lansdell who is an amazing person and mentor. I would especially
like to thank my closest friends, Nicole Hewlett, Sujana Pradhan, Mike Strug,
Emilia Boffi and Britany Brutsche for everything they have done to make this
possible.
Finally, I would like to thank my entire family for their unconditional love
and support. To my grandmother, you have inspired me to go above and beyond
whatever I thought I could achieve and I appreciate everything you have done for
our family throughout the years. To my mother and step-father, your love and
support throughout my entire life has made me the person that I am today. To
my husband and best friend, Ryan Keating, who believes in me no matter what, I
would have not made it through this without you.

vi

TABLE OF CONTENTS

LIST OF TABLES ...........................................................................................................ix
LIST OF FIGURES.......................................................................................................... x
KEY TO ABBREVIATIONS ........................................................................................... xii
CHAPTER 1- INTRODUCTION ...................................................................................... 1
The ubiquitin-proteasome system ............................................................................... 1
Identification of the imidazoline scaffold as an inhibitor of NF-κB through
modulation of the proteasome ..................................................................................... 3
The role of NF-κB in asthma and the proteasome inhibitor as potential steroid
sparing therapy ............................................................................................................. 4
Disruption of NF-κB signaling through proteasome inhibitors as treatment for
multiple myeloma .......................................................................................................... 5
CHAPTER 2- MATERIALS AND METHODS ................................................................. 9
Reagents ........................................................................................................................ 9
Cell culture..................................................................................................................... 9
Animals and husbandry................................................................................................ 9
OVA-induced allergic airway model .......................................................................... 10
Pulmonary function analysis ..................................................................................... 11
Cytokine bead array .................................................................................................... 11
Enzymatic activity assessment .................................................................................. 11
Cytotoxicity measurement ......................................................................................... 12
Development of bortezomib resistant cell lines ....................................................... 12
Bioavailabilty ............................................................................................................... 13
ALT assay .................................................................................................................... 14
LPS challenge .............................................................................................................. 14
ELISA............................................................................................................................ 14
Statistical analysis ...................................................................................................... 15
CHAPTER 3- RESULTS AND DISCUSSION ............................................................... 16
TCH-013 is bioavailable, non-toxic and disrupts the inflammatory cellular profile
of BALF but not airway hyperresponsiveness in an experimental model of
asthma.......................................................................................................................... 16
Modification of the imidazoline scaffold yielded a molecule with increased
efficacy and a noncompetitive binding profile ......................................................... 38
The modified imidazoline is cytotoxic and overcomes bortezomib resistance in
cell culture of multiple myeloma ................................................................................ 45
Compound 43 is bioavailable and attenuates the systemic inflammatory response
in vivo ........................................................................................................................... 49
vii

CHAPTER 4- SUMMARY AND CONCLUSIONS ......................................................... 53
Limitations and future directions............................................................................... 53
Conclusion ................................................................................................................... 54
BIBLIOGRAPHY ........................................................................................................... 56

viii

LIST OF TABLES

Table 1. The role of cytokines in the pathogenesis of asthma ................................ 28
Table 2. Statistical analysis of IL-5 concentration in BALF from select groups of
OVA or vehicle sensitized mice after treatment with 50 mg/kg TCH-013, vehicle or
0.1 mg/kg dexamethasone.......................................................................................... 30
Table 3. Statistical analysis of IL-13 concentration in BALF from select groups of
OVA or vehicle sensitized mice after treatment with 50 mg/kg TCH-013, vehicle or
0.1 mg/kg dexamethasone.......................................................................................... 32
Table 4. Statistical analysis of IL-6 concentration in BALF from select groups of
OVA or vehicle sensitized mice after treatment with 50 mg/kg TCH-013, vehicle or
0.1 mg/kg dexamethasone.......................................................................................... 34
Table 5. Statistical analysis of KC concentration in BALF from select groups of
OVA or vehicle sensitized mice after treatment with 50 mg/kg TCH-013, vehicle or
0.1 mg/kg dexamethasone.......................................................................................... 36
Table 6. Inhibition of the chymotryptic-like activity of purified human 20S
proteasome by compounds 11-33 ............................................................................. 41
Table 7. Inhibition of the chymotryptic-like activity of purified human 20S
proteasome by compounds 34-46 ............................................................................. 43

ix

LIST OF FIGURES

Figure 1. Chemical structures of the proteasome inhibitors TCH-013 and
bortezomib ..................................................................................................................... 7
Figure 2. The classical and alternative pathways of NF-κB ....................................... 8
Figure 3. Serum concentrations of TCH-013 in BALB/c mice at 1, 3, and 12 hours
after IP administration of drug at 150 mg/kg ............................................................ 21
Figure 4. TCH-013 reduces LPS-induced TNF-α production ................................... 22
Figure 5. Total cell counts in BALF from OVA or vehicle sensitized mice after
treatment with 50 mg/g TCH-013, saline or 0.1 mg/kg dexamethasone.................. 23
Figure 6. Eosinophil count in BALF from OVA or vehicle sensitized mice after
treatment with 50mg/kg TCH-013, saline or 0.1mg/kg dexamethasone.................. 24
Figure 7. Lymphocyte count in BALF from OVA or vehicle sensitized mice after
treatment with TCH-013, saline or dexamethasone.................................................. 25
Figure 8. Neutrophil count in BALF from OVA or vehicle sensitized mice after
treatment with 50 mg/kg TCH-013, saline or 0.1 mg/kg dexamethasone................ 26
Figure 9. Monocyte count in BALF from OVA or vehicle sensitized mice after
treatment with 50 mg/kg TCH-013, saline or 0.1 mg/kg dexamethasone................ 27
Figure 10. IL-5 concentration in BALF from OVA or vehicle sensitized mice after
treatment with 50 mg/kg TCH-013, vehicle or 0.1 mg/kg dexamethasone.............. 29
Figure 11. IL-13 concentration in BALF from OVA or vehicle sensitized mice after
treatment with TCH-013, vehicle or dexamethasone................................................ 31
Figure 12. IL-6 concentration in BALF from OVA or vehicle sensitized mice after
treatment with TCH-013, vehicle or dexamethasone................................................ 33
Figure 13. KC concentration in BALF from OVA or vehicle sensitized mice after
treatment with TCH-013, vehicle or dexamethasone................................................ 35
Figure 14. Total lung resistance after methacholine challenge in OVA or vehicle
sensitized mice after treatment with TCH-013, saline or dexamethasone ............. 37
Figure 15. Dose response curve showing the change in velocity of Suc-LLVYAMC hydrolysis at variable concentrations of TCH-013 ......................................... 40
x

Figure 16. Kinetic analysis of CT-L activity inhibition by compound 43 ................ 44
Figure 17. Cell viability of THP-1 wild type cells after 72 hours of treatment with 10
µM imidazoline ............................................................................................................ 47
Figure 18. Cell viability of THP-1 BTZ500 cells after 72 hours of treatment with 10
µM imidazoline or 1 µM bortezomib........................................................................... 48
Figure 19. Compound 43 is present in the serum after IP administration at 150
mg/kg............................................................................................................................ 51
Figure 20. Compound 43 reduces LPS-induced TNF-α production. ....................... 52

xi

KEY TO ABBREVIATIONS

AHR

airway hyperresponsiveness

ALT

alanine aminotransferase

AMC

aminomethylcoumarin

ANOVA

analysis of variance

BALF

broncheoalveolar lavage fluid

BBB

blood brain barrier

BTZ500

bortezomib resistant THP1 cells

CO2

carbon dioxide

D5W

5% dextrose in water

DILI

drug induced liver injury

DMSO

dimethylsulfoxide

ELISA

enzyme linked immunosorbent assay

FDA

United States Food and Drug Administration

h

hours

IC50

inhibitory concentration 50%

ICAM

intracellular adhesion molecule

Ig

immunoglobulin

IFN

interferon

IκB

inhibitor of kappa B

IκK

inhibitor of kappa kinase

IL

interleukin

IP

intra-peritoneal
xii

KC

keratinocyte chemoattractant

KM

Michaelis constant

LC/MS

liquid chromatography/mass spectrometry

LPS

lipopolysaccharide

MCL

mantle cell lymphoma

MM

multiple myeloma

NF-κB

nuclear factor kappa B

OVA

ovalbumin

RA

rheumatoid arthritis

S

svedberg unit

[S]

substrate concentration

SEM

standard error of the mean

TNF-α

tumor necrosis factor alpha

VCAM

vascular cell adhesion molecule

Vmax

maximum velocity

WT

wild type

xiii

CHAPTER 1- INTRODUCTION
The ubiquitin-proteasome system
A key component of biological homeostasis is the targeted degradation of
1

intracellular proteins by the ubiquitin-proteasome system.

