INVESTIGATION OF SMA LL MOLECULE MEDIATED REGULATION OF PROTEASOME ACTIVITY: COMPUTATIONALLY GUI DED DESIGN, SYNTHESIS, AND FORMU LATION OF A THEORETI CAL FRAMEWORK FOR ACTIVITY By Corey L. Jones A DISSERTATION Submitted to Michigan State University i n partial fulfillment of the requirements for the degree of Chemistry Doctor of Philosophy 2019 ABSTRACT INVESTIGATION OF SMA LL MOLECUL E MEDIATED REGULATION OF PROTEASOME ACTIVITY: COMPUTATIONALLY GUI DED DESIGN, SYNTHESIS, AND FORMU LATION OF A THEORETI CAL FRAM EWORK FOR ACTIVITY By Corey L. Jones Proteins undergo constant proteolytic degradation to regulate intracellular processes and maintain biological homeostasis. One of the main intracellular proteolytic pathways involves the proteasome, which is responsible for the degradation of misfolded, oxidatively damaged, and redundant proteins. The age - related decline of the efficiency of this enzymatic pathway leads to accumulation of abe rrant proteins which leads to aggregates and a host of amyloidosis disease state s. Small molecule intervention has been suggested as a viable therapeutic strategy; however, enhancement of proteasome activity is not well understood. This work details effort s and evidence for mechanistic understanding of how small molecule action may en hance and bias proteasome proteolysis and provides a theoretical model for further advancement. iii Dedicated to my parents Ronald and Sharmin Jones, and my sister Haley Jones. iv ACKNOWLEDGMENTS Grad school has been an arduous journey with many high and low points. I doubt such in my life. First, I would like to thank my family. M y parents, Ronnie and Sharmin , for their support and encouragement, and my si ster Haley who called regularly just to check and talk. You rather than try one more reaction. I could not have asked for better lab mates to journey with. Travis Bet hel and Matthew Giletto . We had many great discussions about chemistry and many more about everything else in life, and I like to imagine the halls of the 5 th floor will echo with our laughter for years to come. g choices Travis would play on weekends. I would also like to thank my other group mates for being good people and not making grad school any more difficult than it had to be. Evert Njomen, the bio - lab princess, for our discussions on the bi o data were ver y helpful. Katarina and Shafaat, thank you for having the right try hard attitude. Helping Travis train you two was a rewarding experience in its own right and I look forward to seeing what you do in the future. Grace, Taylor, Sophie and All ison, I have en joyed my slightly more limited time with you all. Keep working and know the journey does end eventually. Beyond the training and education, I think the best thing I have received are lifelong friends who have forged the path with me. Tanne r McDaniel, Tim Shannon, Kristen Gore (now Kristen Shannon) , Yu - Ling Lien, Yukari Nishizawa - Brennan, Brennan Billow, Tyler Walter, Oscar Judd, Kristen Reese, Kelly Aldrich, and Olivia Chesniak . You all have been v an excellent source of information and livel y discussion as well as much needed decompression from the stresses of lab work. I would like to thank Dr. Dan Holmes for his NMR assistanc e and conversation over the years. I would also like to thank Marvey Olson, for her help in dealing with the outside world and for always being a delight to interact with , I hope you are enjoying retirement. I also want to thank Dawn Khun who moved in to fill your shoes and has done an excellent job. Also, Nancy Lavirik as my quick 10 - minute updates would inevitably tur n to hour long you nearly enough. I also want to thank Dr. Chrysoula Vasileiou who has been a great source of conversation and advice throughout the years. Another person MSU gets to ke ep for too little. I would like to thank my committee, Dr. Wulff, Dr. Huang , and Dr. Odom, for many enlightening and fruitful conversations. Finally, I would like to thank Dr. Jetze Tepe . I would like to thank him for the opportunity to work in his lab oratory and the creative freedom he has encouraged. We have had many ups and downs between us but have managed to make it to the end and I look forward to seeing where the lab goes next. I would also commend you if you have read this far. G ood job! vi T ABLE OF CONTENTS LIST OF TABLES ................................ ................................ ................................ ......... viii LIST OF FIGURES ................................ ................................ ................................ ......... ix KEY TO ABBREVIATIONS ................................ ................................ ........................ xvi Chapter 1. The Proteasome and Related Systems ................................ ......................... 1 A. Proteostasis and Aging ................................ ................................ ............................... 1 B. Systems of Degradation ................................ ................................ ............................. 2 C. Autophagy Lysosome System ................................ ................................ .................... 3 D. The Proteasome ................................ ................................ ................................ .......... 4 E. The Ubiquitin - Proteasome Syst em (UPS) ................................ ................................ .. 5 F. Overview of Gating Mechanism ................................ ................................ ................. 8 G. The Ubiquitin Independent Proteasome System (UIPS) ................................ .......... 11 H. Intrinsically Disordered Proteins ................................ ................................ ............. 13 Ch ap ter 2. In Silico Investigation of Proteasome Regulators: Development of Theory of Action and Applications ................................ ................................ ............... 15 A. Introduction ................................ ................................ ................................ ........... 15 B. A Brief Overview of Molecular Docking ................................ ............................. 16 C. Defining Boundary Conditions ................................ ................................ ............. 17 D. Why Chose Vina? ................................ ................................ ................................ . 20 E. Results and Theory of Proteasome Activation ................................ ...................... 24 Chapter 3. Phenothiazine Small Molecule Activators ................................ ................. 26 A. Introduction to Phenothiazine Activators and Chlorpromazine ............................ 26 B. Validation of Docking Model by Rep urposing of Neuroleptic Agent .................. 28 C. Attempts at SAR: Challenges of Irrational Da ta ................................ ................... 38 D. Optimization of Proteasome Agonist Evaluation and Work Flow ....................... 45 E. Construction of New Metrics for Agonism Effectiveness ................................ .... 51 F. Theory Update, Conclusion, and Futu re Outlook ................................ ................. 55 Chapter 4. Efforts Toward Small Molecule Binding Site Identification ................... 61 Chapter 5. Allosteric Inhibition of the 20S Proteasome ................................ .............. 67 A. Design and Synthesis of a n Allosteric Prot easome Inhibitor ................................ 67 B. Computational Insight for New Allosteric Inhibitors ................................ ........... 74 Experimental Section ................................ ................................ ................................ ...... 78 A. Synthetic Methods ................................ ................................ ................................ 78 B . Proteomic Methods ................................ ................................ ............................. 130 C. Proteasome Activity Assays ................................ ................................ ............... 133 vii APPENDIX ................................ ................................ ................................ .................... 146 REFERENCES ................................ ................................ ................................ .............. 193 viii LIST OF TABLES T Table 3.3: Table 3.4: Table of Individual and Combinatio n Table 3.5: All Top - to - .65 ix LIST OF FIGURES Figure 1.1: Overview of Proteasome Structure ................................ ................................ ... 5 Figure 1.2: Cartoon of Ubiquitin (Ub) Cascade ................................ ................................ .. 6 Figure1.3: Depiction of the 19S Subunits with Base (shades of blue) and Lid (multicolored ) ................................ ................................ ................................ ..................... 7 Figure 1.4: Top View of Proteasome CP Depicting the Gate (Yellow) and Intersubunit Pockets (circles) ................................ ................................ ................................ .................. 9 Figure 1.4: 75 ......................... 10 Figure 1.5: Simplified Outline of UPS vs UIPS ................................ ............................... 12 Figure 2.1: Published Utilization of Docking Software 1990 - 2013 ................................ . 20 Figure 2.2: Conceptual Illustration of Energetically Preferred States .............................. 22 Figure 2.3: Graphical Overview of Docking Process and Result ................................ ..... 24 Figure 3.1: Selected Results from HTS of Proteasome Activators ................................ ... 26 Figure 3.2: Graphic of Key Dopamine Receptor I nteractions with CPZ .......................... 28 Figure 3.3: Binding Affinity Values for Top 9 Poses (Table), Dif ferent Orientations with Similar Binding Affinity (Pose 1, 4, and 9), Overlay of Similar Poses (A) and Proposed Anchoring Residue Arg83 (B). ................................ ................................ ......................... 29 Figure 3.4: Overlay of Predicted Binding Modes of CPZ (white) and 3 - 3 (orange). ....... 30 Figure 3.5: Synthesis of First - Generation Validation Compounds ................................ ... 31 Figure 3.6: Dose Re sponse of 3 - 3 (Top), IDP Degradation (B), and Complex Selectivity (C) ................................ ................................ ................................ ................................ ..... 32 Figure 3.7: Synthesis of Lead C andidates ................................ ................................ ......... 34 Figure 3.8: Synthesis of Flexible Ester and Depiction of Decomposition ........................ 36 Figure 3.9: Analogues of Lead Compound 3 - 8 ................................ ................................ 39 Figure 3.10: Synthesis of Tail Analogous ................................ ................................ ......... 41 Figure 3.11: Proposed Activator Structures with Key Interaction Motifs Highlighted .... 43 x Figure 3.12: Synthesis of HbYX Small Molecule Mimics ................................ ............... 44 Figure 3.13: Illustration of Vehicle Stability Issue ................................ ........................... 46 Figure 3.14: Illustration of EC 50 Value Use (A) and Sample Projec t Data (B) ................ 50 Figure 3.15: Comparison of Compound 3 - 8 and CPZ Activities per Active Site ............ 51 Figure 3.16: Selected Compounds Effect on Protein Degradation ................................ ... 54 Figure 3.18: Il lustration of PAProC Analysis and Table of Assignents ........................... 55 Figure 3.19: Illustration of Catalytic Site Conformers ................................ ..................... 56 Figure 3.20: Pocket Preference Difference Among Different 20S Conformers ............... 57 Figure 3.21: Intersubunit Pockets bound ................................ ................................ .......... 58 Figure 4.1: Synthesis of Phenothiazi ne Photoaffinity Label. ................................ ........... 61 Figure 4.2: Diagram of the Binding Site Identification Process ................................ ....... 63 Figure 4.3: Example of Combinatorial Explosion ................................ ............................ 63 Figure 4.4: Synthesis of Lysine Targeting Substrate ................................ ........................ 64 Figure 5.1: Velcade (A) Shown Bound in Catalytic Site Cartoon (B) and Crysta l Structure (C) ................................ ................................ ................................ ................................ ..... 67 Figure 5.2: (A) Indolophakelin Bound in S3 - Sub Domain (B) Hydroxyurea Inhibitor Bound in Active Sit e ................................ ................................ ................................ ......... 69 Figure 5.3: Indolophakelin (A) and Hydroxyurea (B) Bound in Catalytic Site. O verlay of Proposed Compound (C) with Hydroxyurea in Catalytic Site. ................................ ......... 70 Figure 5.4: Generalized Retrosynthetic Analysis of Proposed Compound ...................... 71 Figure 5.5: Synthesis of Fragment A (Compound 5 - 4 ) ................................ .................... 71 Figure 5.6: Synthesis of Allosteric Inhibitor 5 - 13 . ................................ ........................... 72 Figure 5.7: Ele ctrostatic Potential Maps of 5 Catalytic Site in Human (A) and Yeast (B) ................................ ................................ ................................ ................................ ........... 73 Figure 5.8: Published Structures Prepared by Dr. Giletto 177 ................................ ............ 75 Figure 5.9: ................................ ................. 77 Fig ure E.1: Compound 3 - 1 ................................ ................................ ............................... 80 xi Figure E.2: Compound 3 - 2 ................................ ................................ ............................... 81 Figure E.3: Compound 3 - 3 ................................ ................................ ............................... 82 Figure E.4: Compound 3 - 4 ................................ ................................ ............................... 83 Figure E.5: Compound 3 - 5 ................................ ................................ ............................... 85 Figure E.6: Compound 3 - 6 ................................ ................................ ............................... 86 Figure E.7: Compound 3 - 7 ................................ ................................ ............................... 87 Figure E.8: Compound 3 - 8 ................................ ................................ ............................... 88 Figure E.9: Compound 3 - 9 ................................ ................................ ............................... 90 Figure E.10 : Compound 3 - 10 ................................ ................................ ........................... 91 Figure E.11: Compound 3 - 11 ................................ ................................ ........................... 93 Figure E.12: Compound 3 - 12 ................................ ................................ .......................... 94 Figure E.13: Compound 3 - 13 ................................ ................................ .......................... 95 Figure E.14: Compound 3 - 14 ................................ ................................ .......................... 96 Figure E.15: Compound 3 - 15 ................................ ................................ .......................... 97 Figure E.16: Compound 3 - 16 ................................ ................................ .......................... 98 Figure E.17: Compound 3 - 17 ................................ ................................ .......................... 99 Figure E.18: Compound 3 - 18 ................................ ................................ ........................ 100 Figure E.19: Compound 3 - 19 ................................ ................................ ........................ 101 Figure E.20: Compound 3 - 20 ................................ ................................ ........................ 102 Figure E.21: Compound 3 - 21 ................................ ................................ ........................ 103 Figure E.22: Compound 3 - 22 ................................ ................................ ........................ 104 Figure E. 23: Compound 3 - 23 ................................ ................................ ........................ 105 Figure E.24: Compound 3 - 24 ................................ ................................ ........................ 106 Figure E.25: Compound 3 - 25 ................................ ................................ ........................ 107 Figure E.26: Compound 3 - 26 ................................ ................................ ........................ 108 xii Figure E.27: Compound 3 - 27 ................................ ................................ ........................ 109 Figure E.28: Compound 3 - 28 ................................ ................................ ........................ 110 Figure E.29: Compound 3 - 29 ................................ ................................ ........................ 111 Figure E.30: Compound 3 - 30 ................................ ................................ ........................ 112 Figure E.31: Compound 3 - 31 ................................ ................................ ........................ 113 Figure E.32: Compound 3 - 32 ................................ ................................ ........................ 114 Figure E.33: Compound 3 - 33 ................................ ................................ ........................ 115 Figure E.34: Compound 3 - 34 ................................ ................................ ........................ 116 Figure E.35: Compound 4 - 1 ................................ ................................ .......................... 117 Figure E.36: Compound 4 - 2 ................................ ................................ .......................... 118 Figure E.37: Compound 4 - 3 ................................ ................................ .......................... 119 Figure E.38: Compound 4 - 5 ................................ ................................ .......................... 120 Figure E.39: Compound 4 - 6 ................................ ................................ .......................... 121 Figure E.40: Comp ound 4 - 7 ................................ ................................ .......................... 122 Figure E.41: Compound 5 - 4 ................................ ................................ .......................... 123 Figure E.42: Compound 5 - 5 ................................ ................................ .......................... 125 Figure E.43: Compound 5 - 6 ................................ ................................ .......................... 126 Figure E.44: Compound 5 - 7 ................................ ................................ .......................... 127 Figure E.44: Compound 5 - 11 ................................ ................................ ........................ 128 Figure E.45: Compound 5 - 12 ................................ ................................ ........................ 129 Figure E.46: Dose Response of Combination Set 1 ................................ ........................ 134 Figure E.47: Dose Response of Combi nation Set 2 ................................ ........................ 135 Figure E.48: Dose Response of Combination Set 3 ................................ ........................ 136 Figure E.49: Dose Response o f B1 Set 1 ................................ ................................ ........ 137 Figure E.50: Dose Response of B1 Set 2 ................................ ................................ ........ 138 xiii Figure E.51: Dose Response of B1 Set 3 ................................ ................................ ........ 139 Figure E.52: Dose Response of B2 Set 1 ................................ ................................ ........ 140 Figure E.53: Dose Re sponse of B2 Set 2 ................................ ................................ ........ 141 Figure E.54: Dose Response of B2 Set 3 ................................ ................................ ........ 142 Figure E.55: Dose Response of B5 Set 1 ................................ ................................ ........ 143 Figure E.56: Dose Response of B5 Set 2 ................................ ................................ ........ 144 Figure E.57 : Dose Response of B5 Set 3 ................................ ................................ ........ 145 Figu re A.1: 1 H and 13 C NMR Spectra of Compound 3 - 1 ................................ ............... 147 Figure A.2: 1 H and 13 C NMR Spectra of Compound 3 - 2 ................................ ............... 148 Figure A.3: 1 H and 13 C NMR Spectra of Compoun d 3 - 3 ................................ ............... 149 Figure A.4: 1 H and 13 C NMR Spectra of Compound 3 - 4 ................................ ............... 150 Figure A.5: 1 H and 13 C NMR Spectra of Compound 3 - 5 ................................ ............... 151 Figure A.6: 1 H and 13 C NMR Spectra of Compound 3 - 6 ................................ ............... 152 Figure A.7: 1 H and 13 C NMR Spectra of Compound 3 - 7 ................................ ............... 153 Figure A.8: 1 H and 13 C NMR Spectra of Compound 3 - 8 ................................ ............... 154 Figure A.9: 1 H and 13 C NMR Spectra of Compound 3 - 9 ................................ ............... 155 Figure A.10 : 1 H and 13 C N MR Spectra of Compound 3 - 10 ................................ ........... 156 Figure A.11: 1 H and 13 C NMR Spectra of Compound 3 - 11 ................................ ........... 157 Figure A.12: 1 H and 13 C NMR Spectra of Compound 3 - 12 ................................ ........... 158 Figure A.13: 1 H and 13 C NM R Spectra of Compound 3 - 13 ................................ ........... 159 Figure A.14: 1 H and 13 C NMR Spectra of Compound 3 - 14 ................................ ........... 160 Figure A.15: 1 H and 13 C NMR Spectra of Compound 3 - 15 ................................ ........... 161 Figure A.16: 1 H and 13 C NMR Spectra of Compound 3 - 16 ................................ ........... 162 Figure A.17: 1 H and 13 C NMR Spectra of Compound 3 - 17 ................................ ........... 163 Figure A.18: 1 H and 13 C NMR Spectra of Compound 3 - 18 ................................ ........... 164 xiv Figure A.19: 1 H and 13 C NMR Spectra of Compound 3 - 19 ................................ ........... 165 Figure A.20: 1 H and 13 C NMR Spectra of Compound 3 - 20 ................................ ........... 166 Figure A.21: 1 H and 13 C NMR Spectra of Compound 3 - 21 ................................ ........... 167 Figure A.22: 1 H and 13 C NMR S pectra of Compound 3 - 22 ................................ ........... 168 Figure A.23: 1 H and 13 C NMR Spectra of Compound 3 - 23 ................................ ........... 169 Figure A.24: 1 H and 13 C NMR Spectra of Compound 3 - 24 ................................ ........... 170 Figure A.25: 1 H and 13 C NMR Sp ectra of Compound 3 - 25 ................................ ........... 171 Figure A.26: 1 H and 13 C NMR Spectra of Compound 3 - 26 ................................ ........... 172 Figure A.27: 1 H and 13 C NMR Spectra of Compound 3 - 27 ................................ ........... 173 Figure A.28: 1 H and 13 C NMR Spe ctra of Compound 3 - 28 ................................ ........... 174 Figure A.29: 1 H and 13 C NMR Spectra of Compound 3 - 29 ................................ ........... 175 Figure A.30: 1 H and 13 C NMR Spectra of Compound 3 - 30 ................................ ........... 176 Figure A.31: 1 H and 13 C NMR Spec tra of Compound 3 - 31 ................................ ........... 177 Figure A.32: 1 H and 13 C NMR Sp ectra of Compound 3 - 32 ................................ ........... 178 Figure A.33: 1 H and 13 C NMR Spectra of Compound 3 - 33 ................................ ........... 179 Figure A.34: 1 H and 13 C NMR Spect ra of Compound 3 - 34 ................................ ........... 180 Figure A.35: 1 H and 13 C NMR Spectra of Compoun d 4 - 1 ................................ ............. 181 Figure A.36: 1 H and 13 C NMR Spectra of Compound 4 - 2 ................................ ............. 182 Figure A.37: 1 H and 13 C NMR Spectra of Compound 4 - 3 ................................ ............. 183 Figure A.38: 1 H and 13 C NMR Spectra of Compound 4 - 4 ................................ ............. 184 Figure A.39: 1 H and 13 C NMR Spectra of Compound 4 - 5 ................................ ............. 185 Figure A.40: 1 H and 13 C NMR Spectra of C ompound 4 - 7 ................................ ............. 186 Figure A.41: 1 H and 13 C NMR Spectra of Compound 5 - 4 ................................ ............. 187 Figure A.42: 1 H and 13 C NMR Spectra of Compound 5 - 5 ................................ ............. 188 Figure A.43: 1 H and 13 C NMR Spectra of Compo und 5 - 6 ................................ ............. 189 xv Figure A.44: 1 H and 13 C NMR Spectra of Compound 5 - 7 ................................ ............. 190 Figure A.4 5: 1 H and 13 C NMR Spectra of Compound 5 - 11 ................................ ........... 191 Figure A.46: 1 H and 13 C NMR Spectra of Compound 5 - 12 ................................ ........... 192 xvi KEY TO ABBREVIATIONS Arg Arginine Asp Aspartamie ATP Adenosine Triphophate Boc Tert - butoxycarbonyl BTZ Bortezomib CMA Chaperone Mediated Autophagy CP Core Particle CPZ Chlropromazine DIPA Diisoproyl amine DMF Dimethyl Formamide EC 50 Concentration producing 50% of m aximal response EDCI N - (3 - Dimethylaminopropyl) - - ethylcarbodiimide hydrochloride HTS High - throughput - screen IBX 2 - Iodoxybenzoic acid IC 50 Concentration inhibiting 50% of maximal response IDP Intrinsically Disordered Proteins IDPR Intrinsically Disordered Protein Region Lys Lysine MD Molecular Dynamics Ms Mesyl nBuLi n - butyl lithium xvii NMR Nuclear Magnetic Resonance OAc Acetate PA Protein Activator Ph Phenyl PN Proteostasis Network PTM Post Translational Modificaiton QSAR Quanitative Struture Activity RP Regulat ory Particle TBDPS Tert - butyl dimethyl silyl THF Tetrahydrofuran Thr Threonine Ub Ubiquitin Ub Ubiquitin UIPS Ubiquitin Independent Proteasome System UPS Ubiquitin Proteasome System UV Ultra Violet 1 Chapter 1. The Proteasome and Related Systems Multiple projects detailed herein concern the proteasome , and related systems , to varying degrees. To prevent repetitive introduction, this chapter will serve as a general primer to provide perspective , while other chapters will contain a more focused intr oduction to needed topics. A. Proteostasis and Aging Maintaining fidelity among the over 10,000 proteins 1 , in mammalian cells, present at any given time is a h erculean task. Cells maintain this protein homeostasis (proteostasis) via the intervention of the proteosta sis network ( PN ) : a highly coordinated and intricate system whic h acts to rectify proteome imbalance 2 . Exact definitions of what is and is not included in the PN vary between authors; however, the consensus appears to be that the PN incorporates all machinery directly involved in the synth esis ( ca. 279 components 3 ), folding and disaggrega tion (chaperones ~332 components 4 ), or degradation (two canonical pathways ~1388 components 5 ) of proteins. Regulators of posttranslational modifications (PTMs), structural components, and othe r such systems essential for PN function may be considered secondary to the PN depending on the focus of th e work. Due to the vast conformational space available to polyp eptides, folding is inherently error prone 6 . Unhelpfu lly, the protein concentration of a cell is nearly 300g/L, an extremely crowded envir onment which significantly increases the tendency of proteins to aggregate (compared to dilute solutions) 7 . Compounding this proble m is the decreased efficiency of the PN as the system ages 8 . Aging is a complex, and ultimately disheartening, aspect of living cells. A common feature of aged cells is the accumulation of non - native protein aggregates and general loss 2 of proteome fidelity. Recent reviews exhaustively cover proteostasis in aging 9 , dysfunction of associated pathways 8 , role in cardiac health 10 , neurodegenerative diseases 11 - 13 an d many other diseases that appear to be caused by dysregulation of the PN 14 . M any reasons for its decline are given 15 but in brief, age related decline of the PN may be explained by small muta tions to the PN machinery over long time periods leading to decreased efficiency. Thi s decreased efficiency leads acute stressors, such as misfolded or damaged proteins, becoming chronic stressors. Chronic dependence on the PN leads to an increase in the s ynthesis of chaperones and other machinery for proteostasis 16 - 18 . Increased syn thesis leads to more errors and more stress 19 - 20 . A destructive cycle ensues where decreasing capacity leads to more stress, more stress leads to more mutations, more mutations leads to a greater decrease in capacity , which ultimately overcomes the PN resulting in a dysfunctional cellular state 20 . B. Systems of Degradation Damage to proteins is natural and very c ommon in cells and may be induced by a wide range of external stimuli like air pollution 21 - 22 and UV radiation 23 . Likewise, natural cellular processes 24 - 25 produce oxidants and reactive species that damages cellular proteins. These aberrant protein s must be dealt with quickly and selectively to prevent aggregate formation while simultaneously maintaining the proper distribution of necessary protein s (proteostasis) . To achieve this flexibility and allow a wide range of half - lives, ranging from minut e s to days, the eukaryotic PN relies upon two organelles: the lysosome and the 20S proteasome. Lysosomes are closed organelles containing a multitude of digestive enzymes and proteases 26 . The membrane boundary prevents uncontrolled destruction of cellular contents 3 but requires ad ditional pathways for uptake of proteins. These pathways are collectively termed aut o phagy and are discussed below. C. Autophagy Lyso some System - 27 : a special series published in Nature Reviews Molecular C ell Biology in July 2018 covering the topic exhaustively is also available. In gen e ral, all forms of autophagy proceed through the same steps: sequestering proteins, transport to the lysosome, translocation into the lysosome, and finally degradation by t he lysosome. Autophagy appears to be largely non - selective but operates relatively s lowly 28 . Often, autophagy is discussed in the context of three main forms: macroautophagy, microautophagy, and chaperone - mediated autophagy (CMA) depending on the transportation type. Macroautophagy is p r imarily attributed with the destruction of damaged cell organelles, large protein aggregates, and anything else too large for proteasomal degradation 29 . This pathway has been implicated as having key roles in immunity and inflammation 30 ; particula r ly in stress response during times of starvation 31 . The overall process proceeds through the sequestration of doomed species by a membrane termed a phagophore. The resulting double membrane autophagosome transports its cargo and fuses with the lysosome. The autophagosome and its contents are then degraded by lysosomal enzymes. Microautophagy is characterized by the direct and non - selective degradation of cytopl asmic components 32 . Microautophagy complements the other two processes and may be induced through starvation. The general process is the direct invagination and ve sicle 4 scission into the lumen, mediated by autophagic tubes and i s believed to be inv olved in the maintenance of organelle size and membrane homeostasis. Chaperone - mediated autophagy is, by comparison, less studied but is known to be highly selective 33 . The two main chaperones for CMA have been identified as hsc70 an d hsp90 which direct selected proteins to and regulate translocat ion through the lyso somal membrane. Other forms of autophagy are known, such as mitophagy 34 and lipophagy 35 , which is the process of lysosomal degradation of mitoch ondria and lipids respectively, and more are likely to be discove red . H owever, the ov erall pathway is the same: isolation and transport of proteins to the lysosome for degradation. While this chapter has only covered the basics of autophagy ( vide supra ) a nd its utilization by the cell, it is an important pathway to kee p in mind as autopha gy works in tandem with proteasome systems ( vide infra ) 36 - 37 . D. The Proteasom e The importanc e of the proteasome is difficult to overstate as the cell has evolved to utilize its proteolytic power in several specialized tasks including cell cycle regulation, differentiation, the inflammatory response, a large role in immune function and apoptosis 38 . The proteasome comprise 39 and are found within a 40 , biological assembly 41 , role in health 42 , and other topics are available 37 , 43 - 44 , this section will merely outline the proteasome and associated pathways . The human proteasome core particle (CP) is a large, ca. 750 kDa, multic atalytic prote ase 45 - 46 . Each ring is in turn 5 subunits form a hollow cavity and house two sets of three (6 total) distinct proteolytic active sites: chymotrypsin - - - 47 . Access to the interior chamber is limited by the convergence of the N - either end of the core particle (Figure 1.1) . The gate prevents non - selective degradation of cellular c ontents - rings termed intersubunit pockets 48 . These binding domains ar e utilized by a variety protein activators (PA) with a specific PA utilized for specific pathways ( vide infra ). A weak equilibrium exist s between the open and closed forms of the proteasome , heavily favoring the closed form 49 . However, the gate does not open wide enough for properly folded globular proteins to gain access to the catalytic sites 50 - 51 . Figure 1 .1 : Overview of Proteasome Structure E. The Ubiquitin - Proteasome System (UPS) The 20S CP participates proteolytically in two distinct biological pathways distinguished by the presence or absence of ubiquitin. Ubiquitin (Ub) is a 76 - residue protein unique to eukaryotic cells with high levels of conservation 52 . The addition of - Ring Top View (HUMAN) Open vs Closed Gate 150 Å 115 Å Anterior Chambers Gate Hydrolytic Chamber Active Sites 6 ubiquitin to a substrate is termed ubiquit ination or ubiquitylation. Prot ein modification using Ub produces a wide range of effects from tagging a protein for degradation 47 to signal transduction 53 among others 52 , 54 . Ub conjugation is carried out in three steps utilizing three different enzyme types 55 . Gly76 is activated in an ATP dependent manner by a ubiquitin - activating enzyme (E1) forming a thioester linkage with the E1 cysteine. A second enzyme termed the ubiquitin - conjugating enzyme (E2) binds the Ub - E1 complex and transfers the Ub onto itself through trans(thio)esterifi cation resulting in an E2 - Ub complex. Finally, an E3 ubiquitin ligase transfers its bound target protein (most commonly through a lysine residue) to the E2 bound Ub and releases the Ub - protein complex back into the cell, ending the Ub cascade 56 (Figure 1.2) . Figure 1.2: Cartoon of Ubiquitin (Ub) Cascade The cascade is hierarchical with the two E1 enzymes 57 able to bind to multiple E2 enzymes (humans have 35) 58 and each E2 is able to bind to multiple E3 ligases (humans are estimated to have between 500 - 1000) 59 . This tiered system bur ied within a cascade allows tight regulation of Ub and ubiquitinylated systems. Originally it was thought that a multiubiquitin chain was required for protein recognition by the UPS; however, emerging 7 evidence suggests otherwise 60 . Likewise, a ne w level of regulation has been detailed in recent years illustrating the spatial arrangement and linkage specific conformations direct tagged protein outcome and is utilized by the cell for transient PTMs 60 - 61 . To participate in ubiquitin dependent proteolysis, the 20S CP requires endogenous activation 62 . This is achieved via the non - covalent addition of activator caps to special pockets formed between adjacent alpha subunits termed intersubunit pockets (see Section F) . The most common activa tor is the 19S regulatory particle (RP) wh ich is comprised of 19 subunits and split into two main parts (Figure 1.3) : a base and a lid. Figure1.3: Depiction of the 19S Subunits with Base (shades of blue) and Lid (multicolored) The RP base binds directly to the CP while the lid extends outward. Once a Deubiquitylating enzymes recycle the tag back to the cell while ATP hydrolysis, occurring on the lid, drives the unfolding and translocat ion of bound substrate into the proteolytic chamber of the CP. This process is regulated by the motor units that make up the base. Six 8 distinct subunits (Rpt1 - 6) from the AAA (ATPases associated with diverse cellular activities amily of ATPases regulate lid mediated engagement and unfolding 39 , 63 . This process is conformationally complex as the holoenzyme has been reported to exits in 19 distinct states 50 , 64 - 66 . Because of this complexity, the exact mechanism of how endogenous ligands open the proteasome gate is still poorly understood (See Section F). form encountered is a doubly bound CP with two identical 19S RPs gene rating the 26S proteasome. The 26S proteasome has been attributed with at least 80% of protein degradation in growing mammalian cells and widely assumed to automatically degra de any ubiquitinylated protein it encounters 67 ; however, recent evidence suggests the 26S is tightl y regulated and may not automatically degrade any bound protein 43 . Other endogenous caps exist, and the proteasome has been observed to exist as hybrid with two different caps 41 , 63 . In any event, the coordinated action of the 26S proteasome and ubiquitin is widely acknowledged as the main proteolytic pathway for selective degradation of misfolded or redundant structured proteins 68 . F. Overview of Gating Mechanism The proteasome gate is formed by th e convergence of the N - termini of the seven a l pha subunits (Figure 1.4). 