Mutated and misfolded

proteins, as well as those involved in inflammation, gene expression, and cell division
are just a few examples of proteins regulated by this degradation mechanism.

2,3

The human proteasome is a multi-subunit protein complex responsible for the
4

regulation of protein homeostasis through degradation.

The barrel-like structure of the

26S proteasome consists of two 19S regulatory caps that flank four stacked rings of the
20S catalytic core. The 19S cap is a six membered ring consisting of AAA-ATPases
that are responsible for unfolding and recognizing ubiquitinated proteins.

5

These

processed proteins are then shuttled into the center of the 20S catalytic core. The
outermost parts of the 20S unit are composed of the seven membered α-rings, which
are responsible for interaction with the 19S regulatory caps. The center of the barrellike structure is formed by two β-rings that consist of seven subunits each, three of
which are responsible for proteolysis.

6

The unfolded proteins encounter a catalytic

threonine on the β1, β2, or β5 subunit where they undergo an amide cleavage reaction
and are broken down.

7

Each of these subunits has a unique cleavage profile, the β1

has caspase-like activity, the β2 has trypsin-like activity and the β5 has chymotrypsinlike activity.

Disruption of the peptidase activity of these subunits is the target of
8,9

proteasome inhibition.

1

In 2003, the FDA approved bortezomib, the first in class proteasome inhibitor
shown in Figure 1, for the treatment of multiple myeloma (MM).

Since that time,

bortezomib has been approved for the treatment of refractory mantle cell lymphoma
(MCL) and second generation proteasome inhibitors have been developed as
10,11

treatments for malignancies and auto-immune disorders.

Unfortunately, there are

several drawbacks to bortezomib therapy, including dose-limiting peripheral neuropathy,
limited activity in solid tumors, resistance and route of administration restrictions.

12,13

It

has been suggested that alteration of the pharmacokinetics and pharmacodynamics of
proteasome inhibitors will alleviate the restrictions of bortezomib therapy.

14,15

The keystone of the mechanism of bortezomib therapy is the role the proteasome
plays in NF-κB activation.

16,17

Although NF-κB activation can be initiated through two

different pathways, classical and alternative, a common event in these signaling
cascades is the proteasomal degradation of inhibitory proteins (Figure 2).

18,19

Activation of NF-κB has been implicated as a significant promoter of pro-survival/proinflammatory signaling in many different types of cancers and autoimmune
diseases.

20,21

In the classical pathway, illustrated on the left portion of Figure 2, the active
components of NF-κB exist as a complex with an inhibitory protein, IκBα.

Upon

response to the principal activators, LPS, IL-1, or TNF-α, IκKβ is activated and
phosphorylates IκBα which leads to ubiquitination, marking it for proteasomal
degradation.

22,23

When the NF-κB complex encounters the proteasome, the IκB

2

protein is degraded, liberating the active subunits (p65/p50), which then translocate into
the nucleus and initiate transcription of NF-κB mediated genes responsible for innate
immunity, cell survival, and inflammation.

24

The alternative pathway, shown on the right side of Figure 2, which mediates
adaptive immunity and lymphoid development, follows a similar pathway. Activation of
the CD40, LT-β, or BAFF receptors activates IκKα which phosphorylates p100. The
ubiquitination and processing of p100 follows, resulting in translocation of p52/ReB into
the nucleus and subsequent transcription of target genes.

22

The impact of proteasome inhibition on NF-κB signaling results in both antiinflammatory and anti-proliferative effects.

9

As of 2010, all clinically evaluated

proteasome inhibitors exhibited a competitive mechanism of action via irreversible
covalent binding to the catalytic threonine of the β5 subunit.

25,26

This binding profile

results in toxic accumulation of intracellular proteins and disruption of NF-κB activation
in both healthy and malignant cells.

17

Identification of the imidazoline scaffold as an inhibitor of NF-κB through
modulation of the proteasome
In 2004, the same year that Aaron Ciechanover, Avram Hersheko and Irwin Rose
were awarded the Nobel Prize for their discovery of the proteasome, the Tepe
laboratory identified the molecule TCH-013 as a potent inhibitor of NF-κB signaling
(Figure 1).

27,28

Subsequent studies involving this molecule, including kinetic analysis

3

and evaluation of bortezomib cross-resistance, identified the mechanism of action as
noncompetitive proteasome inhibition.

28,29

The unique mechanism of action has attractive therapeutic potential for
autoimmune and malignant diseases. Models of rheumatoid arthritis (RA) and MM in a
xenograft setting were employed to evaluate the effectiveness of this molecule in
vivo.

29,30

In cell culture, TCH-013 was found to induce apoptosis in MM cells, but

exhibited limited cytotoxicity against primary bone marrow stromal cells.

29

Encouraging

results, such as reduced tumor burden and RA disease score, showed efficacy against
both disease states. In addition to inhibiting disease progression, treatment exhibited
limited toxicity, observed as no apparent weight loss or increase in serum alanine
aminotransferase (ALT).

30

These studies indicate that a noncompetitive proteasome

inhibitor is effective in animal models with limited toxicity and have opened a window for
the development of more potent analogs of these molecules and application to other
diseases.

The role of NF-κB in asthma and the proteasome inhibitor as potential steroid
sparing therapy
Asthma, a disease characterized by wheezing, coughing, and shortness of
breath, affected over 24 million Americans in 2009.

31

Steroids remain the primary

therapy for the treatment of this disease; however, the extensive side effects associated
with these drugs (weight gain, indigestion, osteoporosis, and glucose intolerance) have
encouraged

the medical community to search
4

for adjunctive

or alternative

therapies.

32,33

NF-κB has emerged as an attractive therapeutic target for the treatment

of asthma, due to the regulation of many important immunomodulatory and
proinflammatory genes involved in the pathogenesis of the disease.

34,35

Of particular

importance is the role of NF-κB in the recruitment and activation of eosinophils, a
process which has been identified as an important early in the development of
36

asthma.

Attenuation of NF-κB in allergic airway disease has been achieved with mixed
results by administration of proteasome inhibitors, oligonucleotides, mutation of IκBα,
33,37

and mutation of IκK.

Interestingly, administration of a proteasome inhibitor

significantly reduced pulmonary eosinophilia but did not appear to disrupt airway
38,39

remodeling or hyperresponsiveness.

Disruption of NF-κB signaling through proteasome inhibitors as treatment for
multiple myeloma
In 2009, approximately 20,500 individuals were diagnosed with multiple
myeloma, a plasma cell neoplasm with a five year relative survival rate of only 35% and
a sustained remission rate of 3%.