9 Figure 1.4: Top View of Proteasome CP Depicting the Gate (Yellow) and Intersubunit Pockets (circles) Pockets formed between two adjacent subunits are used by endogenous protein act ivators (PA), like the 19S, for binding and ga te opening 69 . PAs ut ilize a conserved three peptide hydrophobic - tyrosine - variable (HbYX) recognition unit , found at the C - terminus of a number of proteasome binding partners including assembly factors 70 - 71 , to bind within these intersubunit pockets . One of the first studies demonstrating the importance of the Hb YX motif was done by Smith et. al. using PAN and archaeal 20S 71 . Systematic mutational studies demonstrate d that no AA substitution was tolerated at the penultimate tyrosine. Additionally , they discovered that lys66 within the - subunit was needed for PA - 20S association. Based on this information the authors concluded that the penultimate tyrosine and a preceding hydrophobic residue were essential for gating, but the terminal AA was only r equired for PA association and played no role in inducing substrate hydrolysis. Forster et al 72 demonstrated gate opening with a different PA, the 11S, which lacks the HbYX motif. In that complex, C - terminal residues form main - chain to main - chain h ydrogen bonding and a salt bridge between the C - terminal carboxylate and Lys 66. Interaction with the Pro17 reverse turn on the proteasome with a small (0.5 - 3.5 Å) movements of each subunit. Four conserved r esidues were identified as crucial to binding and stabilization of the open form: Tyr8, Asp9, Pro17, and Tyr26. Severe reduction in model substrat e degradation was observed in mutant archaeal proteasomes lacking any of these residues 72 . Rabl et al discovered a similar mechanism at play with the proteasomal ATPases in PAN (yeast version of the 19S) 73 . Using x - ray c rystallography, Rabel et al illustrated that 10 PAN also induced the same radial and lateral displacement of the alpha ring reverse loop without contacting Pro17 and instead attributed the displacement to HbYX binding. They proposed association of PAN with th e 20S through HbYX through contacts with Gly34, Lys66, and Leu81 induces the rotation of Pro17 away from the c entral pore and causes stabilization of the open - gate conformation. The importance of Lue81 was confirmed by mutagenesis 72 . C orroborated b y several studies on peptide mimics po sse ssing HbYX tails 71 , 73 , it was believed that the binding of the HbYX motif was sufficient to induce gate opening. However, recent Cryo - EM studies have revealed stabl y b ound eukaryoti c 26S structures with a closed gate 74 . In a more recent publication 75 , substrate engaged human 26S has been described in 7 different states: te rmed E A1 , E A2 , E B , E C1 , E C2 , E D1 , and E D2 . As the 26S progresses from E A to E D , the 1 9S is marked by major conformational changes as the Rpt units (Figure 1.3) insert more tails into the alpha rings (Figure 1.4). Intriguingly, the CP remains largely unchan ged until the final gate opened form E D . Figure 1.4: Rpt Tails Insertion Points Through Each 26S Conformer 75 Unfortunately, the authors did not comment extensively on what factors may be contributing to the opened form. Yet, their publication 75 does illustrate the mechanism of gate openi ng is far more complex than previously thought as 19S binding alone is 11 insufficient to induce an open - gate proteasome , a stark - contrast to previous reports ( vide supra ). G . The Ubiquitin Independent Proteasome System (UIPS) As mentioned above, th e UPS is an ATP - dependent pathway; however, ATP is only required for driving the unfolding of structured proteins. Once a protein has entered into the proteolytic chamber , it is degraded passively . To pass unaided though the gate, a protein must already be unfolde d. Two types of proteins fit this criterion: intrinsically disordered proteins (IDPs) and oxidatively damaged proteins (detail in the next section). 12 Figure 1. 5 : Simpl ified O utl ine of UPS vs UIPS While IDPs and oxidatively damaged proteins are unstructured enough to pass through an open gate 20S CP, the open and closed gate exist in an equilibrium heavily favoring the closed form (Figure 1. 5 ) 76 . Like the UPS pathway, the UIPS also contains specific PAs that bind and open the gate. The two main PAs for this pathway are the 11S (REG/PA28) and PA200. Both of these PAs bind in a similar manner to the 19 S (i.e. 13 utilizing the intersubunit pockets) yet form c omplexes with open ends allowing suitable substrates access to the proteolytic chamber 69 . H. Intrinsically Disordered Proteins Understanding IDPs as a br oad class of functionally important proteins began in earnest in the mid - 1990s with a bioinformatics study 77 on the emerging complete genome sequences. Analysis revealed that disordered regions were actually common in eukaryotic proteins 77 - 78 , with some estimates proclaiming that 44% of human protein - coding genes have disordered segments of at least 30 residues 79 . Today it is known that IDPs play indispensable roles in numerous cellular processes like signa ling, transcription /translation, and cell cycle progression 77 - 78 , 80 - 82 . The recent awareness in IDP res earch publications would suggest the se complexes are a recent discovery; yet, the reality is that IDPs have been reported periodically over the past 80 years 77 , 83 - 84 . A plausible explanation of this confusion, proposed by Uversky 85 , is a traditional lack of a un ifying terminology. IDPs have been previ ously described as floppy, pliable, flexible, partially folded, natively denatured, and many more 86 . It has also been suggested that the term IDP is not ideal; however, it is the currently recognized umbrella term found in literature. Additionally, a special issue of Chem Rev. covers classification comprehensively 87 . The accepted definitions, derived from common use in the field, may be listed as 87 - 89 : Any functional protein or protein domain possesse d of a unique 3D structure described by minimal fluctuation around their equilibrium Ramachandran angles is termed a 14 Any functional protein or protein domain that exists as a dynamic ensemble lacking specific equilibrium Ramachandran ang les with backbone atomic positions that naturally undertake non - cooperative conformational changes is termed an protein or region ( IDP or IDPR) . IDP/IDPRs are characterized by low sequence complexity and biased amino acid co mposition (preference for highly charged and hydrophilic residues) 89 . The lack of hydrophobic/bulky residues results in a relatively flat energy surface and existence as a 90 . D isordered regions leverage their high conformational freedom to maximize potential binding partners. Specificity is generally achieved per partner by multiple low affini ty contact points , and the entropic loss keeps the binding interaction transient. As the disordered domain gets larger, the increase in surface area of binding begins to compensate for the loss of freedom and binding time increases in permanence. The main functional features described above exist on a continuum , though may be mutually exclusive. As such, a single protein can be comprised of multiple disordered regions that belong to different functional classes, offering immense conformational variabi lity a nd adaptability. This is exploited by the cell to facilitate regulation through varied PTMs, and recruitment/localization of different binding partners. Recent reviews on IDP function 78 , 86 , 91 - 92 , role in cellular signaling 80 , 93 - 95 , advantages in protein - protein interactions 82 , regulation and disease 95 - 99 , empirical studies using single molecule methods 100 , NMR spectroscopy 101 , methods of characteriz ing conformational ensembles 102 , their identification 103 and specifics of IDP netwo rk interactions in great detail 104 are available and will not be discussed here. 15 Chapter 2. In Silico Investigation of Proteasome Regulators: Development of Theory of Action and Applications A. Introduction Previou s work in the Tepe Lab detailed the diversity - oriented synthesis of imidazoline scaffolds as potent proteasome inhibitors 105 . However, Dr. T heresa Lansdell discovered, in a non - standard assay, that one of the more potent compounds could exhibit enhancement of proteasome activity (i.e. proteasome agonism) . I found this dual abi lity to be both a proteasome inhibitor and activator, regardless of assay conditions, to be highly intriguing. I decided to investigate further with the aim of offering a plausible explanation of this odd behavior. For this, I turned to in silico methods , specifically molecular docking. Molecular docking, hereafter called docking, generally refers to the computational species. Most often, the two species are a protein - lig and pair. A bit more formally, the docking problem may be stated a s: Given the atomic coordinates of two species, predict In principle, only the structural information of the two species is required for docking. In prac tice however, docking is often complemented with biological or other empirical information to aid refinement. One of the first practical suggestions for docking was posited by Crick in the early 1950s 106 . He proposed that complementarity in helical coiled coils could b e modeled as knobs fitting holes. Yet, the first program written to 107 . calculating a smooth 3D contour about a molecule 108 was critical for the development of docking algorithms. The mid - 1980s would see the field begin to flourish with advent of the f irst docking program by Kuntz and 16 coworkers called DOCK 109 . Today, co mputer - aided drug discovery/design (CADD) plays an increasingly central role in the quest for small molecule therapeutics 110 . CADD encompasses a number of powerful methods to aid hit - to - lead campaigns including molecular dynamics (MD), protein - ligand and protein - protein docking, homology modeling, quantitative structure - activity (QSAR), and many others 111 . Reviews discussing 110 , methods of MD implementation 112 , and detailed discussion docking software 113 - 114 and potential pitfalls 115 are available. B. A Brief Overview of Molecular Docking Many docking programs are available today (click2drug.org and Wikipedia maintain impressive lists). In all cases, d ocki ng attempts to predict the orientation and conformation of ligand (called a pose ) complexed with its binding partner, most often a small molecule - protein complex. Generally, docking has two aims: accurate structural mod elling and correct predicti on of activity. Unfortunately, this far easier said than done. Identifying key molecular features responsible for biological recognition is a difficult task empirically, let alone predictively . As such, docking is routinely impl emented in a multi - step pro cess with each successive step adding complexity 116 . The first step is generation of a pose space. Dozens of implementations exist for this single step, but the idea is to cover as many conformations of the ligand of interest as possible in a reasonable amount of time. The search algorithm dictates how a ligand will be divided (if it is) and how the program will proceed in attempting to identify geometrically complementary surface i nteractions. Ligand - protein complexes with good complementarity are re - evaluated with a scoring function. The scoring function, depending on a particular implementation, will re - evaluate each pose on the basis of a more rigorous 17 geometric and other criteri a. The other criteria are heavily dependent on program used and may be purely energetic (force - field based) or purely knowledge - based, wherein poses are evaluated with how well they match similar structures in data banks . Finally, top scoring poses are e valuated a final time with a ranking algorithm which is usually attempts to account for as many factors as possible (entropy, solvation/desolvation energies, rotational freedom, etc) to yield an accurate binding affinity. A general summation of the preced ing paragraph is thus: all docking programs follow the same basic process. First is make multiple conformations of the ligand, bind each to the protein of interest, evaluate each and rank them in order of best to worst. The details of this three - step proce ss vary widely from program to program and even a brief description of each point would become a several page review of the field, which many are already provided 110 , 114 - 118 . Further, as the field has progressed , an d computational power has become more available, the once clear - cut denominations have blurred and mixed. Because of this complexity, interested parties are referred to th e an excellent, and brief, basic overview to docking from Jurgen 119 or for more detailed discussion of components, the one from Haleprin 120 . Ins tead of recapitulating those publications , I will discuss my chosen program in greater detail and why I beli eve to be a good choice compared to other options in the next section. C. Defining Boun dary Conditions At the outset of this project, compounds capable of enhancing proteasomal activity were sparse and an explanation for what an interaction may be occurring was non - existent. Additionally, a single paper reported transforming proteasome activ ators to inhibitors 121 t hough offered no mechanistic explanation. I midazoline TCH - 165 was unique as it 18 presented both of these activities without structural modification to the molecule . Intrigue d, I wanted to determine how such a thing was possible and believed my best chance wa s docking. After a long literature investigation, I d iscovered - docking program , and the success of such an endeavor would come down to accurate definition of boundary conditions and interpretation of results . Therefore, I defined the following boundary conditions. Equipment/Funds. Practically speaking, I would begin docking on my la ptop, a dual - core system with 8 GB of RAM. An excellent laptop for heavy student multitasking; however, it certainly was not meant for the computational complexity of molecular docking. The program would also have to be free as I personally could not affor d the monetized options and without positive results it something expensive. Available Crystal Structure. Fortuitously, the first human proteasome structure was published shortly after the start of this project 122 . From another project (see Chapter 4), I knew that species were different enough that accurate modelling would require a human crystal structure. Yet , t he resolution was limited to h this level of resolution is very near the 3 Å cutoff where only general protein contours can be made out. T his meant that during the searching analysis, the amino aci d side chains The lack of discrete side chain positions was problematic for two reasons. First, a crystal structure is a rigid image of a conformational ensemble. The proteasome in s olution is undoubtedly transitioning between energetica lly near conformers , which are lost on a 19 single structure . Many docking programs attempt to obviate this limitation by allowing side chains of interest to flex and move around a bound ligand and allow SAR progress as sidechains of interest in the available form could be identified. However, a static structure of clearly defined secondary structure - chains casts doubt on the calculation. Second, many docking programs weight hydrogen bond ing quite heavily based on directionality 120 . Chemically this seems reas onable; however, I balked at this as without sub 1 Å resolution (very rare for proteins) , positioning of hydrogens is pure guess - work and again, it is a non - dynamic system. I believed this would unfair ly weigh t potentially unwanted poses on the basis of a randomly found and completely uncertain H - bonding interaction. , and , thankfully , I was not the only one who thought this way (s ee next section). Finally, the sheer size of the prote asome rendered many server - based offerings used by other groups untenable and meant flexible side chain docking was unlikely due to computational expense . Also, the lab had numerous imidazoline scaffol ds to compare with and docking each manually would take an incredible amount of time. Consequently , I also sought some way to manage a robust workflow. 20 D. Why Chose Vina? Figure 2.1 : Published Utilization of Docking Software 1990 - 2013 Between 1990 and 2013, AutoDock was the most utilized docking program in the published literature. However, this lion share of utility is not correlated to performance 115 . AutoDock and GOLD are some of the earliest programs written and hav e had more time in the market to be assimilated by interested parties. Additiona lly, price is an unfortunate motivator for popularity as free options (AutoDock, GOLD, etc . ) continue to be overrepresented in the scientific literature regardless of suitabili ty to the problem at hand 115 . Despite this, I did look at AutoDock first. However, at the time , limitations on number of atoms , rotatable bonds , and grid map size were unacceptable limitations. Additionally, AutoDock employed a genetic search algorithm and a force - field based scoring method 116 . Neit her of th ese are particularly troublesome in the scheme of docking as a whole ; h owever, I had reservations due to my literature readings. In my readings, many purely calculation - based scoring methods were theoretically the best (i.e. , theoretically this 21 sh ould work!) , but performance was inconsistent . My thoughts on this are that despite a probably excellent physics - based force field, the simplifying assumptions imposed for usability purposes (time vs accuracy) hobbles the utility of these systems at the mo ment. For example, AutoDock applies the same energy potential for all hydrogen bonds, as opposed to different terms for different types, and then weights the contribution based on directionality 123 . The scoring calculation is very physics - based, based on the AMBER force field (ff) used to predict protein folding . AMBER c alc ulates the potential energy of the system by summing the contributions from v an der Waals energy, geometry (sterics), torsional energy, and covalently bound elements atom by atom 124 . This means, AMBER will calculate each individual H - bond ind ividually; however, AutoDock simply assigns a number to this interaction regardless of other factors , which seems counter - intuitive to the goal. T his is just one example of a very non - ph ysics - based simplification for the sake of computation time 123 . Other programs were considered as well though , I eventually decided to use AutoDock Vina. AutoDock Vina (hereafter referred to as Vina) had many attractive features. Vina used a united atom type scheme wherein each atom is assigned a type with a corresponding set of symmetric interaction functions based on interatomic distance 123 . This greatly reduces computational expense as representing the group . For example, instead of a methyl group representing a carbon with requisite functions app lied. In my opinion, this is conceptually brilliant as the author, Dr. Trott 123 , has shifted the focus from energies (force field and th e like) to chemical potentials. This is only a qualitative difference as force fields concern themselves with well 22 dept h of a potential while a chemical potential is also concerned with the shape of the potential. This is a difficult thing to describe s o o bserve Figure 2.2 . Figure 2. 2: Conceptual Illustration of Energetically Preferred States Docking methods that rely on energetic calculations for scoring are primarily g potential possible. However, as illustrated above, this often neglects the shape of the potential well this global minimum occurs in. In the above case , the global minimum is within a well with very sheer and narrow sides , which is entropically very disf avored 125 . Entropic effects are often ignored until the final ranking procedure; however, ranking does not eliminate calculated poses, it simply ranks them. A binding complex so entropically unfavored should be culled during initial scoring which Vina does. The scoring function is approach to the scoring function. - empirical, and a full d escription has yet to be published. What has been published 123 explains the process as follows: 23 Initial population of a pose space is generated (i.e. the program makes lots of copies with different conformations). The set of conformations is then evaluat ed for clashes or other disfavored interactions and are culled, reducing the set of conformations required to be docked. After initial docking, the bound conformation is immediately locally optimized using a quasi - Newtonian method 126 . In other similar programs, the compound would be randomly mutated and rescored. The two scores would be compared , the be tter score kept , and reiterated until the program is satisfied no other conformation is better. Vina mutates (the derivative in each dimension). Chemical ly, the gradient of the scoring function is the total force acting on the ligand and so accounts for more than just the sum of good vs bad interactions, it accounts for which direction is the bad coming from and the random change is then made in an effort to optim ize these interactions based on empirical considerations (i.e. a methyl group is more likely to be X distance than Y from group Z based on published structures) . After a set of locally minimized structures are generated, Vina then ranks each and as signs bi nding affinity values. A few final notes that need to be mentioned is that Vina does not explicitly utilize charged states nor hydrogens , though utilizes symmetric hydrogen bonding (i.e. directionality is ignored) . Chemical intuition may initially balk at this; however, it is a surprisingly useful implementation choice for my system in particular. Many programs implicitly use a single protonation state of the protein in question , and it is a non - trivial task to account for changes in protonation st ate of e very atom in a protein. The atom - type explicitly. Hydrogens are often ignored in most cases anyway , and , once again, the atom - 24 can H - b parameter to hydrogen bonding - capable group. However, by treating them implicitly, directionality is no longer a factor , and the goal becomes minimizing the distance between an H - bonding group on the ligand with that on the protein. In recent years, V ina has become one of the most cited programs in use for its incredible ability to quickly identify empirical binding modes. E. Results and Theory of Proteasome Activation I eventually was able to set up Vina on my laptop. To begin, TCH - 165 was drawn in relaxed using the buil - in MM2 force field and the relaxed structure converted to PDB coordinates. The PDB coordinates were converted to pdqt file format (required for Vina). The p r otea some crystal structure was accessed online from the PDB databank (code: 4R3O) . Organics and waters were removed, and the protein was converted to pdbqt format. Due to previously mentioned constraints, docking was done in stages ( F igure 2.3 ) . In the fi rst stage, unbiased docking was achieved by including the whole proteasome in the search space and allow ing unbiased conformational search. Figure 2.3 : Graphical Overview of Docking Process and Result 25 e program looks for a solution, search space. After the run complete d , I was amazed to find no binding modes were predicted exclusively on the alpha ring . I shr an k the search space and resubmitted the docking at a higher exhaustiveness (80 was the highest achievable on my laptop due to memory constraints). The final predicted binding mode (shown in yellow in the above figure) rested within an intersubunit pocket. O ther binding modes were predicted; however, The criteria for this identification was difficult to put together , though obvious now. The form of the p ro teasome crystal structure available was the close inactive form (see Ch. 1 for detail) and all the data suggested an active open form. As such, I was limited to an induced mechanism of action based on the available information and not on biochemical theo ry . Seeking an induced conformational change, I examined each predicted pose for potential interactions that would result in opening the gate (i.e. pulling this amino acid would pull this beta sheet, which would create space for the alpha helix to move bac k, which would affect gating residues). After settling on a subset of the predicted poses, I found that all of them resided in an intersubunit pocket formed by adjacent alpha subunits. Binding in the same pocket would plausibly behave as a competitive inh ib itor of the 19S cap resulting in an increase in ubiquitinylated proteins in cells, a published result 105 , while simultaneously mimicking the 19S activity and inducing an open form of the proteasome. 26 Chapter 3 . Phenothiazine Small Molecule Activators A. Introduction to Phenothiazine Activators and Chlorpromazine With a workin g theoretical frame - work in hand, the lab felt confident that proteasome activators of different scaffolds could be found. A high through put screen (HTS) was performed on the NIH Clinical Collection and Prestwick libraries . The compounds were ranked based on EC 50 values , and we were able to successfully identify several hit candidates ( Figure 3.1). Figure 3.1: Selected Results from HTS of Proteasome Activators 27 An EC 50 value represents the concentration at which a molecul e produces 50% of its maximal eff ect, in this case hydrolysis of peptides. The bioassay we use to evaluate is a kinetic assay where the rate h ydrolytic release of a fluorescent molecule bound to idealized peptides is taken from the linear portion of the cu rve (see Section D for detail) . S e veral recurring core structures stood out , phenothiazine chief among them, with activities in the low micromolar range . The phenothiazine core structure is quite old, originally prepared by Bernthsen in 1883 and is considered a privileged structure in me dicinal chemistry as they have been found to have insecticidal, antiseptic 127 - 129 , anthelmi ntic, anti - cancer 130 , antiemetics 131 , and antioxidants 132 . The identified phenothiazine compounds are all 1 st generation antipsychotics. These compounds are believed to bi nd to D 2 receptors and prevent access by the endogenous ligand (dopamine) 132 - 134 . Chlorpromazine (CPZ) was selected to be the hit compound due to good reproducibility in bioassays. The goal then became removal of the natural dopamine antagonism with preservation, if not improvement of, proteasome activity. Structural features required for the dopamine antagoni sm were fairly well known in the literature 135 - 136 (Figure 3.2). Despite this, I was unwilling to purse a traditional SAR project for several reasons. First, phenothiazines have long been known in the literature with many synthetic routes to desired structures available in the literature 137 . Synthesis would be non - trivial; however, there was very little room to pursue a worthwhile methodology. Second, due to th 138 , a group change could destroy both proteasome and dopamine activity while giving it some other unwanted activity . Next, random gene ration of analogues aimed at removal of dopamine activity while relying on bioassay evaluation 28 to determine if the change is good or bad, then making another random change equates to fumbling in the dark, and I would prefer a flashlight. Figure 3.2: Graphic of Key Dopamine Receptor Interactions with CPZ Finally, even a successful SAR campaign may end with a potent compound but no mechanistic insight as to how the effect is achieved. This could result in requiring a new SAR campaign i f the compound has off - target effects that have to be removed. Instead, I decided to use this opportunity to validate the docking model empirically in hopes of gaining some mechanistic insight while meet ing the requirements of the lab. B. Validation of Docki ng Model by Repurposing of Neuroleptic Agent CPZ was subjected to the sa me docking procedure detail ed in C hapter 2. Likewise, it displayed an impressive preference for the alpha ring intersubunit pockets and found lodging within an intersubunit pocket, su ggesting a similar mechanism of action to that of the imidazoline scaffol ds. T he binding affinities calculated by Vina (Figure 3.3, Table) had a very narrow range of values, despite some poses possessing very different orientations (Figure 3.3, Pose 1 vs P ose 2). Overlaying multiple binding poses (Figure 3.3A) revealed 29 prefere nce for the tail to be oriented downward toward the center of the enzyme. Investigating this area revealed a proximate arginine residue (Arg83, Figure 3.3B ) that was postulated to be the key residue anchoring the tail downward. As the terminal amine tail w as known to be key in binding to the dopamine receptor (Figure 3.2), it was targeted for replacement first. Figure 3.3: Binding Affinity Values for Top 9 Poses (Tabl e), Different Orientations with Similar Binding Affinity (Pose 1, 4, and 9), Overlay of Similar Poses (A) and Proposed Anchoring Residue Arg83 (B). 30 Two analogues were proposed based of the above model. A carbon analogue compound 3 - 1 (Figure 3.5) was propos ed as a negative control as replacement of the terminal nitrogen with a carbon would be expected to remove dopamine activity as well as its interaction with Arg83 (Figure 3.3B). Replacing the terminal amine with a sulfonate tail (Figure 3.5, compound 3 - 2 ) would instead be expected to make a powerful salt bridge with Arg83 w hile being unable to bind to the dopamine receptor. However, when both compounds were submitted to docking investigation both had no predicted poses within the intersubunit pocket. Accord ing to the current theory, both compounds would be expected to be ina ctive as proteasome agonists. Curious to know if the sulfonate was a bad choice or if a secondary factor was to blame, two additional analogues containing a longer chain of four (Figure 3 .5, compound 3 - 3 ) and five carbons (Figure 3.5, compound 3 - 5 ) respectively docked against the proteasome . Intriguingly, only compound 3 - 3 , four carbon linker , was predicted to b i nd in the same pocket, and incredibly in the same pose as CPZ (Figure 3.4)! Figure 3.4: Overlay of Predicted Binding Modes of CPZ (white) and 3 - 3 (orange). 31 This was a fantastic result as the synthesis of all four analogues ( Figure 3. 5 ) and biological testing would allow swift determination of how trust - worthy the predictive power of docking could be in the absen ce of a known binding site. Figure 3. 5 : Synthesis of First - Generation Validation Compounds Compound 3 - 1 was generated by treatment of the phenothiazine core with sodium hydride in tetrahydrofuran (THF) followed by addition of the alkyl halide. The reaction proceeded fairly smoothly; however, purification was unfortunately a challenge. The extreme similarity between product and starting material resulted in co - elut ion regardless of solvent polarity. Selective precipitation of the starting phenothiazine core with chloroform ultimately provided the alkylated core. Synthesis of the 3 - 2 and 3 - 3 proceeded smoothly with treatment of deprotonated phenothiazine with the app ropriate sul t one which precipitated upon cooling. Synthesis of 3 - 5 was a little more involved requiring alkylation 32 of the phenothiazine core to give 3 - 4 followed by Finkelstein conversion of the ter minal halide to an alkyl iodide before displacement with s odium sulfite to attain 3 - 5 . The results were outstanding 139 . As predicted, compounds 3 - 1,2 , and 5 were inactive in testing while compound 3 - 3 showed excellent dose - response up to 8 - fold over proteasome alone (set to 100% i n Figure 3.6A ) and activating all three catalytic sites (Figure 3.6 A , yellow, red and purple lines ). Figure 3.6: Dose Response of 3 - 3 ( Top ) , IDP Degradation (B), and Complex Selectivity (C) Compound 3 - 3 was also investigated for its ability to enh ance degradation of an IDP (Figure 3.6B). From left to right in Figure 3.6B, lane one illustrates a control for where alpha synuclein ( synca ) resides on the gel. Lane two is the effect the proteasome alone on synca digestion (the smaller fragments below th e synca level). Lane 3, 4, and 5 show Log[M] 3 - 3 B C 33 increasing digestion (absence of fragments) as t he dose of 3 - 3 increases. Lane 6 contains a negative control , BTZ. BTZ is Bortezomib, a proteasome inhibitor which prevents the 20S from degrading any proteins and resul ts in no change from the synca control in Lane 1. The final lane uses CPZ as a positiv e control to ensure validity of the assay and also demonstrates the increase in 20S proteolysis induced by compound 3 - 3 at the same dosage (lane 4 vs lane 7). GAPDH is an added protein used to ensure equal loading of sample . As each lane has an approximate ly equal amount of GAPDH, we can be confident that the differences in synca is due to proteasomal degradation and not simply different amount of protein. GAPDH, as a stru ctured protein is not susceptible to degradation by the 20S CP (see Chapter 1 for deta ils). Improvement in IDP proteolysis strongly supported the theory that proteasome stimulation could be a viable therapeutic strategy (see Chapter 1). 