40,41

NF-κB, which is dysregulated in MM, controls

transcription of many of the key pathogenetic factors, including IL-6, ICAM-1, VCAM1.

18,42

Inhibition of the NF-κB pathway, and these mediators, has been achieved by

the aforementioned proteasome inhibitor bortezomib.

43,44

Despite successful

disruption of the NF-κB pathway and amelioration of disease pathogenesis, bortezomib
5

has limited clinical potential due to its narrow therapeutic window, the development of
resistance mechanisms and limited bioavailability.

12,45

The chronic treatment of the human leukemia cell line, THP-1, with bortezomib
lead to development of a proteasome inhibitor resistant strain.

46

A mutation in the

binding site of bortezomib and overexpression of the β5 subunit was identified through
47

genetic and proteomic analysis to be the mechanism for developed resistance.

Recent studies indicate that noncompetitive proteasome inhibitors are able to overcome
resistance by circumventing direct interaction with the mutated and overexpressed β5
subunit.

25,29

The development of non-toxic proteasome inhibitors for the treatment of
malignancies and autoimmune disorders is crucial. The data presented herein suggests
that a modified imidazoline scaffold is an effective and non-toxic proteasome inhibitor
that can overcome bortezomib resistance and has therapeutic potential for the
treatment of multiple myeloma and inflammatory conditions.

6

N

O

HN

N

N

OH

NH
N

O

O O

B
OH

TCH-013

bortezomib

Figure 1. Chemical structures of the proteasome inhibitors TCH-013 and
bortezomib.

7

Figure 2. The classical and alternative pathways of NF-κB. Under normal
conditions, NF-κB is bound to the IκB inhibitory proteins in the cell cytoplasm.
Extracellular signals begin the activation process by inducing phosphorylation of IκB.
The phosphorylated IκB proteins are then ubiquitinilyated and subsequently degraded
by the proteasome. This process releases the active NF-κB which then translocates
into the nucleus. The classical activation pathway, on the left of this figure, is
represented with TNF signaling. The alternative pathway, on the right of this figure, is
represented with LT-β signaling. Reprinted by permission from Macmillan Publishers
48
Ltd: NATURE IMMUNOLOGY, copyright 2011.

8

CHAPTER 2- MATERIALS AND METHODS
Reagents
Bortezomib was purchased from Selleck Biochemicals (Houston, TX), fluorogenic
substrates and purified proteasome particles from Boston Biochem (Cambridge, MA).
All antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA) or
Cell Signaling Technology (Danvers, MA) and ELISA kits from R&D Systems
(Minneapolis, MN).

Cell culture media and supplements were purchased from Life

Technologies (Grand Island, NY), all other chemicals were purchased from SigmaAldrich (St. Louis, MO).

Cell culture
The human cell line RPMI-8226 was purchased from American Type Culture
Collection (Manassas, VA); THP-1 and THP-1/BTZ500 were a kind gift from Dr.
Oerlemans of the VU University Medical Center, Amsterdam, The Netherlands.47 Cell
lines were maintained in RPMI-1640 media supplemented with 10% fetal bovine serum,
100 U/mL penicillin, 100 mg/mL streptomycin, 1 mM sodium pyruvate, and 0.2 mM Lglutamine. Cells were cultured at 37 ̊C, 5% CO2.

Animals and husbandry
Female, immune competent BALB/c mice were bred in the McCormick laboratory
at Michigan State University or purchased from Charles River (Wilmington, MA). The
animals were given access to water and food (Teklad 7904) ad libitum and were
9

maintained in a pathogen free environment. Animal rooms were maintained at 21-24º C
with 40-55% relative humidity, and were set on a 12 hour light/dark cycle.

All

procedures involving animals were performed with approval from the Michigan State
University Institutional Animal Care and Use Committee in accordance with all policies
and guidelines.

OVA-induced allergic airway model
Induction of allergic airway disease was achieved as previously described.

49

In

short, mice were randomly assigned to either vehicle or OVA treatment groups. The
vehicle group received an IP injection of 250 µL saline, while the OVA group received
OVA/alum (20 µg/1 mg in 250 µL saline).

Ten days after sensitization, OVA animals

were injected with a boost of OVA (20 µg in 250 µl saline) and vehicle animals received
saline alone. One week following the boost, animals were challenged with OVA or
saline via inhalation for three consecutive days. Animals were administered vehicle
(30:70 propylene glycol:D5W), TCH-013 (50 mg/kg), or dexamethasone (0.1 mg/kg),
one hour prior to each nebulizer challenge. Pentobarbital sodium (50 mg/kg) was used
to anesthetize mice 48 hours after final challenge, after which a midline laparotomy was
performed. Broncheoalveolar lavage fluid (BALF) was collected and analyzed for total
and differential cell counts using a hemacytometer and Diff-Quick reagent (Baxter,
Deerfield, IL).

10

Pulmonary function analysis
Mice were anesthetized with an IP injection of pentobarbital sodium (50 mg/kg)
after which time they were subsequently intubated, and ventilated with a small animal
ventilator (SAV, flexiVent; SCIREQ, Montreal, Quebec, Canada). Ventilator settings
were set at an initial frequency of 120–150 respirations/minute and at a volume of 1.5
mL/kg.

An Aeroneb nebulizer (Aerogen) was used to administer variable doses of

methacholine, and flexiVent pulmonary function testing system was used to measure
total lung resistance (SnapShot-150; SCIREQ).

Following data collection, the mice

were euthanized and BALF was collected as described above.

Cytokine bead array
Cytokine content of BALF was analyzed using Cytometric Bead Array kits and
reagents (BD Bioscience, Franklin Lakes, NJ) according to manufacturer's suggested
protocol. In brief, BALF samples are analyzed using a flow cytometer after they have
been mixed with the cytokine-specific antibody coated beads. The presence of specific
cytokines is based on the specific fluorescent signature of each bead.

Samples

supplied by the manufacturer were used to calculate standard curves for each cytokine.

Enzymatic activity assessment
The fluorogenic substrate Suc-LLVY-AMC was used to measure chymotrypticlike proteasome activities using 1 nM purified 20S proteasome.

50

The rate of cleavage

of fluorogenic peptide substrates was determined by monitoring the fluorescence of
released aminomethylcoumarin (AMC) using a SpectraMax M5e multiwell plate reader
11

at an excitation wavelength of 380 nm and emission wavelength of 460 nm.
Fluorescence was measured at 37̊ C every minute over a 30 minute period and the
maximum increase in fluorescence per minute was used to calculate specific activities
of each sample. IC50 was calculated by non-linear regression analysis using GraphPad
prism software (La Jolla, CA).

KM, and Vmax values were calculated from

Michaelis−Menten analysis using variable concentrations of Suc-LLVY-AMC and
imidazoline.

Cytotoxicity measurement
Cell viability was assayed 72 hours after drug treatment using the CellTiter 96®
AQueous One Solution Cell Proliferation Assay (Promega Corporation, Madison WI)
according to manufacturer specifications. Drugs were dissolved in DMSO and added to
cell culture to reach a final concentration of 10 µM or 5 µM drug and 0.5% DMSO. The
absorbance of formazan was measured at 490 nm on a SpectraMaxM5e microplate
reader. Background absorbance was subtracted from each measurement and viability
was calculated using the vehicle control as 100%. Drug potency was expressed as
IC50 and was calculated by non-linear regression analysis using GraphPad prism
software (La Jolla, CA).

Development of bortezomib resistant cell lines
The human cell line THP-1 cells were a kind gift from Dr. Oerlemans of the VU
University Medical Center, Amsterdam, The Netherlands.