3 - 3 was also invest igated for its effect on the 26S proteasome (Figure 3.6C). Excitingly, only the 20S co re particle showed increased proteolysis in the presence of 3 - 3 but had no effect on the 19S (Figure 3.6) ; strongly support ing the docking model that binding is occurring utilizing the same binding site as the 19S. Despite the inarguable success of compound 3 - 3 , it still had a few undesirable features. The permanently charged tail is unsuitable for cell permeability 140 , at very high concentrations inhibition of the proteasome was observed (a property of detergents 141 ) . Additionally, polymorphism of the compound was suspected as different batches possessing identical s pectral data performe d differently under the same assay conditions. As such, a more drug - like molecule was sought to become a new lead structure to explore a docking guided SAR. Toward that end, a number of potential compounds were designed with the aim to further validate the docking model as well as yield a suitable lead compound . 34 Figure 3.7: Synthesis of Lead Candidates Compound 3 - 7 was proposed to investigate the flexibility of Arg83. Docking predicted the 3 - 7 would be inactive; however, as flexibility in protein side changes could not be simulated (see Chapter 2 for detail), such an induced change could not be checked computational ly. Synthesis of 3 - 7 was therefore purs u ed via alkylating the core scaffold with propargyl bromide to yield 3 - 6 . The terminal alkyne was deprotonated using nBuLi , and the resultant anion used to trap CO 2 giving the desired product 3 - 7 . From the activity di fferences between compounds 3 - 2,3, and 5 , it was known that tail length had a strong effect on the ability to stimulate proteasome activity. A ligand with less conformational freedom often binds more effectively to a protein binding pocket due 35 to lower ent ropic penalty 125 , 142 . As such, we sought to lower the number of rotatable bonds while preserving favorable interactions with Arg83. Compound 3 - 9 was expected to offer a more rigid system than 3 - 3 while preserving the ability to interact with Arg83 . Synthesis was achieved through b enzylation of the core structure followed by hydrolysi s provided the desired compound with minimal issue. We were also interested in exploring the tolerance of functionality at the end of the tail. As stated above, the conformational flexibility may be a detriment; however, the excellent activity of the sulf onate analogue made us unwilling to completely abandon the structure without a n alternative. An analogue containing an amide tail (compoun d 3 - 11 ) was generated by alkylating the phenothiazine core with valeronitrile (compound 3 - 10 ) followed by acid catalyz ed hydrolysis. A morpholine tail was also explored as a mimic for the active tail of several of the original active compounds (Figure 3.1, entry 1,4, and 5). Synthesis was pursed in a similar manner to 3 - 5 except displacement of the terminal iodide was don e using morpholine to give 3 - 12 . It is also worth noting that the flexible ester and carboxylic acid were obvious additions to this explo ration. However, during the course of the synthesis of these compounds, it was found that this compound would be unsui table. As previously mentioned, purification of these compounds was incredibly challenging /frustrating . Often, the resultant product mixtu re would be purified via column chromatography using gravity to isolate a mixture of phenothiazine starting material a nd product. This mixture would then have to be suspended in chloroform and chilled overnight to precipitate out phenothiazine to give a pr oduct. In the case of the flexible ester tail, the acidity of the silica gel resulted in some amount of hydrolyzed pro duct. The carboxylate, in solution, then 36 began catalyzing the decomposition of the product into more phenothiazine starting material and a volatile side product (Figure 3.8). Figure 3.8: Synthesis of Flexible Ester and Depiction of Decomposition This particular process was challenging to determine as after filtration and drying, it simply appeared as though the precipitation was incomplete. However, due to an impossible mass balance (I had isolated more phenothiazine fro m this process than should be possible based on crude mass), I investigated on a small scale with deuterated chloroform and was able to iden tify the lactone in the mixture. Synthetically, this is easily remedied . However, this decomposition pathway is like ly to be far more prevalent under assay conditions, and even more so in a cellular assay. Because of this, it was agreed that pursuit of thi s compound should be abandoned due to it being unsuitable for biological testing. 37 Desired compounds in hand, they were submitted for biological evaluation ( Table 1 ) . 20S D2R Compound R EC 50 (µM) Max Fold %Inhibiti on Ki (µM) CPZ 9.9 20 77.9 0.48 3 - 1 >25 -- 0 >250 3 - 2 >25 -- -- -- 3 - 3 6.3 8 - 4.5 >250 3 - 5 >25 -- -- -- 3 - 7 >25 -- - 2 >250 3 - 8 15.6 10 2 >250 3 - 9 6.4 2 - 3 1 >250 3 - 12 8.9 4 74.5 2.97 20S Assays per formed By Evert Njomen Dopamine Activity Assays Performed by Dr. Benita Sjogren Table 3. 1 : Tabulated Depiction of Bioassay Results . 139 Unfortunately, though not entirely surprising, 3 - 12 was a poten t dopamine antagonist and limit s functionality options for this position (i.e. no basic amine functions lest they regain unwanted off target effects). Compound 3 - 7 was completely inactive, sugg esting Arg83 does not have a great deal of motion available to it as 3 - 9 was quite potent. The amide derivative 3 - 11 (Figure 3.7) was likewise inactive in the bio assay screen but was not tested for dopamine activity. T wo active candidate compounds ( 3 - 8 and 3 - 9 ) were tested by Evert Njomen in a manner similar to 3 - 3 ( protein assay Figure 3.6B) and 3 - 8 was chosen as the lead compound due to superior ability to degrade IDPs 139 . 38 C. Attempts at SAR: Challenge s of Irrational Data The beginning of this project was an inarguabl e success. We had demonstrated the utility of a docking - based theory for identification of proteasome agonists (literature first) , found a new scaffold capable of such agonism (the phenothiazines , literature f irst ), illustrated its ability to remove IDPs implicated in commo n neurodegenerative pathologies in cells ( offering a new strategy for targeting such pathologies ) , and demonstrated w e could remove undesirable off target effects. Our interest then turned to making a more drug - like proteasome agonist. With little literat ure precedence, we arbitrarily decided that any compound unable to produce 2 - hydrolytic activity) would be considered inactive as a rou gh guide to aid in optimization. Many strategies were discussed; however, i n the course of synthesizing the previously discussed compounds and other unpublished attempts (not detailed herein), the phenothiazine core was found to be intermittently thermo a nd photo - sensitive depending on group attachments, though no clea r pattern emerged to predict this. Additionally, the nucleophilicity of the ring sulfur interfered with derivatization steps attempted on the sulfonate analogues. Many other points against co ntinued use of the phenothiazine core can be give n ; however, the consensus was the first priority should be its removal. As removal of the core structure was desired, investigation began there with the goal of logically transitioning to new chemical space. Using the candidate 3 - 8 ester tail, several analogues were gene rated via benzylation . A general procedure was discovered to give acceptable yield and ease of purification which was used in the generation of all benzylation reactions afterward. Using DMF as a solvent, phenothiazine would be deprotonated using sodium hy dride to give a red to red/orange solution. The desired 39 e lectrophile would then be added as a single portion and stirred at room temperature wrapped in foil for 16 hours (Figure 3.9) . The initial strategy was two - fold. First, the ring substituent was exami ned for its effect on activity. Figure 3.9: Analogues of Lead Compound 3 - 8 The chloro - substituent was replaced with hydrogen to probe for a potential halogen bond ( 3 - 13 ). Trifluoromethyl was examined as it has literature precedence for increasing cell permeability and also act s as a powerful electron withdrawing group , though lacks - donor ability (which the chlorine possesses) 143 - 144 . The thio - ether ( 3 - 16 ) was examined because it was the core structure that displayed greater activity in the original HTS (Figure 3.1 entry 1). The methoxy substituent ( 3 - 15 ) was desirable as it had been shown to greatly increase activity of the previously disc ussed imidazoline scaffolds. We also examined the requirement of the sulfur atom. The ethyl linkage ( 3 - 1 7 ) was chosen over a single methylene unit because two methylene allowed minor flexibility while preserving the overall geometry 3 - 8 without the sulfur atom. Excising the sulfur entirely ( 3 - 18 ) allows the phenyl rings to expand and greatly changes the shap e of the molecule. For t he next discussion of bioactivity results , it is important to note here the intentional omission of collected bio data about to be discussed. It has been omitted for 40 narrative coherence and will be discussed in detail in the next s ection (Section D) in great detail . For now, activity results will be discussed in general terms. Surprisingly, 3 - 15 and 3 - 18 were reported to be ina ctive while the remainder of the analogues had approximately the same potency. This was very difficult to rationalize using docking as the uncertainty in the binding mode meant several different poses could be the active one and each compound bound within an intersubunit pocket; although not all in the same one. The result was suggestive that the tail had g reater effect in activating the proteasome than the attached core structure. To evaluate this theory, both the phenothiazine (Figure 3.10, center left) and the iminodibenzyl (Figure 3.10, center right) scaffolds were used (Figure 3.10). However, early in this investigation it was clear something was wrong. Both compounds were performing approximately the same in in vitro tests. Also, equal potency was re ported for the para - methyl ester (Figure 3.9, compounds 3 - 8 and 3 - 17 ), the meta ester (Figure 3.10, comp ounds 3 - 2 1 and 3 - 2 2 ), and even the unsubstituted benzene ring s ( Figure 3.10, compounds 3 - 19 and 3 - 20 ) provoked 2 - fold activities, albeit at high EC 50 v a lues (>25). These results (all behaving basically the same) were both unexpected and frustrating. The a bsence of the ester moiety was expected to drastically decrease activity of the compounds and allow the determination of which core was more suitable to explore. The size of the intersubunit pockets was suspected to be a contributing factor as the amount o f available binding surface made them oddly accommodating. 41 Figure 3.10: Synthesis of Tail Analogous Expanding the structure outward was debated; however, the project goal of maintaining drug - like propert ies limited mass addition to approximately 120 (as the average masses were ~380 and most sources claim a molecular weight of 500 should be the goal 145 - 146 ) which would not go far in expanding into the pocket. Further, the on/off nature of the analogues made docking refinement impossible. Nine predicted binding poses across 3 binding sites meant an argument could be made for any site. A d istribution of activities was needed to make proper progress as on/off was simply to o extreme to effective ly build more from theory. As I was unwilling to generate analogues through random synthesis of whatever was in the organic cabinets, I took inventor y of what we had discovered and its relationship to the known literature. Despite SAR stagnation, a few key points could be glea n ed: Core Structure requires some rigidity (compound 3 - 18 vs everything else) Tail Length is important but variable (compound 3 - 7 vs 3 - 9 and 3 - 3 vs 3 - 4/5 ) 42 Some measure of hydrophobicity is required alon g an edge of the molecule (performance of phenothiazine and iminodibenzyl core vs others) Ring substitution apparently does not matter (compound 3 - 13 vs 3 - 8 ) On top of my own work, l iterature at the time offered a few additional suggestions. Mutational studies on the C - terminal HbYX motifs ( Chapter 1) demonstrated the necessity of a penultimate tyrosine residue and provided evidence that the C - terminus carboxylate was required for ass ociation but not activity of the 19S - 20S complex 70 . This provide s a possible explanation for the remarkable similarity of activity between ester, carboxylate, and the lack thereof should it be aid ing in orientation but not effect. I also went back to the original HTS list in an effort to see if a functional group suggestion could be found. Each potent compound possessed a tertiary amine tail (which everyone noticed); however, what now stood out was the pKa similarity between the ter tiary amines, approximately 10 for the protonated analogue, and the required phenolic tyrosine pKa (9.6). Additionally, Trader and co - workers published the identification of two new proteasome stimulators AM - 404 and MK - 8 86 with strikingly similarity to ou r published compound and the back - bone tyrosine found in HbYX motifs (Figure 3.11) 147 - 148 . 43 Figure 3.11: Proposed Activator Structures with Key Interaction Motifs Highlighted As the phenolic tyrosine had been demonstrated to be necessary for activity 149 , the obvious course of action seemed to be attachment to the phenothiazine core. By the same notion, attachment of the benz yl phenol at the nitrogen of an indole with a C2 carboxylate would make a very near mimic for the endogenous HbYX tails. As the meth yl ester appeared to be providing some stimulatory activity to our previous compounds, I was curious to see its effect on an indole. Likewise, with a C2 carboxylate, but also with a thioether in the C3 position. This compound would be an effective mimic fo r 3 - 13 and allow more investigation of the phenothiazine core. Synthesis of the indole analogues (Figure 3.12) was done in multiple steps. Nucleophilic attack on an activated disulfide gave the indole thioether 3 - 23 . After purification , this was benzyla te d with our general procedure (sodium hydride in DMF followed by electrophile) to yield the target compound 3 - 24 . 44 Figure 3.12: Synthesis of HbYX Small Molecule Mimics The electrophile 3 - 25 was synthesized via benzyl ic bromination in freshly distilled chloroform and coupled with anionic phenothiazine to yield 3 - 28 which was hydrolyzed to yield the desired compound 3 - 29 . Instead of installing the C2 carboxylate, the commercially available C2 indole ester was benzylated to give the phenolic precursor 3 - 26 . T he indole ester was also benzylated with the 3 - 8 tail to see if the diester species w ould be active. Unfortunately, only compound 3 - 28 was active though, as its hydrolyzed product was inactive, 3 - 28 is an unsuitable compound for further optimization as it would be expected to hydrolyze to the inactive compound under cellular conditions. C oncomitantly, with the above synthetic and computational hurdles, periodic complaints about the reproducibility o were raised. Some would 45 be minor variance expected in any biological system; however, others raised serious concerns as some compounds activity would drop below our 2x threshold. A scaffold that sometime s would be disastrous. As accurate and reliable bioactivity was crucial to the success of my project, I transitioned into the b io - lab to investigate assay conditions . D. Optimization of Proteasome Agonist Evaluation and Work Flow The original protocol for the biological assay most commonly performed by our lab is as follows: Purified 20S proteasome is dissolved in a suitable buffer to maintain its biological activity. Drug is added , and the mixture is allowed to sit for 10 - 15 minutes . An ideal ized peptide bound to 7 - aminomethylcoumarin (AMC) is then quickly added , and the rate of fluorescence measured over time at 37 °C for 1 hour. R ate is determined by taking the slope of the line of through the linear region of the relative fluorescent unit ( RFU) vs time graph . By varying the concentration of drug applied and keeping all other concentrations constant, a dose - response curve of activi ty vs concentration can be drawn, and different drugs compared for their effect on the proteasome , most commonly based on their EC 50 value . An EC 50 value is the effective concentration (EC) that produces half of the maximum response ( 50% ) and graphically i s the inflection point of the dose - response curve. After being taught the procedure and demonstrating competency , I began testing tracked the cause to be an unstable veh icle (Figure 3.13, left). This discovery was distressing as the effect of a drug is now variable and a positive o r negative determination was nearly random. For example, an example compound that adds 0.02 to the rate of 46 hydrolysis may measure <20% activity increase (Figure 3.13, Trial 2, blue block) in one trial while measuring double the activity in another (Figure 3.13, Trial 3, blue block). Figure 3.13: Illustration of Vehicle Stability Issue A systematic investigation into the factors that can affect the proteasome background activity was undertaken. Determination of effects was a time - consuming process, as eac h change had to be subjected to the full protocol to determine outcome. Numerous f actors such as salt concentration, cation source (i.e. potass ium vs sodium), presence/absence of salt in buffer, as well as resistivity of the water were examined , and a summary of findings and corrective actions are depicted below ( Table 2 ). With greater understanding of factors effecting proteasome background act ivity, attention turned to modification of the protocol. Some changes made were by necessity. For example, ma ny unusable data points are generated while the proteasome, drug, and substrate (fluorogenic peptide) cocktail warm to assay temperature due to the changing pH of the medium , and the fact kinetics of chemical reactions tend to increase with increasing temp erature. 47 Table 3. 2: Summary of Sources o f Error and Corrective Actions As such, incubation of drug and enzyme at assay temperature beforehand addition of substrate and immediate reading was an obvious modification. However, more chang es needed to be made. The original protocol performed a full d ose - response on every c ompound, limiting each plate to a maximum 4 - 6 compounds. As the synthetic section of the lab expanded and purification of compounds was becoming easier due to lab acquisition of a medium pressure liquid chromatography instrument (MPL C, colloquially referre d to as an auto - column) , analogue generation was rapidly progressing. This required a more efficient work flow to accommodate the newfound analogue backlog. To achieve this, a pre - screening protocol was generated. By checking each co mpound at three concent rations, Error Source Problem Correction Buffer Variable pH with Temperature Instability of solvated Tris (~14 days) Daily pH change Buffer is to be made fresh and pH adjusted daily to a pH that will give 7.4 at 37 C. After 2 weeks buffer is disposed of, sooner if required. Water MiliQ water was not stabl y at high resistivity. Cascadingly effects pH, protein folding, etc. Accounted for >2 - fold a ctivity changes in the same batch of 20S proteasome MiliQ water is obtained from a different lab with reproducible and stable water supply. Salt effects Addition or removal of salt greatly affects the activity of the proteasome and was sometimes added, s ometimes not. This has wide ranging effects. water and sodium chloride gave the most consistence results across multiple assays and so was chosen to be in all in vitro assays pH probe pH probe was found to be inconsistent/faulty. Probe replaced. Enzyme Equilibrium Immediate data collection gives wide variety of outputs due to the changing pH (Tris) and changing kinetics as the enzyme warms. Incubate drug and Enzyme at 37 C before addition of substrate. 48 in duplicate, up to 13 compounds could be screened simultaneously. Further, discussion with a SpectraMax technician identified other variables that could optimize performance : lowering the filter cutoff to more than 5 nm bel ow emission and volume in wells being key incorporations . Volumes were reduced to confident minimums allowing 50% reduction in enzyme usage. These changes allowed the same amount of enzyme to be utilized for twice as many assay plates with each plate conta ining >2x the usual num ber of compounds. A screening protocol was also established with : ( 1) a three - point screen graded on a pass/fail basis to determine active compounds ( 2) active compounds being titrated to determine EC 50 points , and ( 3) most potent an d drug - like compounds b eing introduced to a cellular assay as a final evaluation. Using this protocol, I re - tested all of my compounds ( Table 2 ) as well as began testing compounds for other lab members . Disappointingly, the HbYX mimics were inactive. Never theless, active compoun ds were carried forward to obtain EC 50 values. Once again, despite good data the values were rather flat and still had the appearance of on/off regulation. Having now been so involved in the evaluation of compounds, I proposed a chan ge to the goals of the project ( vide infra ) . To this point, the assays looked exclusively at the chymotrypsin - like activity of the apter 1 for detail). This was done as the majority of the literature supported the idea that this site was the most important to targe t 150 - 153 . However, that conclusion was drawn by looking specifically at inhibition , not activa tion , and the endogenous 20S CP the other sites be examined for thei r effect? Also, with a stable assay came stable EC 50 values and with multiple compounds to draw on, a glaring issue with the EC 50 metr ic was brought to the fore. 49 Cmpd R R1 A/In Cmpd R R1 A/In 3 - 1 Cl In 3 - 15 A 3 - 8 A 3 - 16 A 3 - 9 Cl In* 3 - 19 Cl A 3 - 10 Cl In 3 - 21 Cl A 3 - 11 Cl In 3 - 28 Cl In 3 - 13 H A 3 - 29 Cl In 3 - 14 A 3 - 30 Cl A Cmpd R R1 A/In Cmpd R R1 A/In 3 - 17 Cl A 3 - 22 H In 3 - 20 H In 3 - 31 H In 3 - 27 In 3 - 24 In Table 3.3 : Table of Screened Stimulator Compounds As mentioned previously, an EC 50 value is calculated from the dose - response curve and enables comparison of multiple compound s potency (Figure 3.1 7 A ). Notice, however, that the assumption is that all compounds involved (the three lines) are capable of producing a maximal effect. 50 Figure 3.1 4 : Illustration of EC 50 Value Use (A) and Sample Project Data (B) Unfortunately, project data (Figure 3.17 B ) demonstrates that this assumption can be misleading at times . According to the EC 50 superior to the black line. Method s exits to account for such partial agonism; however, they all rely on knowing what 100% agonism is. For proteasome activity, this is an unknown quantity. Over reliance on EC 50 values was also beginning to show in the literature as compounds were beginning to be published with low micromolar EC 50 values but producing less than 50% increase in proteasome activity 147 - 148 , 154 . This gap in our knowledge base is the real barrier to improved compound activities , and we really needed to know if we should concern ourselves with the other active sites and what metrics translate to a biological system . With so many unkno wns, SAR could not realistically advance until more As such, efforts were aim ed at determining what factors translate to the protein level . A B EC 50 51 E. Construction of New Metrics for Agonism Effectiveness Fi rst, I checked our lead compound and chl orpromazine for activity in each of the three catalytic sites and the mixture (Figure 3.18) . Figure 3.1 5 : Comparison of Compound 3 - 8 and CPZ Activities per Active Site ivity is limited to only the chymotryptic - like N ote Evert Njomen tested CPZ for all three sites ori ginally , and my data matches hers). Intriguingly, compound 3 - 8 produced the greatest effect on the t rypsin - As the project goals had now changed to exploratory as opposed to performance oriented, on ly the phenothiazine compounds , along with some newly synthesized phenothiazine analogues, were subj ect to the screening protocol for each site and the mixture to give a pure data set ( Table 3.4 ; note that inactive compounds are omitted). Additionally, as the lab was strongly opposed to interpret activity based on EC 50 values, we decided to look at a dif ferent metric: the AC 200 . AC 200 is the concentration of drug that produces double the Fold Activity 52 Table 3.4: Table of Individual and Combination Activities activity of the enzyme. compound can be reliabl y compared. Fold M is another new metric that stands for maximal fold activity (i.e. the maximum effect this compound can produce is the listed fold over unactivated proteasome). Finally, significant differences in activity across multiple domains co uld be seen. Many differe nt ranking orders can be offered by simple arranging compounds from best to least in a desired bracket (Figure 3.30). However, determining which one to follow would be decided by protein degradation studies. To that end, Evert Njom en took a selection of my - synuclein (Figure 3.21). Caspase 1 Trypsin 2 Chymotrypsin 5 Combo 1:1:1 Cmpd R R 1 R 2 AC 20 0 Fold M AC 200 Fold M AC 200 Fold M AC 200 Fold M 3 - 8 Cl H CO 2 CH 3 2.7 7 2.6 15 1.9 12 1.4 9 3 - 14 CF 3 H CO 2 CH 3 X X 2.8 6 13.5 2 1.9 9 3 - 16 SEt H CO 2 CH 3 X X 2.2 3 X X 1.4 3 3 - 13 H H CO 2 CH 3 X X 4.7 5 1 5.4 2 4.4 4 3 - 30 Cl H CO 2 (CH 3 ) 3 X X X X X X X X 3 - 21 Cl CO 2 CH 3 H 9.7 4 1.9 8 2.4 6 2.4 6 3 - 9 Cl H CO 2 H 10 2 10 2 >30 2 10 2 3 - 31 Cl CO 2 H H X X X X X X X X 3 - 32 Cl NO 2 H 1.2 12 1.6 8 1.1 9 1.1 8 3 - 33 Cl NH 2 H 3.8 10 9.2 10 6.5 9 3.6 8 3 - 34 Cl OCH 3 H 1.3 4 5.4 2 X X 1.5 4 3 - 19 Cl H H X X 3.1 2 2.8 3 1.5 2 CPZ Chlorpromazine X X X X 7 >4 13.5 4 53 AC 200 Fold Combo Combo 3 - 32 3 - 32 3 - 32 3 - 32 3 - 8 3 - 13 3 - 8 3 - 8 3 - 8 3 - 34 3 - 21 3 - 8 3 - 32 3 - 16 3 - 33 3 - 33 3 - 16 3 - 8 3 - 16 3 - 21 3 - 33 3 - 14 3 - 32 3 - 32 3 - 34 3 - 33 3 - 8 3 - 19 3 - 21 3 - 19 3 - 21 3 - 21 3 - 19 3 - 21 3 - 14 3 - 33 3 - 13 3 - 31 3 - 14 3 - 19 3 - 21 3 - 9 3 - 19 3 - 14 3 - 34 3 - 32 3 - 13 3 - 14 3 - 14 3 - 31 3 - 13 3 - 13 3 - 16 3 - 33 3 - 16 3 - 13 3 - 33 3 - 16 3 - 34 3 - 9 3 - 14 3 - 8 3 - 19 3 - 9 3 - 13 3 - 19 3 - 33 3 - 31 3 - 19 3 - 21 3 - 34 3 - 30 3 - 9 3 - 14 3 - 9 3 - 34 3 - 9 3 - 34 3 - 9 3 - 16 3 - 30 3 - 13 3 - 31 3 - 16 3 - 31 3 - 9 3 - 31 3 - 34 3 - 31 3 - 30 3 - 30 3 - 30 3 - 30 3 - 30 3 - 30 3 - 31 Table 3.5 : All Top - to - Bottom Ranking Combinations The results are quite interesting. From left to right in Figure 3.21, once again is synca control next to a vehicle control. We see t hat each active compound is indeed capable of enhancing synca degradation. The two standout observations are lanes 6, 7, and 8 holding compounds 3 - 13, 33, and 32 respectively. Compounds 3 - 13 and 33 are able to completely remove synca while compound 3 - 32 appears to preferentially remove digested pieces first as they are absent in the 1 hour treatment (top image) and barely observable in the 2 hour treatment (bottom image), presumably this is because asyna has begun to be d egraded again. Ranking these compounds on the basis of this degradation would likely be 3 - 33, 13, 8, 32/14 based only on the disappearance of synca . Which of course does not match any of the possibilities listed above (Figure 3.20). Note these results are bizarre as compound 3 - 13 , for example, is by every metric an inferior compound to compound 3 - 8 ; h owever, it performs as well as if not better. The different site stimulation of each site compelled me to look deeper. 54 Figure 3. 16 : S elected Compounds Effect on Protein Degradation oteins and would that explain the somewhat bizarre results of the digestion? To investigate this, I took the gene sequence from the variant used in the above a ssay and subjected it to PAProC analysis. PAProC is an online server - based service which computat ionally predicts which sites will be cleaved by the human 20S proteasome. Inspecting these predicted sites, I assigned them a likely catalytic site. For exampl e, prediction of cleavage at an acidic residue would be assigned specificities 40 (Figure 3.2 2). cleavage s 155 and a compelling proposal can be made. If what has been presented so far is true, then to degrade synca (not its degradation products but the 55 protein itself) as most useful (as hi Figure 3. 18 : Illustration of PAProC Analysis and Table of Assignents This would explain the pronounced ability of 3 - 13 to degrade synca as it is unable to en re 3.1 8 ) . The resulting degradation products appear to be cleared best by overall activation of the proteasome, in which case the Fold M activity of the combination (Figure 3.20, column 5) matches the best for cle arance of these species. F. Theory Update, Con clusion, and Future Outlook Elated at the breakthrough we discovered in the previous section (i.e. how to effect change at the protein level) , the obvious question remains , how can a small molecule produce such d ifferent effects? The proteasome active sit es contain a conserved catalytic triad of Asp17, Lys33, and Thr01 (Figure 3. 19 A ). Proteolysis requires the concerted action of all three amino acid s 156 . By examining recent crystallographic data 50 , 75 , 122 , 157 , I found that the catalytic sites undergo a range of changes varying from nearly identical to markedly different (Figure 3. 19 B). For the sake of illustration, assume the magenta form of the catalytic si te shown is active and the yellow form is inacti ve. 56 Figure 3. 19 : Illustration of Catalytic Site Conformers These two forms likely exist in a dynamic equilibrium and our small molecules must be biasing inactive conformer of Bas ed on biochemical theory, conformational trapping is more likely . Fortuitously, solved crysta l structures of the 20S CP possessing an open gate portion were recently published this year 75 and access to the powerful high - performance computational cluster (HPCC) here at MSU allowed high exhaustiveness docking to compar e both the open and closed forms (Figure 3.2 0 ). 57 Figure 3.2 0 : Pocket Preference Difference Among Different 20S Conformers The remarkable change in binding pocket preference strongly suggests a mechanism of trapping an existing conformation and not bi nding followed by a conformational change (recall the original theory was pigeon - holed into this theory due to available data). Unfortunately, despite many forms of the crystal structures available with intersubunit pockets bound to Rpt tails (see Ch apter 1), no investigation of key interactions has been done. Below I provide my own thoughts on what interac tions may be present and suggestions for future directions of this project (Figure 3.2 1 ). Excellent work published on 26S dynamics 41 , 62 , 67 , 75 , 158 allows for great understanding of substrate recognition, unfolding, and translo cation events; however, due to pocket geometry, a variety of PA - CP interactions may be plausibly proposed resulting in limite d mechanistic understanding of which residues are key to generate an open gate CP. Among the 11 states listed above only the C - term ini of Rpt3 is generally unchanged whereas Rpt5 goes through minor conformational changes and Rpt1, 2, and 6 display high var iability between states limiting us to only a few general observations. 58 Figure 3.2 1 : Intersubunit Pockets bound R pt3 appears to operate as an anchor for the 19S base as strong hydrogen bonding interactions are present at multiple points along the bottom crystalline fo rms despite large conformational changes in the lid and minor changes in the alpha ring geometry. This binding dynamic is the same in the other HbYX containing Rpt2 and 5 (i.e. hydrogen bonding network along the back and bottom of one subunit and front of the adjacent subunit). Rpt5 displays minor conformational changes through the substrate processing process while Rpt2 possess es even more varied forms while preserving the number of contacts if not the same contacts . 59 However, Rpt1 and 6, lacking the HbYX m otif, begin changing this commonality. Rpt1 binds most similarly to Rpt2, 3, and 5; however, it also bridges off to attack mo re accepting a hydrogen bond from L27. In Rpt6, t his multi - subunit binding breaks down R cited as the most important subunit i n gati ng as it possesses the greatest amount of electron density over the CP opening. Perhaps this extreme binding interaction with the Rpt6 tail is required to induce the conformational swing that moves the N - termini away from the CP opening; however, no clear indication of how this is achieved is currently available. The above brief discussion provides a number of possible interacti ons and key residues utilized by numerous endogenous proteasome activators while also offering general comments about the apparent pocket geometries. I would suggest future endeavors utilize docking to provide a starting point for small molecule diversific ation, as was done at the beginning of this project. With the new metrics provided, reliable analogue differentiation can be achiev ed and progress on small molecule proteasome activators should increase. It is also strongly suggested that molecular dynamic s be pursued as potential binding modes can be checked on their effect on the proteasome gate computationally. This would be a key contribution as intersubunit binding is most likely based on current information; however, this does not preclude the possibi - ring/small molecule event and a computational simulation demonstrating stabilization of an open gate conformer would en able swift identification of necessary small molecule structural feat ures. 60 This work has illustrated how complicated proteas ome activation can be and the huge amount of work necessary in the future to elucidate more potent activators. Clearly, potency, in the case of activation, is extremely case dependent and a general activator may be untenable. Scientifically, this is the be st - case scenario as it implies selective targeting of IDPs is possible based on their degradation sites and fragment products. Prac tically speaking, this is the worst - case scenario since knowledge of all metrics provided will be necessary for continued pro gress , as the current data suggests there is no - one - size - fit - all metric to gauge progress. 61 Chapter 4. Efforts Toward Small Molecule Binding Site Identification Concomitantly with investigative efforts on factors that translate to the protein level, a new effort aimed at identification of the proteasome binding site / sites was initialized . Diazirine photoaf finity labels are often used for identification of protein - ligand binding pockets 159 . The diazirine is installed in an accommodating branch of the des ired ligand 160 . Light of an appropriate wavelength (variable) irradiates the ligand - enzym e mixture generating a carbene in a bound pocket. The carbene then inserts into a proximate residue 161 - 162 . Since the benzyl tail of the phenothiazine compounds had thus far shown remarkable insensitivity to substitution, I decided to install the photo - labile group in the para position and replace the methyl ester (Figure 4.1 , 4 - A ) . Figure 4.1: Synthesis of Phenothiazine Photoaffinity Label. Synthesis of the benzyl tail proceeded following a literature precedence 162 . Due to 62 mmol scale with protection of the commercially available starting material to give protected alcohol 4 - 1 . Lithiu m/h alogen exchange a nd quench with ethyl trifluoroacetate proceeded smoothly to afford 4 - 2 . The trifluromethy group offered a convenient handle through which to track reaction progress via fluorine NMR. Imine formation followed tosyl protection yielded the O - tosyl protected h ydroxyl imine in moderate yield ( 4 - 3 ). Condensation of ammonia enable d nucleophilic displacement of the weakened N - O bond to give diaziridine in 4 - 4 . This compound was oxidized with iodide in m ethanol to yield the diazirine 4 - 5 . After t his, great care was taken to ensure reactions would take place in darkness. TBDS deprotection with TBAF gave the alcohol 4 - 6 in excellent yield. The final two steps were performed in a one - pot two step procedure. Crude spectral data indicated the presence of desired end produ ct. However, during purification, the diazirine decomposed (confirmed by FNMR). During repetition of the route , further research and discussion with the metabolomics core s on how identification of a binding site is done prompted abandon ment of the diazirin e target. Binding site identification is ultimately done via computational search. A simulated fragmentation is performed generating a list of poss ible fragment products (Figure 4.2, red dots). The covalently bound enzyme (20S CP in th is case) will be deg raded into smaller fragments using a peptidase producing thousands of fragments (Figure 4.2, blue dots) and analyzed via mass spectrometry. The two fragment libraries generated are then compared to each other as a computer searches for fragmentation patter ns that match the prediction. Clearly, as the size of the protein increases, the number of fragments that have to be checked also increases. 