12

Cells were maintained in

RPMI-1640 media supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100
mg/mL streptomycin, 1 mM sodium pyruvate, and 0.2 mM L-glutamine. Cells were
cultured at 37° C, 5% CO2. BTZ500 cells were maintained in complete media with 500
nM bortezomib and were given a 72 hours washout period before the cell viability
assay. Drugs were dissolved in DMSO and added to cells to reach a final concentration
of 10 µM drug and 0.5% DMSO. Cytotoxicity was assayed after 72 hours as described
above.

Bioavailabilty
To determine bioavailability of the compounds, liquid crystal mass spectroscopy
29

(LC/MS) was performed on serum samples as previously described.

In short: female,

BALB/c mice received 50 mg/kg of drug in 30% propylene glycol: D5W via
intraperitoneal injection. Blood was drawn via saphenous vein collection into 75mm
hematocrit tubes (Drummond; Broomall, PA) at 1, 3, and 6 hours post imidazoline
injection.

Isolated serum was processed and supernatant was transferred to a

polypropylene microplate.

LC/MS analysis to determine drug concentration was

performed in the MSU Mass Spectrometry Core under the direction of Dr. Dan Jones
using a Waters LCT Premier mass spectrometer in conjunction with a Waters Atlantis
dC18 column. Positive mode electrospray ionization was used to analyze supernatant
volumes injected in concentrations of 10 µL. Purified drug standards were analyzed to
determine retention times and response factors.

13

ALT assay
To assess hepatotoxicity, serum was collected from animals treated with an
extended dosing regimen of TCH-013 to assess hepatotoxicity after subacute exposure
(13-19 days).

Animals were treated with vehicle or TCH-013 (50 mg/Kg and 150

mg/Kg) via intraperitoneal injection. Drug was administered on days 1-5, 8-12, and 1519; on days 6-7, and 13-14 animals were not treated. The animals were sacrificed
when distress was observed by laboratory staff according to IACUC guidelines; length
of treatment ranged from 13-19 days. Blood was collected via cardiac puncture and
serum samples were analyzed for ALT according to manufacturer specifications using
Infinity™ ALT reagent (Thermo Fisher Scientific, Middletown, VA).

LPS challenge
Immune competent BALB/c mice were treated with one IP injection of TCH-013
(50 mg/kg in 30:70 propylene glycol:D5W) 1 hour prior to dosing with 1 mg/kg LPS
(E.coli -055B5). Animals were sacrificed 2 hours after LPS administration and blood was
collected by cardiac puncture. Serum was collected and analyzed for TNF-α using an
ELISA procedure.

ELISA
The presence of TNF-α in serum was assessed using the commercially available
mouse TNF-α Quantikine ELISA Kit (R&D Systems, Minneapolis, MN). ELISA was
performed according to manufacturer specifications. Blood was collected as described
above following LPS challenge protocol.

14

Statistical analysis
Statistical analysis was performed using GraphPad Prism for Windows,
GraphPad Software, San Diego California USA, www.graphpad.com.
All data are presented as mean ± standard error of the mean (SEM). For the
comparison of two groups, statistical significance was defined by a p value of 0.05 or
less as determined by Student’s t test. A one-way ANOVA with post-hoc analysis using
Tukey’s multiple comparison test was used to determine the significance between
multiple groups unless otherwise noted.

15

CHAPTER 3- RESULTS AND DISCUSSION
TCH-013 is bioavailable, non-toxic and disrupts the inflammatory cellular profile
of BALF but not airway hyperresponsiveness in an experimental model of asthma
Prior to evaluation in an animal model of experimental asthma, TCH-013 was
evaluated for bioavailability and toxicity in vivo. Immune competent BALB/c mice were
treated with a single dose of TCH-013 (150 mg/kg), administered intraperitoneally, in a
vehicle comprised of 30:70 propylene glycol:D5W. Serial blood draws at 1, 3, and 12
hours were performed to determine the level of TCH-013 in the serum after dosing.
Figure 3 represents LC/MS analysis of the serum obtained; indicating that IP
administration of the compound produces blood levels in the micromolar range (n > 3).
Studies to determine organ distribution, blood brain barrier (BBB) permeability,
metabolism, and elimination characteristics are needed to further investigate the
pharmacokinetic properties of TCH-013.
Although no significant weight loss was reported with administration of TCH-013,
further assessment of the compounds toxicity was warranted.

30

Discontinuation of

candidate during the drug development process is most commonly caused by drug
induced liver injury (DILI).

51

The standard biomarker used to monitor DILI, alanine

aminotransferase (ALT), was measured in animals receiving IP TCH-013 at either 50
mg/kg (BID for 19 days) or 150 mg/kg (BID for 13 days) to assess the compounds liver
toxicity.

30,51

These studies found no significant increase in serum ALT between the

treatment groups and the vehicle groups (p > 0.05, n ≥ 4) indicating TCH-013 is not
hepatotoxic, as measured by serum ALT.
16

Further studies including liver histology,

organ function, and evaluation of carcinogenesis are necessary to conclude that this
compound has a clinically acceptable toxicity profile.
The inflammatory cytokine, TNF-α, has been implicated as an important mediator
52

in the pathogenesis of asthma.

In order to determine if TCH-013 could ameliorate

inflammatory response in vivo and if there was indication for application in the treatment
of asthma, a lipopolysaccharide (LPS) model of systemic inflammation monitored by
serum TNF-α was employed.

LPS has been used previously to produce a robust

inflammatory response that can be monitored by serum TNF-α.

30

Immune competent

BALB/c mice were treated with an IP injection of 50 mg/kg TCH-013 one hour prior to
administration of 1 mg/kg LPS. Two hours after LPS administration, animals were
sacrificed and serum was collected.

Figure 4 illustrates that TCH-013 significantly

reduced serum TNF-α in response to LPS when measured by ELISA (p < 0.05, n > 4).
TCH-013 was found, through LC/MS analysis, to be present in the serum of these
animals in concentrations of 4.6 ± 1.0 μM. The results of this study indicate that these
compounds have in vivo efficacy against systemic inflammation and thus may be useful
in an experimental model of asthma.
Cellular infiltrate and resultant cellular response are considered chief mediators
in the pathogenesis of asthma, their unique roles are outlined in Table 153-55. Analysis
of the bronchoalveoar lavage fluid (BALF) indicates that treatment with TCH-013
decreases the presence of inflammatory cells.

BALF was collected immediately

following the flexi-vent procedure 48 hours after the last OVA or saline challenge and
total and differential cell counts were obtained.

Figures 5-9 illustrate that OVA

sensitized mice treated with vehicle exhibited a significant increase in total cells,
17

eosinophils, neutrophils and lymphocytes, when compared to vehicle control (p < 0.05,
n ≥ 5). Dexamethasone significantly attenuated this response in total cells and in all of
the inflammatory cell types, and TCH-013 reduces the BALF concentrations of total
cells, macrophages, eosinophils and lymphocytes (Figures 5-9, p < 0.05, n ≥ 5). Since
eosinophils are considered a hallmark of allergic airway disease by causing epithelial
damage and airway hyperresponsiveness (AHR), the attenuation of their response
indicates potential clinical application for these compounds.

53,54

In addition to inflammatory cell quantification, cytokine evaluation of the BALF
was also employed. Cytokines are secreted proteins that are responsible for many
56

components of the immune response, especially cell trafficking and activation .
Inflammatory cytokines important to the pathogenesis of asthma were measured in the
BALF using the BD Cytometric Bead Array (CBA).

57

IL-4, IFN-γ, and TNF-α were

measured but were below the limit of detection. Figures 10-13 show that ovalbumin
sensitization induced a significant increase in IL-5, IL-6, IL-13 and KC in the vehicle
treated animals (p > 0.05, n ≥ 7). Neither TCH-013 nor dexamethasone treatment
significantly reduced BALF cytokine levels.

A complex statistical analysis was

completed on these samples using ANOVA with Bonferroni’s multiple comparison test,
ANOVA with Tukey’s multiple comparison test, and with Student’s t-test (Tables 2-5). A
plausible explanation for the apparent disconnect between the BALF cellular profile and
the BALF cytokine profile, is the time point at which the BALF was taken. At 48 hours
after final ovalbumin challenge, many of the cytokines will be well beyond their peak
response, measurements of these cytokines at various time points would yield a better

18

58

picture of the cytokine profiles.

These studies did not evaluate the presence of cells

in the lung parenchyma; if their properties and numbers were not affected by the drug
treatments, they could be responsible for secreting cytokines as well.
To assess the effect of TCH-013 on airway hyperresponsiveness, mice were
mechanically ventilated 48 hours after final OVA challenge and total lung resistance
was measured.

Ovalbumin sensitized mice treated with vehicle exhibited airway

hyperresponsiveness characterized by an increase in total lung resistance after
treatment with methacholine (p < 0.05, n ≥ 5).

Treatment with TCH-013 or

dexamethasone did not significantly affect airway hyperresponiveness in ovalbumin
sensitized mice or in the control mice (Figure 14). This result was unexpected because
of the robust decrease in inflammatory cells found in the BALF. There has been some
debate in the literature about the relationship between NF-κB to the cellularity of the
33,37

lavage fluid and airway remodeling in asthma.

In previous studies with TCH-013, it

was shown that the compound lacks cytotoxic activity against bone marrow stromal
cells.

29

It is possible that the inflammatory cells retained their ability to induce AHR and

that the decreased number of cells was enough to initiate the pathogenic process.
Co-treatment with TCH-013 and a steroid, such as dexamethasone, would be
necessary to determine if these proteasome inhibitors could be used as an adjunctive
steroid sparing therapy. A more thorough analysis with particular attention paid to the
relationship of the inflammation and pathogenesis to NF-κB inhibition via the
proteasome is needed to determine the clinical potential for the imidazolines in allergic
airway disease.

19

More recent studies involving bortezomib in an experimental model of asthma
exhibited similar results to our studies, showing a decrease in cellularity without
resultant changes in airway remodeling. These studies focused on the plasma cell
depleting and IgE modulating effects of bortezomib.

38

In future studies involving TCH-

013, these factors should be monitored so that the proteasome inhibitors can be
compared across studies.

20

1.0

0.5

12

3

0.0

1

TCH-013 Concentration (µM)

1.5

hours post IP injection

Figure 3. Serum concentrations of TCH-013 in BALB/c mice at 1, 3, and 12 hours
after IP administration of drug at 150 mg/kg (n >3). Animals were given a single IP
dose of TCH-013 in 30:70 propylene glycol:D5W and blood was drawn at 1, 3, and 12
hours post administration. LC/MS analysis was performed on the samples to determine
the quantity of drug present.

21

TNF-α (pg/mL)

2500
2000
1500

*

1000
500

-0
13

LP
S

+

+

TC

H

Ve
hi
cle

S
LP
LP
S

TC

H

-0
13

0

Figure 4. TCH-013 reduces LPS-induced TNF-α production. Animals were given an
IP dose of 50 mg/kg TCH-013, followed one hour later by an IP dose of 1 mg/kg LPS,
and two hours later serum was collected. Concentration of TNF-α in serum was
measured using an ELISA. Serum concentrations of TNF-α after LPS challenge were
significantly reduced by 50mg/kg IP dose of TCH-013 when compared to LPS + vehicle
(p < 0.05, n = 5).

22

600000

cells/mL

+
400000

200000

*

*

ve
hi
cl
e

ve
hi
cl
e

+

ve
hi
ve
cl
hi
e
c
+
le
de
+
xa
01
m
3
et
ha
so
O
VA
ne
+
ve
hi
O
cl
VA
O
e
V
+
A
de
+
xa
01
m
3
et
ha
so
ne

0

Figure 5. Total cell counts in BALF from OVA or vehicle sensitized mice after
treatment with 50 mg/g TCH-013, saline or 0.1 mg/kg dexamethasone. Treatment
with OVA induced a robust and statistically significant increase in total cells (+p < 0.05,
n =8). In OVA sensitized mice, treatment with TCH-013 or dexamethasone significantly
reduced total cellularity of BALF when compared to vehicle (*p < 0.05, n ≥ 7).

23

250000

cells/mL

200000

+

150000
100000
50000

*
*

ve
hi
cl

ve
hi
cl

e

+

ve
hi
ve
cl
hi
e
e
c
+
l
e
de
+
xa
01
m
3
et
ha
so
O
VA
ne
+
ve
hi
O
cl
VA
O
e
V
+
A
de
+
xa
01
m
3
et
ha
so
ne

0

Figure 6. Eosinophil count in BALF from OVA or vehicle sensitized mice after
treatment with 50mg/kg TCH-013, saline or 0.1mg/kg dexamethasone. Treatment
with OVA induced a robust and statistically significant increase in total cells (+p < 0.05,
n = 7). In OVA sensitized mice, treatment with TCH-013 or dexamethasone significantly
reduced eosinophilia of BALF when compared to vehicle (*p < 0.05, n ≥ 7).

24

cells/mL

9000

+

6000

*
3000

*

ve
hi
cl
e

ve
hi
cl
e

+

ve
hi
ve
cl
hi
e
c
+
le
de
+
xa
01
m
3
et
ha
so
O
VA
ne
+
ve
hi
O
cl
VA
O
e
V
+
A
de
+
xa
01
m
3
et
ha
so
ne

0

Figure 7. Lymphocyte count in BALF from OVA or vehicle sensitized mice after
treatment with 50 mg/kg TCH-013, saline or 0.1 mg/kg dexamethasone. Treatment
with OVA induced a robust and statistically significant increase in total cells (+p < 0.05,
n = 7). In OVA sensitized mice, treatment with TCH-013 or dexamethasone significantly
reduced lymphocytosis of BALF when compared to vehicle (*p < 0.05, n ≥ 6).

25

12000

cells/mL

+
8000

*

4000

ve
hi
cl
e

ve
hi
cl
e

+

ve
hi
ve
cl
hi
e
cl
+
e
de
+
xa
01
m
3
et
ha
so
O
VA
ne
+
ve
hi
O
cl
VA
O
e
V
+
A
de
+
xa
01
m
3
et
ha
so
ne

0

Figure 8. Neutrophil count in BALF from OVA or vehicle sensitized mice after
treatment with 50 mg/kg TCH-013, saline or 0.1 mg/kg dexamethasone. Treatment
with OVA induced a robust and statistically significant increase in total cells when
compared to vehicle alone (+p < 0.05, n = 7). In OVA sensitized mice, treatment with
TCH-013 or dexamethasone significantly reduced neutrophilia of BALF when compared
to vehicle (*p < 0.05, n ≥ 6).

26

200000

+
cells/mL

150000
100000
50000

ve
hi
cl
e

ve
hi
cl
e

+

ve
hi
ve
cl
hi
e
c
+
le
de
+
xa
01
m
3
et
ha
so
O
VA
ne
+
ve
hi
O
cl
VA
O
e
V
+
A
de
+
xa
01
m
3
et
ha
so
ne

0

Figure 9. Monocyte count in BALF from OVA or vehicle sensitized mice after
treatment with 50 mg/kg TCH-013, saline or 0.1 mg/kg dexamethasone. Treatment
with OVA induced a robust and statistically significant increase in total cells (+p < 0.05,
n = 7). There was not a significant decrease in the BALF monocytes of the OVA
sensitized mice treated with TCH-013 or dexamethasone BALF when compared to
vehicle (p > 0.05, n ≥ 5).