63 Figure 4.2: Diagram of the Binding Site Identification Process However, what was non obvious is the nuance of fr agment searching. A protein sequence, call it ABCD, would weigh the same regardless of which amino acid (AA) it is bound to. Identifying a covalent modification requires looking at the sequence and its fragments (Figure 4.3). Now a larger protein will have larger fragments (i.e. ABCDEFGHIJ, etc), requiring more patterns to be predicted and checked for. This problem is exacerbated if no knowledge is available to limit search parameters. Figure 4.3: Example of Combinatorial Explosion In our example with AB CD, a covalent modification to a single AA requires comparison and identification of >56 possible patterns. However, if we know it is binding 64 to A only, this is reduced to 14 patterns. The 20S CP has >6000 amino acids and countless fragmentation possibilit ies. As a photoaffinity label can, in principle, bind to any AA , every possibility would have to be checked. Docking models can make suggested amino acid targets ; however, we feel this to be a disingenuous protocol to follow. Despite good evidence we are b inding in an intersubunit pocket, we still cannot rule out other allosteric binding sites and this would artificially limit possibilities. However, I was advised by the metabolomics core that , without some way to limit the possibilities, identifying the bi nding site would be extremely unlikely. Several review articles discuss selective peptide modification 163 - 164 targeting different AAs in a variety ways. Howe ver, as docking did su ggest binding in an intersubunit pocket , w e elected to target a lysine residue. Lysine appears in every pocket at multiple points and we believed this would give us the best chance of covalently binding in a pocket. A brute force syn thesis was undertaken to test the validity this idea (Figure 4.4 ). Figure 4.4: Synthesis of Lysine Targeting Substrate The phenothiazine core was benzylated and hydrolyzed as detailed in Chapter 3. The N - hydroxysuccinamide (NHC) derivate was made using peptide - coupling reagent EDCI to give the lysine targeting compound 4 - 7 . Compound 4 - 7 was incubated with human proteasome for 1 hour at 37 °C before being frozen in a - 80 °C freezer for an additional hour. This was taken to t he meta bolomics core where it was thawed, digested with trypsin 65 (i.e. the 20S complex was broken into many peptide fragments ), and subjected to mass spectrometry and computational analysis (Figure 4.5). Table 4. 1 : List of Identified Fragments Fifty - one protein s were identified with <1% chance of being misidentified , after deconvolution (see Experimental for Detail), and a bound fragment was discovered near the C - terminal end of attached to Lys242 (Figure 4.6)! 66 Table 4.2 : Identified Bound F ragment Our initial euphoria diminished after it was discovered this linkage is most likely spurious. The C - the surrounding space, away from the proteasome. This area is so di sordered, it does not appear in the proteasome crystal structures. We therefore believe this binding to be solution phase chemistry and not a potential binding mode. Additionally, the fragmentation of the proteasome resulted in a surprisingly low ge expected/predicted to fragment further a number of ways (i.e. from one - end to the other and vice - versa ). Low coverage means a large number of these expected fragments were not found and could be one reason why only this spurious, e xterior binding site was found. The low coverage most likely results from incomplete digestion by trypsin (according to Dr. Whitman) . At the time of writing, optimism is high as this is th e first compound from the lab to be identified and investigation is ongoing on how a more likely binding site can be determined. 67 Chapter 5. Allosteric Inhibition of the 20S Proteasome A. Design and Synthesis of an Allosteric Proteasome Inhibitor Antineoplastic activity, through proteasome inhibition, has made the pro teasome itself a high value target in the treatment of certain cancers 153 . V (Figure 5.1A) , an FDA approved drug, is used in the treatment of multiple myeloma (MM), mantel cell lymphoma, and acute allograft rejection 150 . Efficacy is achieved via the formation of a the active site of the proteasome 150 (Figure 5.1B and C) . Figure 5.1: Velcade (A) Shown Bound in Catalytic Site Cartoon (B) and Crystal Structure (C) proteins, required to maintain cellular homeostasis 165 . The excellent response of MM cells to this tactic is attributed to the prolific output of proteins by these cells, inflicting considerable stress on the degradation pathways within . D erailment of the proteasome degradation pathway results in apoptosis via the unfolded protein response 105 . The success 68 same competitive mechanism 166 . However, this binding mechanism has led to these agents exhibiting permanent abrogation of global protein degradation, lack of specificity, low systemic distribution, resistance, and severe off - target effect s 150 . As a consequence, greater than 97% of patients become intolerant or resi stant to treatment 167 . These severe side - effects and abysmal prognosis indica te the strong need for mechanistically distinct in hibitors. Dr. Hewlett, of the Tepe lab, synthesized 168 a natural product analogue that was discovered 169 to inhibit the prot easome through an unprecedented binding mode 170 . This exclusive and non - covalent bind ing mode br o k e with the dogma of proteasome inhibi tion 171 - 172 . Various interactions a re available; however, most striking is the hydrogen bonding network made possible through the specific orientation of the 5 - 6 fused ring 173 (Figure 5.2A). It has been shown that structurally related compounds of this family, 174 ; implicati ng the 6 - 5 guanidine ring as a potentially useful scaffold for more potent inhibitors. The lab was interested in expanding this S3 - sub domain binding interaction to another part of the catalytic site. To establish the validity of such an approach, an exi sting inhibitor anchored in another domain would have to be modified for such a purpose. Fortuitously, Groll and co - workers reported an ideal inhibitor, unique in being anchored in - sub domain, though not exclusively 171 . The optimized s tructure (Figure 5.2B ) is an alkynyl hydroxyurea mated to a 3 - substituted phenyl ether, all features identified as crucial for potency 151 . 69 Figure 5.2: (A) Indolophakelin Bound in S3 - Sub Dom ain (B) Hydroxyurea Inhibitor Bound in Active Site Upon inspection, we believed replaceme nt of the adamantyl group with a more hydrophilic motif would enable access to the hydrogen bonding network found in the S3 - s ubdomain. re 5.3A/B), a proof - of - concept structural analogue of the hydroxyurea inhibitor w as propo sed (Figure 5.3 C ) . The goal would be to demonstrate the utility of interaction with the S3 - sub domain as an anchor point from which a structurally distinct compound c ould be built (i.e. replacement of the hydroxurea alkyne motif). 70 Figure 5.3: Indolophakelin (A) and Hydroxyurea (B) Bound in Catalytic Site. Overlay of Proposed Compound (C) with Hydroxyurea in Catalytic Site. Retrosynthetic analysi s is shown below (Figure 5.4). The general target is conveniently divided by Sonogashira coupling into two fragments. Fragment B is achievable through literature methods 151 . Fragment B could be obtained through either a Horner - Wadsworth Edmunds (HWE) condensation, if a double bond was preferred, or Bucherer - Bergs synthesis if the shorter carbon unit was re quired. Prec ursors to both are conveniently available through aldehyde FB - 1 . FB - 1 would be synthesized though oxidation of alcohol FB - 2 generated from alkylation of the commercial ly available m - iodophenol ( FB - 3 ). 71 Figure 5.4: Generalized Retrosynthe tic Analysis of Proposed Compound In the forward case, synthesis of alkyne hydroxyurea 5 - 4 (Figure 5.5) went according to the literature method beginning with mesylation of propar gyl alcohol ( 5 - 1 ). Displacement of the activated alcohol 5 - 2 with hydroxylami ne followed by treatment with potassium cyanate and acid provided the desired fragment A ( 5 - 4 ) as a white solid. Figure 5.5: Synthesis of Fragmen t A (Compound 5 - 4 ) 72 Synthesis of Fragment B and coupling to Fragment A is depicted below ( Figure 5.6). Figure 5.6: Synthesis of Allosteric Inhibitor 5 - 13 . Alkylation of m - iodophenol with 3 - bromo - propanol afforded the termi nal alcohol 5 - 5 in good yield at gram scale. Oxidation to the aldehyde 5 - 6 was convenient ly performed using 2 - iodoxybenzoic acid (IBX, 5 - 10 ) in refluxing ethyl acetate. IBX was generated using a literature procedure from 2 - iodobenzoic acid 5 - 9 175 . With the aldehyde 5 - 6 in hand, hydantoin 5 - 7 was converted to the HWE ylide precursor 5 - 8 . Compound 5 - 6 was then converted to the unsaturated coupling partner 5 - 12 following a literature HWE preparation and to the saturated hydantoin 5 - 11 using Bucherer - Bergs conditions. Unfortunately, the Bucherer - Bergs gave too little yield to carry forward. As such, onl y 5 - 12 was coupled with 73 hydroxyurea 5 - 4 . Unfortunately, the desired linke r compound was n ot formed. Likely causes would be the high chelation potential of the hydantoin to either the copper or palladium species. A screen of different ligand sets would have to be undertaken to correct for this; however, the project was shortly h ereafter ended. Further screening was avoided because I began to look closer at the system. The crystal structures that the original analysis w a s based upon (Figure 5.2) were of yeast proteasome. The assay was performed with human proteasome. I copied the coordinates of t he original hydroxurea inhibitor and copied them to the human proteasome map (Figure 5. 7 A, gold compound) for comparison purpose s. Figure 5. 7 : Electrostatic Potentia l Maps of 5 Catalytic Site in Human (A) and Yeast (B) The differences in the two maps ar e most apparent in the S3 - sub pocket. The yeast proteasome contains a generously positive surface (blue shading, Figure 5.8B), presumably from the all the basic amino acids our hydantoin was supposed to take advantage of. Unfortunately, this same area is m ore neutral (green shading) and even slightly negatively charged deeper in the pocket of the human proteasome (Figure 5.8A). By comparing their sequences against each other, I f ound there is very low sequence similarity be tween yeast and human proteasome , which would account for this binding difference and attribute 74 nearly all the activity to the right half of the molecule (note the remarkable similarity of the S1 pocket in Figur e 5. 7 ). If this were true, further effort wou ld inevitable be in vain. To answ er this, I turned to computational docking methods . Once the docking workflow was established, I ran the proposed linker against human proteasome and found it had no predicted b inding modes in the catalytic site and, short ly thereafter, used docking to for m a framework for small molecule activation which pivoted my path away from proteasome inhibitors . B. Computational Insight for New Allosteric Inhibitors While I my personal proje cts moved away from the inhibition side of proteasome activities, I nonetheless still aided collaborators in their inhibitory endeavors. In collaboration with the Gaczynska group 176 , which studies proteasome inhibition, a series of analo gs based on SAR optimized motif (Figure 5.8, B1 ) was performed by Dr. Matthew Giletto (Tepe Group) to further their studies. Several of th ese published compounds 177 are depicted below (Figure 5.8). I was asked to rationalize a mechanism o f action and explain t he unusual behavior of the pipecolic acid enantiomers (Fi gure 5.8, C, D, and E ). In most cases, the potenc y between two enantiomers is an order of magnitude (or more) or their individual activities sum to that of the racemate 178 . 75 Figure 5.8: Published Structures Prepared by Dr. Gilet t o 177 The large siz e of the human proteasome can make docking studies complicated due to the large search space and numerous possible binding pockets. A dditionally, a completely exhaustive search is infeasible with the hardware ava ilable to the lab as the large number of rot atable bonds in the pipecolic ester structures increases the computational expense. To overcome these limitations, docking was undert aken in three parts: 76 1) W hole 20S proteasome The entirety of the proteasome was searched with a low exhaustiveness (30 - 40) i n multiple individual runs (on average ~5 times). This sampling revealed more binding modes within the alpha ring than the beta ring s. 2) Alpha and Beta Rings After (1), the search space was reduced, and exhaustiv eness increased (~60). In two separate runs, each compound was docked against the beta rings, and then the alpha rings. The average binding affinity difference was ~1.2 kcal/mol suggesting a large preference for the alpha ring system. 3) Alpha Ring Site refi nement Finally, the search space was limited to the minimum space necessary to encompass just the alpha ring and as high an exhaustiveness as possible on our hardware: Center: ( 135.9681, - 38.0296, 65.3754) Dimensions (Angstrom): X: 122.94 6 Y: 138.030 Z:53.18 4 Exhaustiveness: 80 Remarkably, t he most potent compounds gathered in only a few of the intersubunit pockets (red circles , Figure 5.9 ). While, the less active compoun ds preferred either a different binding pocket or no preference. This was an intriguing mechanism of action, intersubunit pocket binding for inhibition; however, after the crystallography data published this year 75 is acco unted for, the result makes more sense 75 . 77 Fig ure 5.9: Binding Modes of Dr. Gil The new data (discussed more in Chapter 1), demonstrated that 19S intersubunit binding could occur without inducing a gate o pened form 75 , implying other mechanisms of actio n for binders within this site. The deeply penetrating pipecolic analogous were therefore proposed to be inducing/trapping a non - active proteasome form through alpha ring confor mer adjustment. The enantiomers bind to two different subuni ts of the alpha rin g. An unusual result , but in the absence of other empirical data , a reasonable explanation for the observed results. 78 Experimental Section A. Synthetic Methods Reactions were carr ied out in flame - dried glassware under a nitrogen atmosphere . Reagents and solv ents were purchased from commercial suppliers and used without further purification. Anhydrous THF was distilled over benzophenone and sodium immediately prior to use. All react ions were magnetically stirred. Yields refer to chromatograp hically and spectro scopically pure compounds unless otherwise noted. Infrared spectra , where applicable, were recorded on a JASCO Series 6600 FTIR spectrometer. 1 H and 13 C NMR spectra were recorde d on a Varian Unity Plus - 500 or 600 spectrometers, as noted in the experimental for each compound. Chemical shifts are reported relative to the residue peaks of the solvent ( CDCl 3 : 7.26 ppm for 1 H and 77.0 ppm for 13 C) (DMSO - d 6 : 2.50 ppm for 1 H and 39.5 pp m 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. Due to the hydrophobicity of a number of these compounds, small quantities of solvents were u n - removable in some cases. These instances are label ed in accordance with liter ature precedent 179 . HRMS were obtained at the Mass Spectrometry Facility of M ichigan State University with a Micromass Q - ToF Ultima API LC - MS/MS mass spectrometer. Purification of compounds was achieved in most cases using laboratory medium pressure liqu id chromatogram (MPLC) on silica gel (20 - 40 microns) . Standard method is gradie nt elution from 0 - 50% ethy acetate in hexanes over 45 minutes. Deviations from this will be noted per compound but were otherwise general. Attachment of substituted benzyl group s was performed through a general procedure (detaile d below). Any deviations ar e listed where appropriate in the text of the compound. 79 General Benzylation Procedure : Heterocyclic core (phenothiazine, iminodibenzyl, indole, etc) was dissolved in anhydrous DMF at room temperature under an inert atmosphere in a round bottom flask . Sodi um hydride (1.1 eq) was added as a single portion, vigorous bubbling should be observed, and the mixture allowed to stir at room temperature for 0.5 h wrapped in foil. Substitut ed benzyl derivate (1.5 eq) is added as a single por tion and allowed to react a t room temperature in the dark (i.e. wrapped in foil) for 16 h. After 16 h, the reaction is diluted with ether ( ca. 2 solvent volumes) and poured into separatory funnel containi ng a 10% wt/wt solution of LiBr in DI water. The eth er layer is carefully washe d 2x with LiBr (aq) and then Brine (1x solvent volume), dried over sodium sulfite and concentrated in vacuo . Crude material was then purified using MPLC standard methods. 80 Figure E . 