27

Cytokine

Role

IL-4

Eosinophil growth
Increase TH2 cells
Increase IgE

IL-5

Eosinophil maturation
Inhibits apoptosis
Increase TH2 cell
AHR

IL-6

T and B Cell growth factor
Increase IgE

IL-13

Eosinophil activation
Inhibition of apoptosis
Increase IgE

IFN-γ

Activate endothelial
macrophages

KC

Recruitment of macrophages and neutrophils

TNF-α

Activate epithelium, endothelium, APC, monocytes and
macrophages

cells,

AHR

Table 1. The role of cytokines in the pathogenesis of asthma.

28

epithelial

cells,

and

10

IL-5 (pg/mL)

8

+

6
4
2

ve
hi
cl
e
ve
+
ve
h
ve
ic
hi
hi
l
e
cl
cl
+
e
e
T
+
C
de
H
-0
xa
13
m
et
ha
so
O
VA
ne
+
ve
O
VA
hi
O
cl
VA
+
e
T
+
C
de
H
-0
xa
13
m
et
ha
so
ne

0

Figure 10. IL-5 concentration in BALF from OVA or vehicle sensitized mice after
treatment with 50 mg/kg TCH-013, vehicle or 0.1 mg/kg dexamethasone.
Treatment with OVA induced a statistically significant increase in IL-5 (+p < 0.05, n ≥ 8).
However, treatment with neither TCH-013 nor dexamethasone decreased IL-5
concentration in the BALF (p > 0.05, n ≥ 7).

29

ANOVA
with ANOVA with Tukey’s T test,
Bonferroni’s multiple multiple comparisons P value
comparisons
test, test, adjusted P value
adjusted P value
OVA + vehicle vs. OVA
+ TCH-013

0.9497

0.9497

0.5196

OVA + vehicle vs. OVA
+ dexamethasone

0.9987

0.9987

0.7519

OVA + TCH-013 vs.
0.9996
OVA + dexamethasone

0.9996

0.8264

Table 2. Statistical analysis of IL-5 concentration in BALF from select groups of
OVA or vehicle sensitized mice after treatment with 50 mg/kg TCH-013, vehicle or
0.1 mg/kg dexamethasone (n ≥ 7).

30

IL-13 (pg/mL)

8
6

+

4
2

ve
hi
cl
e
ve
+
ve
h
ve
ic
hi
hi
l
e
cl
cl
+
e
e
T
+
C
de
H
-0
xa
13
m
et
ha
so
O
VA
ne
+
ve
O
VA
hi
O
cl
VA
+
e
T
+
C
de
H
-0
xa
13
m
et
ha
so
ne

0

Figure 11. IL-13 concentration in BALF from OVA or vehicle sensitized mice after
treatment with 50 mg/kg TCH-013, vehicle or 0.1 mg/kg dexamethasone.
Treatment with OVA induced a statistically significant increase in IL-13 (+p < 0.05, n ≥
8). However, treatment with neither TCH-013 nor dexamethasone decreased IL-13
concentration in the BALF (p > 0.05, n ≥ 7).

31

ANOVA
with ANOVA with Tukey’s T test
Bonferroni’s multiple multiple comparisons
comparisons
test, test, adjusted P value
adjusted P value
OVA + vehicle vs. OVA
+ TCH-013

0.1478

0.0977

0.0456

OVA + vehicle vs. OVA
+ dexamethasone

>0.9999

0.9700

0.6276

OVA + TCH-013 vs.
>0.9999
OVA + dexamethasone

0.7606

0.2652

Table 3. Statistical analysis of IL-13 concentration in BALF from select groups of
OVA or vehicle sensitized mice after treatment with 50 mg/kg TCH-013, vehicle or
0.1 mg/kg dexamethasone (n ≥ 7).

32

IL-6 (pg/mL)

4
3

+

2
1

ve
hi
cl
e
ve
+
ve
h
ve
ic
hi
hi
l
e
cl
cl
+
e
e
T
+
C
de
H
-0
xa
13
m
et
ha
so
O
VA
ne
+
ve
O
VA
hi
O
cl
VA
+
e
T
+
C
de
H
-0
xa
13
m
et
ha
so
ne

0

Figure 12. IL-6 concentration in BALF from OVA or vehicle sensitized mice after
treatment with 50 mg/kg TCH-013, vehicle or 0.1 mg/kg dexamethasone.
Treatment with OVA induced a statistically significant increase in IL-6 (+p < 0.05, n ≥ 8).
However, treatment with neither TCH-013 nor dexamethasone decreased IL-6
concentration in the BALF (p > 0.05, n ≥ 7).

33

ANOVA
with ANOVA with Tukey’s T test
Bonferroni’s multiple multiple comparisons
comparisons
test, test, adjusted P value
adjusted P value
OVA + vehicle vs. OVA
+ TCH-013

0.1961

0.1242

0.0927

OVA + vehicle vs. OVA
+ dexamethasone

0.0711

0.0514

0.0745

0.9571

0.3089

OVA + TCH-013 vs.
>0.9999
OVA + dexamethasone

Table 4. Statistical analysis of IL-6 concentration in BALF from select groups of
OVA or vehicle sensitized mice after treatment with 50 mg/kg TCH-013, vehicle or
0.1 mg/kg dexamethasone (n ≥ 7).

34

50

+

KC (pg/mL)

40
30
20
10

ve
hi
cl
e
ve
+
ve
hi
ve
cl
hi
hi
e
cl
cl
+
e
e
T
+
C
de
H
-0
xa
13
m
et
ha
so
O
VA
ne
+
ve
O
VA
hi
O
cl
VA
+
e
T
+
C
de
H
-0
xa
13
m
et
ha
so
ne

0

Figure 13. KC concentration in BALF from OVA or vehicle sensitized mice after
treatment with 50 mg/kg TCH-013, vehicle or 0.1 mg/kg dexamethasone.
Treatment with OVA induced a statistically significant increase in KC (+p < 0.05, n ≥ 8).
However, treatment with neither TCH-013 nor dexamethasone decreased KC
concentration in the BALF (p > 0.05, n ≥ 7).

35

ANOVA
with ANOVA with Tukey’s T test
Bonferroni’s multiple multiple comparisons
comparisons
test, test, adjusted P value
adjusted P value
OVA + vehicle vs. OVA
+ TCH-013

0.3926

0.2185

0.1160

OVA + vehicle vs. OVA
+ dexamethasone

0.0767

0.0549

0.0719

0.9018

0.2824

OVA + TCH-013 vs.
>0.9999
OVA + dexamethasone

Table 5. Statistical analysis of KC concentration in BALF from select groups of
OVA or vehicle sensitized mice after treatment with 50 mg/kg TCH-013, vehicle or
0.1 mg/kg dexamethasone (n ≥ 7).

36

Resistance (cmH2O/mL)

15

10

vehicle + vehicle
vehicle + TCH-013
vehicle + dexamethasone
OVA + vehicle
OVA + TCH-013
OVA + dexamethasone

+

5

0
0

20

40

60

Methacholine Dose (mg/mL)

Figure 14. Total lung resistance after methacholine challenge in OVA or vehicle
sensitized mice after treatment with 50 mg/kg TCH-013, saline or 0.1 mg/kg
dexamethasone. OVA sensitization induced airway hyperresponsiveness, measured
as resistance using a Flexivent protocol, significantly when compared to vehicle treated
mice (p < 0.05, n ≥ 5). Neither TCH-013 nor dexamethasone treatment of the OVA
sensitized mice significantly reduced airway hyperresponsiveness when compared to
OVA sensitized mice treated with vehicle.

37

Modification of the imidazoline scaffold yielded a molecule with increased
efficacy and a noncompetitive binding profile
In order to optimize the imidazoline scaffold for proteasome inhibition, each of the
functional groups on the parent molecule were manipulated and each daughter
molecule underwent evaluation in vitro. The ability of the compounds to inhibit the
chymotryptic-like (CT-L) activity of the 20S proteasome was determined in a purified
enzyme system by measuring the rate of hydrolysis of the fluorogenic substrate SucLLVY-AMC. Fluorescence increase was measured at 37°C over 30 minutes and the
linear portion of the curve was used to measure rates of hydrolysis in response to
varying concentrations of drug. Velocity was plotted against log[drug] to generate a
dose response curve, from which IC50 values were determined. Figure 15 shows a
curve representative of each curve obtained from the enzyme assay used to calculate
IC50 values.
Building on the parent molecule, TCH-013, domains R1-R3 were the first to be
evaluated as potential sites of improvement. A structure-activity analysis led to the
determination that, although these groups were necessary for activity, manipulation led
to only a modest increase in potency (Table 6). The development of the most potent
imidazolines was achieved by synthesizing a compound with the optimal structural
features of the compounds from Table 6 and manipulation of the R4 group (Table 7).
Evaluation of these derivatives led to the discovery of compound 46, the first identified
noncompetitive proteasome inhibitor with nanomolar potency.
38

26

Although the molecule retained the core structural features of TCH-013, the
adjustment of the functional groups necessitated evaluation of the kinetic profile of the
derivative compounds.

In order to determine if these newly identified molecules

retained the characteristic noncompetitive binding profile of TCH-013, compound 43
was evaluated. Although slightly less potent than compound 46, the favorable solubility
properties as well as the significantly increased potency made compound 43 the ideal
candidate for this assessment.

Consistent with the reports of TCH-013, the

Lineweaver-Burk plot of compound 43 suggests the lines of best fit intersect on the Xaxis (1/[S]), this pattern is accepted to be representative of a noncompetitive inhibition
mechanism (Figure 16).

26,29

39

Figure 15. TCH-013 inhibits the CT-L activity of the human 20S proteasome. The
hydrolysis of the fluorogenic substrate Suc-LLVY-AMC was used to measure the CT-L
activity of purified human 20S proteasome. IC50 values were calculated using
GraphPad Prism for Windows, GraphPad Software, San Diego California USA,
www.graphpad.com.

40

R1

R2

R3

IC50(µM)

11

H

Phenyl

Phenyl

>10

12

Acyl

Phenyl

Phenyl

>10

13

Benzoyl

Phenyl

Phenyl

>10

14

Tosyl

Phenyl

Phenyl

>10

15

CH2-2-furan

Phenyl

Phenyl

7.04a

1

Benzyl

Phenyl

Phenyl

2.58

16

4-CH3O-benzyl

Phenyl

Phenyl

2.73

17

4-F-benzyl

Phenyl

Phenyl

3.71

18

4-Cl-benzyl

Phenyl

Phenyl

2.02

19

4-Br-benzyl

Phenyl

Phenyl

1.90

20

Benzyl

4-pyridine

Phenyl

>10

21

Benzyl

2-furan

Phenyl

6.13

22

Benzyl

4-NO2-phenyl

Phenyl

6.71

23

Benzyl

4-NH2- phenyl

Phenyl

3.52

24

Benzyl

4-CF3- phenyl

Phenyl

4.81

25

Benzyl

4-Cl- phenyl

Phenyl

2.23

26

Benzyl

4-NH(SO2Ph)-phenyl

Phenyl

1.08

27

Benzyl

4-SCH3

Phenyl

2.77

28

Benzyl

4-SO2CH3-phenyl

Phenyl

>10

29

Benzyl

4-NHBenzyl-phenyl

Phenyl

0.47

30

Benzyl

Phenyl

4-Br-phenyl

3.19

31

Benzyl

Phenyl

4-CH3O-phenyl

1.27

32

Benzyl

4-CN-phenyl

4-CH3O-phenyl

>10

33

Benzyl

4-CONH2-phenyl

4-CH3O-phenyl

>10

Table 6. Inhibition of the chymotryptic-like activity of purified human 20S
proteasome by compounds 11-33 (n >2, an = 1). The hydrolysis of the fluorogenic
substrate Suc-LLVY-AMC was used to measure the CT-L activity of purified human 20S

41

Table 6 (cont’d) proteasome. IC50 values were calculated using GraphPad Prism for
Windows, GraphPad Software, San Diego California USA, www.graphpad.com.

42

R4

IC50 (µM)

34

H

1.97

35

Ac

2.17

36

Bz

1.44

37

0.63

38

0.58

39

0.53

40

1.08

41

0.91

42

1.22

43

0.30

44

0.54

45

0.37

46

0.13

Table 7. Inhibition of the chymotryptic-like activity of purified human 20S
proteasome by compounds 34-46 (n > 2). The hydrolysis of the fluorogenic substrate
Suc-LLVY-AMC was used to measure the CT-L activity of purified human 20S
proteasome. IC50 values were calculated using GraphPad Prism for Windows,
GraphPad Software, San Diego California USA, www.graphpad.com.

43

20

Vehicle
1.25 µM
2.5 µM
5 µM

1/v

10

-0.2

0.2
-10

0.4

1/[S]

Figure 16. Kinetic analysis of CT-L activity inhibition by compound 43. Human
20S proteasome was treated with varying concentrations of the substrate Suc-LLVYAMC and TCH-013. Hydrolysis of the substrate Suc-LLVY-AMC was used to measure
the CT-L activity. A Lineweaver-Burk plot was generated and illustrates a pattern of
noncompetitive inhibition. Graphs were generated using GraphPad Prism for Windows,
GraphPad Software, San Diego California USA, www.graphpad.com.

44

The modified imidazoline is cytotoxic and overcomes bortezomib resistance in
cell culture of multiple myeloma
Although significant advances have been made in multiple myeloma treatment,
the disease remains incurable and almost all patients experience disease relapse.

59

As

a method to evaluate the resistant cancer, a model of MM resistance was developed
using the human leukemia cell line, THP-1. The mechanism of bortezomib resistance in
this model has been attributed to a mutation in the active site of the β5 subunit and the
over expression of this mutated protein.

47

Molecules that act via a noncompetitive

mechanism may be successful in overcoming bortezomib resistance, because this
25

resistance mechanism is focused on the active site.

The optimized imidazoline compounds were screened for their cytotoxicity
against the wild type (WT) and bortezomib resistant (BTZ500) strains of the THP-1 cell
line in culture. In the wild type cells, treatment with 10 μM imidazoline resulted in
cytotoxicity 72 hours after drug administration (Figure 17).

In the BTZ500 cells,

treatment with 1 μM bortezomib treatment exhibited only a modest cytotoxic effect
leaving >67% of cells viable. However, the modified imidazoline scaffolds retained their
activity at 10μM and overcame resistance, similarly to their parent molecule (Figure
18).29
These results indicate that the imidazolines are capable of overcoming
bortezomib resistance in vitro and act as further evidence that the compounds bind via a
distinct mechanism. Further studies elucidating IC50 values for each of the compounds
in cell culture will allow for the calculation of resistance factors and more thorough
45

evaluation of the cytotoxicity profile. In addition, to assess imidazoline effectiveness in
clinical resistance, primary cells obtained from patients with refractory disease should
be treated with the imidazolines and evaluated for cytotoxicity.

46

% Viability
(treatment/vehicle)

100
80
60
40
20

46

45

44

43

0

Figure 17. Cell viability of THP-1 wild type cells after 72 hours of treatment with 10
μM imidazoline. The imidazolines exhibit cytotoxicity against the human leukemia cell
line THP-1 after 72 hours of treatment (n = 3).

47

% Viability
(treatment/vehicle)

100
80
60
40
20

46

45

44

43

Bo
rte
zo
m

ib

0

Figure 18. Cell viability of THP-1 BTZ500 cells after 72 hours of treatment with 10
μM imidazoline or 1 μM bortezomib. The imidazoline compounds exhibit cytotoxicity
and overcome bortezomib resistance in the human leukemia cell line THP-1 (n = 3).

48

Compound 43 is bioavailable and attenuates the systemic inflammatory response
in vivo
After determining that compound 43 had a noncompetitive profile and
successfully overcame bortezomib resistance, we set out to determine if the lead
molecule was bioavailable and could disrupt the systemic inflammatory response.
Intraperitoneal administration of 150 mg/kg of compound 43 in vehicle
(30:70 propylene glycol:D5W) resulted in serum concentrations in the high nanomolar
range, above the compounds reported IC50 of 300 nM (Figure 19). These data confirm
that the imidazolines, epitomized by compound 43, are bioavailable when administered
via IP injection. This preliminary study should be followed up by complex
pharmacokinetic studies to determine the comprehensive bioavailability characteristics
of compound 43.
Continuing the evaluation of compound 43, the same LPS challenge model of
systemic inflammation used to evaluate the parent compound TCH-013 was employed.
BALB/c mice with no immunodeficiency were treated with an injection of 50 mg/kg TCH013 intraperitoneally and one hour later were administered 1 mg/kg LPS. Animals were
sacrificed and serum was collected 2 hours after LPS treatment. The results indicate a
50 mg/kg dose of compound 43 significantly reduced serum TNF-α when compared to
the vehicle control (Figure 20). LC/MS analysis of serum from these animals revealed
compound 43 at 3.47 ± 0.41 µM, well above the IC50 of the compound.
Identification
noncompetitive

of

compound

proteasome

43

inhibitor

as
that

a

nanomolar

can

potency,

successfully

bioavailable,

reduce

systemic

inflammation is the first step toward developing this optimized molecule into a
49

pharmaceutical candidate. Preliminary data from collaboration with Dr. Terrance Tang
at University of Waterloo, ON, CA, showed that oral administration of compound 43 at
100 mg/kg significantly reduces tumor size in a murine xenograft model of multiple
myeloma. This is further evidence that these molecules should be approached for their
chemotherapeutic potential.

50

0.8
0.6
0.4
0.2

24

12

3

0.0
1

Compound 43 concentration (µM)

1.0

hours post IP injection

Figure 19. Compound 43 is present in the serum after IP administration at 150
mg/kg (n = 4). Animals were given a single IP dose of compound 43 in 30:70 propylene
glycol:D5W and blood was drawn at 1, 3, and 12 hours post administration. LC/MS
analysis was performed on the samples to determine the quantity of drug present.

51

TNF-α (pg/mL)

2500
2000
1500

*

1000
500

43

om
po
un
d

Ve
hi
cl
e
+

LP
S

LP
S

+

C

LP
S

C

om
po
un
d

43

0

Figure 20. Compound 43 reduces LPS-induced TNF-α production. Animals were
treated with an IP dose of 50 mg/kg of compound 43, one hour later animals were
given an IP dose of 1 mg/kg LPS, and two hours later serum was collected.
Concentration of TNF-α in serum was measured using an ELISA. Compound 43
significantly reduces serum TNF-α following LPS challenge when administered at 50
mg/kg IP (p < 0.05, n > 7).

52

CHAPTER 4- SUMMARY AND CONCLUSIONS
Limitations and Future Directions
There were several limitations in these studies, of particular note are the complex
mechanisms involved in the pathogenesis of asthma and the elusiveness of the
imidazoline binding site.
It is well documented that the commonly used OVA-induced allergic airway
disease is not directly representative of the human specific disease, asthma.

60

Although this model is helpful for studying the pathogenesis of acute airway
inflammation, this model has limitations in its predictive value for drug development
purposes.

61

In addition, the pathogenesis of asthma is complex and multi-factorial,

masking the true effects that the manipulation of NF-κB may have on specific parts of
the disease.

62

Further evaluation of the proteasome inhibitor in an experimental model

of asthma should be undertaken in conjunction with steroid therapy and with a
bronchodilator.

Great care should also be taken to determine the effects of the

imidazolines on IgE and plasma cells in allergic airway disease. It appears that, even
though there may be potential for application of these compounds to asthma; focus
should be placed on evaluation of their chemotherapeutic properties.
Although β5 subunit mutation and overexpression has been conclusively linked
to bortezomib resistance, the molecular basis for variable clinical response to
bortezomib is not well understood.

63

The evaluation of these compounds and the

contribution of NF-κB in primary cells from patients with resistant disease would be

53

helpful to determine if these compounds have potential to overcome multiple bortezomib
resistance mechanisms.
In addition, a significant limitation of these studies is the need for further
determination of NF-κB inhibition and specificity. Although previous studies have shown
that the imidazolines inhibit NF-κB, determination of specific pathway components (IκB,
phospho-IκB, and nuclear p65) in both the asthma and bortezomib resistance models is
essential.29
Compound 43 has shown promising results; however, evaluation of the optimized
imidazolines should be expanded. Comprehensive bioavailability and toxicity studies
would be the first step toward moving this molecule further along the drug development
process. Evaluation of these compounds in the bone marrow microenvironment and in
primary cultures from patients with multiple myeloma is essential to complete before this
molecule can be considered for clinical potential.
The most significant limitation to these studies is the elusiveness of the
imidazoline binding site on the proteasome. Despite evidence indicating that these
compounds are noncompetitive inhibitors, determination of the exact binding site is
needed to complete the conclusion regarding the mechanism of action of these
compounds.

Multi-disciplinary experiments are currently underway in the Tepe

laboratory in pursuit of binding site identification.

Conclusion
All clinically approved proteasome inhibitors act through a competitive
mechanism and result in modulation of NF-κB activity. Although these molecules have

54

potent anti-cancer and anti-inflammatory properties, they also exhibit dose limiting
toxicity and pharmacokinetic issues. The studies presented in this thesis show that the
noncompetitive proteasome inhibitor, TCH-013, is bioavailable and not hepatotoxic.
This compound was also shown to have anti-inflammatory properties, as evidenced by
its ability to reduce TNF-α in the LPS challenge and to reduce the BALF cellularity in an
experimental model of asthma. In addition, we showed that efficacy of this molecule
could be enhanced by manipulating the functional groups while maintaining its
noncompetitive binding profile and anti-neoplastic characteristics. The result of these
studies is the discovery of compound 43, a noncompetitive proteasome inhibitor that is
able to overcome bortezomib resistance, is bioavailable, and is effective in an animal
model of systemic inflammation.

These studies illustrate that these imidazoline

proteasome inhibitors may be possible alternatives to the classical competitive agents.
Administration of the imidazoline proteasome inhibitors to the complex systems
of inflammation and neoplasia clearly modulates the outcomes of these processes, but
the precise intracellular mechanism of action needs to be further elucidated. It is my
hope that the results and discussion presented in this thesis inspire researchers to
further investigate the activity of the imidazolines, with particular emphasis on
illuminating how the compounds affect specific intracellular process and inflammatory
cascade in vivo.

55

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