1: Compound 3 - 1 2 - C hloro - 10 - (4 - methylpentyl) - 10H - phenothiaz ine A solution of 2 - chloro - 10H - phenothiazine (0.467 g, 2 mmol) in THF is added dropwise to a suspension of sodium hydride ( 60% wt/wt, 0.080 g, 2 mmol) at room temperature. The mixture is allowed to stir at room tem p for 30 minutes. 1 - Bromo - 4 - methyl penta ne (0.146 mL, 1 mmol) was added neat, dropwise. After stirring for 2 hours, the solution was poured into saturated bicarbonate solution ( ca. 50 mL) and extracted into ethyl acet ate (3x 50 mL). The combined organic la yers were washed with brine ( ca. 50 mL) and dried over sodium sulfate and concentrated in vacuo to give a purple solid , which was purified via MPLC to give the final product as a white solid (85.1 mg, 26.8%). 1 H NMR ( 500 MHz, CDCl 3 7.20 7.10 (m, 2H), 7.02 (d, J = 8.2 Hz, 1H), 6.93 (td, J = 7.5, 1.1 Hz, 1H), 6.88 (dd, J = 8.1, 2.0 Hz, 1H), 6.85 (dd, J = 8.2, 1.1 Hz, 1H), 6.81 (d, J = 2.0 Hz, 1H), 3.78 (t, J = 7.2 Hz, 2H), 1.82 - 1.73 (m, 2H), 1.59 - 1.51 (m, 1H), 1.34 - 1.24 (m, 2H), 0.88 (d, J = 6.6 Hz, 6H). 13 C NMR (126 MHz, CDCl 3 122.9, 122.2, 115.8, 115.8, 47.9, 36.2, 27.8, 24.8, 22.7. HRMS (ESI) m/z : [M+H] + Calcd for C 18 H 22 ClNS: 318.1083; Found 318.1082 ATIR: CH (2952 cm - 1 ), aromatic CH (3056 cm - 1 and 3176 cm - 1 ) 81 Figure E . 2: Compound 3 - 2 3 - (2 - C hloro - 10H - phenothiazin - 10 - yl)propane - 1 - sulfonate 2 - chloro - 10H - phenothiazine (0.981 g, 4.2 mmol) was added as a solution in anhydrous THF (10 mL), to a round bottom flas k charged with sodium hyd ride (0.160 g, 4 mmol) and THF (15 mL). The solution was then heated to reflux for 1 hour to give a bright red solution , which was cooled to near room temperature before addition of 1,3 - propane sultone (0.41 mL. 4 mmol). The solution immedi ately becomes ye llow and forms a white precipitate. The solution was stirred for 1 hr at reflux . White solids form ed upon cooling were washed with THF (100 mL) and diethyl ether (100 mL) before being left to dry in air overnight (985 mg, 65%). 1 H NMR (500 MHz, DMSO - d 6 7.21 (ddd, J = 8.6, 7.3, 1.6 Hz, 1H), 7.16 7.12 (m, 2H), 7.07 (dd, J = 8.3, 1.5 Hz, 2H), 7.01 6.93 (m, 2H), 3.99 (t, J = 7.2 Hz, 2H), 2.53 (t, J = 7.3 Hz, 2H), 1.96 (tt, J = 8.4, 6.5 Hz, 2H). 13 C NMR (126 MHz, (DMSO - d 6 146.3, 144.0, 132.5, 128.1 , 127.8, 127.2, 123.1, 122.9, 122.4, 122.1, 116.3, 115.7, 48.5, 45.7, 22.7. HRMS (ESI) m/z : [M+H] + Calcd for C 15 H 115 ClNO 3 S 2 356.0182; Found 356.0182 . ATIR: Aromatic CH (3427 cm - 1 ), CH (2950 cm - 1 , very weak) RSO 3 - (1049 cm - 1 ) 82 Figure E . 3: Compound 3 - 3 4 - (2 - C hloro - 10H - phenothiazin - 10 - yl)butane - 1 - sulfonate 2 - chloro - 10H - phenothiazine (3.5 g, 15 mmol) was added as a solution in anhydrous THF (10 mL), to a round bottom flask charged with sodium hydride (0.6 g, 15 mmol) and THF (15 mL). The solution was then heated to reflux for 1 hour to give a bright red solu tion , which was cooled to near room temperature and injected with 1,4 - butane sultone (1.54 mL, 15 mmol). The s olution was then refluxed for 24 hours. Upon cooling, title compound precipitated from solution as an off white solid (4.6 g, 78%) and may be used without further purification. Further purification can be achieved if desired by taking a portion of the compound and refluxing in benzene overnight ( ca. 12 h) wi th a D ean - S tark trap. The b enzene solution was then frozen , and the solvent sublimed off to g ive clean compound. 1 H NMR (500 MHz, (DMSO - d 6 7.10 (m, 2H), 7.06 7.01 (m, 2H), 6.99 6.93 (m, 2H), 3.84 (t, J = 6.7 Hz, 2H) , 2.42 (t, J = 7.4 Hz, 2H), 1.74 - 1.62 (m, 4H). 13 C NMR (126 MHz, (DMSO - d 6 32.5, 128.1, 127.8, 127.2, 123.2, 122.9, 122.5, 122.0, 116.3, 115.7, 50.9, 46.5, 25.5, 22.6. HRMS (ESI) m/z : [M+H] + Calcd for C 16 H 17 ClNO 3 S 2 370.0338; Found 370 . 0344 . ATIR: Aromatic CH (3427 cm - 1 ), CH (2950 cm - 1 , very weak) , RSO 3 - (1049 cm - 1 ) 83 Figure E . 4: Compound 3 - 4 2 - Chloro - 10 - (5 - chloropentyl) - 10H - phenothiazine To an oven dried round bottom flask charged with sodium hydride (600 mg, 15 mmol) was adde d 2 - chloro phenothiazine (2.3 g, 10 mmol) as a solution in anhydrous THF (20 mL), dropwise at room temperature. An additional 20 mL of THF was added and the solution brought to reflux for one hour. The solution, now red to orange, was cooled to room temper ature and 1 - bromo - 5 - choropentane (1.58 mL, 12 mmol) was added in a single por tion. The solution was allowed to stir at room temperature for 12 hours. The solution, now with brown solids, was poured into a separatory funnel containing an equivalent volume o f sodium bicarbonate and extracted with diethyl ether (2x 50 mL). The organic layers were dried over sodium sulfate and concentrated to dryness before being applied to the MPLC for purification to give a dark oil as the final product (1.44 g, 42%). 1 H NMR (500 MHz, CDCl 3 ) 7.24 7.13 (m, 2H), 7.04 (d, J = 8.2 Hz, 1H), 6.98 (td, J = 7.5, 1.1 Hz, 1H), 6.91 (dd, J = 8.2, 2.1 Hz, 1H), 6.88 6.83 (m, 2H), 3.82 (t, J = 7.0 Hz, 2H), 3.52 (t, J = 6.6 Hz, 2H), 1.88 1.72 (m, 4H), 1.66 1.49 (m, 2H). 13 C NMR (1 26 MHz, CDCl 3 ) 146.5, 144.5, 133.2, 128.0, 127.6, 127.5, 125.0, 123.7, 123.0, 122.3, 115.9, 115.8, 47.2, 44.9, 32.2, 26.1, 24.3. This compound (18.5 mmol) was dissolved in acetone (150 mL). Finely ground sodium iodide ( ca. 50 g, 333mmol) was added and th e mixture vigorously stirred. The mixture was refluxed for 3 days. The mixture was then placed in a - 20 C freezer 84 for 4 hours and then filtered through a medium frit. Solids were washed with acetone (2x100 mL) and the filtrate concentrated to dryness to gi ve the product as a waxy brown solid in quanti tative yield (7.6 g). The iodo - products were used without further purification for the preparation of compound 3 - 5 . 85 Figure E . 5 : Compound 3 - 5 5 - (2 - C hloro - 10H - phenothiazin - 10 - yl)pentane - 1 - sulfo nic acid 2 - Chloro - 10 - (5 - iodopentyl) - 10H - phenothiazine (343 mg, 0.8 mmol) was added to a mixture of acetone and water (1:1) and sodium sulfite (403.2 mg, 3.2 mmol) in a round bottom flask giving a milky white sol ution. Mixture was allowed to stir overnight (ca. 12 hr) to give a clear yellow solution with a white precipitate. The solution is concentrated to dryness in vacuo and residue swirled with dichloromethane (30 mL) and solids collected on a medium frit. Soli ds washed with acetone (30 mL) to give the p roduct as an off white solid. Solid is collected and dried overnight on a high vacuum line to give the title compound (207 mg, 66%). 13 C NMR (126 MHz, DMSO - 127.8, 127.2, 123.3, 1 23.0, 122.6, 122.1, 116.3, 115.7, 51.4, 46.5 , 26.1, 25.6, 24.9. 1 H NMR (500 MHz, DMSO - 7.17 (m, 1H), 7.17 7.09 (m, 2H), 7.04 7.00 (m, 2H), 6.98 6.93 (m, 2H), 3.83 (t, J = 7.0 Hz, 2H), 2.42 2.26 (m, 2H), 1.65 - 1.60 (m, 2H), 1.58 1.46 (m , 2H), 1.42 - 1.36 (m, 2H). ATIR: OH (from hyd rate, 3428 cm - 1, broad), Aromatic CH (2930 cm - 1), CH (2850 cm - 1) HRMS (ESI) m/z: [M - H] - 17 H 17 ClNO 3 S 2 382.0338; found: 382.0336. 86 Figure E.6: Compound 3 - 6 2 - Chloro - 10 - (prop - 2 - yn - 1 - yl) - 10H - phenothiazine 2 - Chlor o - 10H - phenothiazine (0.467 g, 2 mmol) was added as a solution in anhydrous THF ( ca. 10 mL), to a round bottom flask charged with sodium hydride (0.076 g, 1.9 mmol). The solution was stirred one hour to give a reddish - brown solution before the addition of p ropargyl bromide (80% in Toluene, 0.24 mL, 2.2 mmol) in a single portion. The solution was stirred for 12 hours and then concentrated to dryness in vacuo before being placed on a high vacuum line for a pproximately 4 hours. Crude material was suspended in c hloroform and cooled to - 20 C overnight. The starting material precipitates out and was removed by filtration. The filtrate was concentrated to dryness to give thick, dark oil as product (0.300 g, 58.1 %) which was used in the next step without further puri fication. 13 C NMR (126 MHz, cdcl 3 ) 145.4, 143.7, 133.5, 127.8, 127.6, 127.2, 123.5, 123.2, 122.9, 122.1, 115.3, 115.2, 78.6, 75.1, 38.8. 1 H NMR (500 MHz, CDCl 3 ) 7.23 - 7.18 (m, 3H), 7.13 (dd, J = 7.6, 1.4 Hz, 1H), 7.02 (d, J = 8.2 Hz, 1H), 6.98 (ddd, J = 7.8, 6.7, 2.0 Hz, 1H), 6.93 (dd, J = 8.2, 2.0 Hz, 1H), 4.48 (d, J = 2.4 Hz, 2H), 2.51 (t, J = 2.4 Hz, 1H). HRMS (ESI) m/z : [M] - Calc d for C 15 H 10 ClNS 271.0222; Found 271.0228. 87 Figure E.7: Compound 3 - 7 4 - (2 - Chloro - 10H - ph enothiazin - 10 - yl) but - 2 - ynoic acid A solution of 2 - chloro - 10 - (prop - 2 - yn - 1 - yl) - 10Hphenothiazine (0.338 g, 1.25 mmol) in THF was cooled to - 78 C in an acetone/dry ice bath and allowed to stand for one hour in a round bottom flask . A solution of nBuLi (2.5 M in hexanes, 0.52 mL, 1.31 mmol) was added dropwise and allowed to stir for ca. 20 min. An excess of solid carbon dioxide was added and the round bottom flask sealed. The reaction was allowed to stir for 2 hours before warming to 10 C. Solution was then po ured into a small beaker containing 10% HCl solution (pH ~2) and extracted into ether. The organic layer was adjusted with 10% NaOH to a pH of 11, organic layer discarded, and the aqueous laye r acidified to 2 by the addition of 10% HCl. The aqueous layer w as then extracted into ether, washed with brine, dried using sodium sulfate, and concentrated in vacuo to give the product as a light brown solid (0.078g, 20%). 1 H NMR (500 MHz, CDCl 3 ) 7.16 (d, J = 7.8 Hz, 1H), 7.09 (d, J = 7.6 Hz, 1H), 7.05 (d, J = 8.1 Hz, 1H), 7.00 (d, J = 7.9 Hz, 2H), 6.95 (t, J = 7.6 Hz, 1H), 6.91 (dd, J = 8.1, 1.9 Hz, 1H), 4.60 (s, 2H). 13 C NMR (126 MHz, CDCl 3 ) 156.0, 144.9, 143.1, 133.6, 127.9, 127.8, 127.4, 12 3.9, 123.3, 123.3, 122.2, 115.0, 115.0, 83.9, 77.8, 38.7. HRMS (ESI) m/z : [M+H]+ Calcd for C 16 H 11 ClNO 2 S 316.0189; Found 316.0201. ATIR CO 2 H (br, 3400 cm - 1 2700 cm - 1 ), - C=C - CO2 (2236 cm - 1 ) 88 Figure E. 8 : Compound 3 - 8 Methyl 4 - ((2 - chloro - 10H - phenothiazin - 10 - yl)methyl)benzoate 2 - chloro - 10H - phenothiazine (0.583 g, 2.5 mmol) was added as a solution in anhydrous THF (ca. 15), to a round bottom flask charged with sodium hydride (0.090 g, 2.25 mmol). The mixture was stirred at roo m temperature for 1 hour followed by addition of 4 - bro momethyl benzoate (0.458 g, 2 mmol). Upon addition, the reaction becomes a brown - orange solution and was covered in foil before stirring for 4 days, after which the solution was green. The solution was poured into a seperatory funnel containing diethyl eth er and turned purple. Saturated sodium bicarbonate was added and the aqueous layer (brown in color) was discarded. The organic layer was washed with brine, dried over sodium sulfate and concentrated in vacuo to give the crude product. The crude was suspend ed in chloroform and placed in a - 20°C freezer overnight to precipitate out unreacted starting material. The solution was decanted, and the chloroform concentrated in vacuo to give the pure product as a white solid (0.700 g, 92%). A sample was applied to a n MPLC (Hexane: Ethyl Acetate gradient) for analytical purity. 1 H NMR (500 MHz, CDCl 3 J = 8.3 Hz, 2H), 7.42 7.35 (m, 2H), 7.09 (dd, J = 7.6, 1.6 Hz, 1H), 7.00 (d, J = 8.2 Hz, 1H), 6.89 (td, J = 7.5, 1.2 Hz, 1H), 6.85 (dd, J = 8.2, 2.0 Hz, 1H) , 6.60 6.54 (m, 3H), 5.09 (s, 2H), 3.91 (s, 3H ) 13 C NMR (126 MHz, CDCl 3 45.7, 143.7, 141.6, 133.3, 130.3, 89 130.3, 129.5, 127.6, 127.6, 127.2, 126.8, 123.3, 122.7, 115.8, 115.7, 52.6, 52.3. . HRMS (ESI) m/z : [M+H] + Calcd for C 21 H 17 ClNO 2 S 382.0669; Found 382.0670 ATIR Aromatic CH (3100 cm - 1 ), CH(2949 cm - 1 , 2922 cm - 1 ) - CO 2 R (st, s harp, 1716 cm - 1 ) 90 Figure E. 9 : Compound 3 - 9 4 - ((2 - C hloro - 10H - phenothiazin - 10 - yl)methyl)benzoic acid Methyl 4 - ((2 - chloro - 10H - phenothiazin - 10 - yl)methyl)benzoate (0.400 g, 1.05 mmol) was added to a soluti on of 10% NaOH and methanol (1:1) and refluxed for 2 hours. The reaction was extracted with ether and the organic layer discarded. The aqueous layer was ac idified with 10% HCl to pH 2 and extracted into ether (50 mL), washed with brine (50 mL), dried over sodium sulfate. The resulting solution was concentrated in vacuo to give a white solid (0.376 g, 97%). 1 H NMR (500 MHz, CDCl 3 J = 8.2 Hz, 2H), 7.43 (d, J = 8.1 Hz, 2H), 7.10 (dd, J = 7.6, 1.5 Hz, 1H), 7.06 6.95 (m, 2H), 6.93 6.81 (m, 2H), 6.62 6.50 (m, 2H), 5.12 (s, 2H). 13 C NMR (126 MHz, CDCl 3 133.3, 131.0, 128.5, 127.7, 127.6, 1 27.3, 126.9, 1 23.5, 123.4, 122.8, 122.3, 115.8, 115.7, 52.6. HRMS (ESI) m/z : [M] - Calcd for C 20 H 1 3 ClNO 2 S - 366.0356; Found 366.0356 ATIR CH Aromatic (2995 cm - 1), CH (2912 cm - 1 ), CO2H (w, br, 3433 cm - 1 ), (w, 1610 cm - 1 ) 91 Figur e E. 10 : Compound 3 - 10 5 - (2 - chloro - 10H - phenothiazin - 10 - yl)pentanenitrile To a suspension of sodium hydride (200 mg, 5 mmol) in anh ydrous THF, was added a solution of 2 - chloro - 10H - phenothiazine dropwise at room temperature in a round bottom flask . Solution was stir red for 0.5 hours before addition of valeronitrile (0.58 mL, 5 mmol) in a single portion. The mixture was allowed to stir for 48 hours before being concentration to dryness, extracted into ether, washed with brine (3 x 50 mL), concentrated in vacuo and dis solved in chloroform to remove as much starting material as possible , filtered and concentrated to give the final product that was used without further purification (21%, 131 mg) . *Special Note: The above procedure was repeated after the lab obtain ed a med ium pressure liquid chromatogram ( MPLC ). However, after brine wash, the crude material was concentrated in vacuo then appl ied to the MPLC using a very slow gradient of hexane:ethyl acetate giving superior yield (1.3g, 88%) . The NMR spectra attached is from this run and not the original. 1 H NMR (500 MHz, CDCl 3 ) 7.12 (m, 2H), 7.05 (d, J = 8.2 Hz, 1H), 6.97 (td, J = 7.5 , 1.2 Hz, 1H), 6.91 (dd, J = 8.2, 2.1 Hz, 1H), 6.86 (dd, J = 8.1, 1.2 Hz, 1H), 6.82 (d, J = 2.1 Hz, 1H), 3.88 (t, J = 6.4 Hz, 2H), 2.3 2 (t, J = 7.1 Hz, 2H), 1.96 1.88 (m, 2H), 1.80 1.71 (m, 2H). 13 C NMR (126 MHz, CDCl 3 ) 7.8, 92 127.6, 125.5, 124.3, 123.3, 122.7, 119.4, 116.0, 46.2, 25.6, 22.7, 16.9. HRMS (APCI) m/z : [M+H] + 17 H 16 ClN 2 S 3 15.0717; Found 315.0720. 93 Figure E. 11 : Compound 3 - 11 5 - (2 - C hloro - 10H - phenothi azin - 10 - yl)pentanamide 5 - (2 - chloro - 10H - phenothiazin - 10 - yl)pentanenitrile was dissolved in concentrated sulfuric acid and sti rred for 2.5 hours in a round bottom flask . The solution was then poured into an ice - water mixture and basified with concentrated ammonium hydroxide. Mixture was then extracted with EtOAc (3x 100 mL), was hed with brine (1 x 100 mL), dried over sodium sulfi te, and concentrated in vacuo to give the title compound ( 95 % ). 1 H NMR (500 MHz, CDCl 3 ) 7.10 (m, 2H), 7.02 (d, J = 8.1 Hz, 1H), 6.93 (td, J = 7.5, 1.1 Hz, 1H), 6.88 (dd, J = 8.2, 2.0 Hz, 1H), 6.8 5 (dd, J = 8.2, 1.1 Hz, 1H), 6.81 (d, J = 2.0 Hz, 1H ), 3.84 (t, J = 6.6 Hz, 2H), 2.21 (t, J = 7.3 Hz, 2H), 1.88 1.78 (m, 2H), 1.78 1.70 (m, 2H). 13 C NMR (126 MHz, CDCl 3 ) 127.60, 124.98, 123.75, 123.11, 1 22.46, 115.97, 115.95, 47.05, 35.29, 26.13, 22.91. H RMS (APCI) m/z : [M+H] + 17 H 18 ClN 2 OS 333.0823; Found 358.1387. 94 Figure E. 12: Compound 3 - 12 4 - (4 - (2 - Chloro - 10H - phenothiazin - 10 - yl) butyl) morpholine (5) 2 - Chloro - 10 - (4 - iodobutyl) - 10H - phenothiazine was added to a neat solution of morpholine and gently refluxed for 2 hours. The solution was poured into separatory funnel containing 0.5M HCl solutio n. The aqueous layer was extracted with EtOAc (2x 100 mL), and the combined organic layers washed with brine (100 mL), dried over sodium sulfite and concentrated in vacuo to give the title compound in quant itative yield. 1 H NMR (500 MHz, CDCl 3 ) 7.15 (ddd, J = 8.1, 7.4, 1.6 Hz, 1H), 7.12 (dd, J = 7.7, 1.5 Hz, 1H), 7.02 (d, J = 8.1 Hz, 1H), 6.93 (td, J = 7.5, 1.2 Hz, 1H), 6.88 (dt, J = 8.2, 1.8 Hz, 2H), 6.83 (d, J = 2.0 Hz, 1H), 3.86 (t, J = 6.9 Hz, 2H), 3.6 6 (t, J = 4.6 Hz, 4H), 2.36 (dd, J = 14. 9, 7.7 Hz, 6H), 1.85 (tt, J = 7.7, 6.2 Hz, 2H), 1.62 (p, J = 7.3 Hz, 2H). 13 C NMR (126 MHz, CDCl 3 ) 146.6, 144.7, 133.3, 128.0, 1 27.7, 127.5, 124.9, 123.7, 123.0, 122.6, 115.9, 115.9, 67.1, 58.3, 53.8, 47.3, 24.4, 23 .6. HRMS (ESI) m/z : [M+H]+ Calcd for C 20 H 24 ClN 2 OS 375.1298; found: 375.1306. ATIR: Aromatic CH (3100 cm - 1), CH (2945 cm - 1), CH (2846 cm - 1). 95 Figure E. 13: Compound 3 - 13 M ethyl 4 - ((10H - phenothiazin - 10 - yl)methyl)benzoate Ge neral procedure. 101.3 mg, 15% yield as a waxy off - white solid . 1 H NMR (500 MHz, CDCl 3 J = 8.3 Hz, 2 H), 7.41 (d, J = 8.6 Hz, 2 H), 7.12 (dd, J = 7.6, 1.6 Hz, 2 H), 7.04 6.93 (m, 2 H), 6.89 (t, J = 7.5 Hz, 2 H), 6.60 (dd, J = 8.2, 1.1 Hz, 2 H), 5.12 (s, 2 H), 3.91 (s, 3H). 13 C NMR (126 MHz, CDCl 3 ) 166.8, 144.3, 142.3, 130.1, 129.1, 127.3, 127.0, 126.8, 123.5, 122.8, 115.4, 52.5, 52.1. HRMS (APCI) m/z : [M+H] + 21 H 1 8 NO 2 S : 348 .1058 Found: 348.1099 ATIR: CH 2 (2 843, 2882 cm - 1 ), aromatic CH (30 04, 3 059 cm - 1 ) , carbonyl (s, 1713 cm - 1 ) 96 Figu re E. 1 4 : Compound 3 - 1 4 M ethyl 4 - ((2 - (trifluoromethyl) - 10H - phenothiazin - 10 - yl)methyl)benzoate General procedure. 257.1 mg, 31% yield as a sticky white semi - solid that discolors on standing to green - white . 1 H NMR (500 MHz , CDCl 3 ) 8.02 (d, J = 8.3 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 7.19 (dd, J = 8.0, 0.9 Hz, 1H), 7.14 7.10 (m, 2H), 7.02 (ddd, J = 8.1, 7.4, 1.6 Hz, 1H), 6.92 (td, J = 7.5, 1.2 Hz, 1H), 6.80 (d, J = 1.7 Hz, 1H), 6.64 (dd, J = 8.2, 1.1 Hz, 1H), 5.14 (s, 2H), 3.91 (s , 3H). 13 C N MR (126 MHz, CDCl 3 ) 166.8, 144.9, 143.7, 141.4, 130.3, 129.6 (q, 2 J CF = 32.1 Hz, 1C), 129.5, 127.8, 127.3, 127.2, 126.8, 124.0 (q, 1 J CF = 274.2 Hz, 1C), 123.5, 122.9, 119.4 (q, 3 J CF = 3.9 Hz, 1C), 115.9, 111.7 (q, 3 J CF = 3.7 Hz, 1C), 52.5, 52 .2. HRMS (APCI) m/z : [M+H] + 22 H 1 7 F 3 NO 2 S + 41 6.0932 ; Found 41 6.0835 ATIR: CH ( 2957 cm - 1 ), aromatic CH (30 09 cm - 1 ) , carbonyl (s, 1713 cm - 1 ) 97 Figure E. 15: Compound 3 - 15 M ethyl 4 - ((2 - methoxy - 10H - phenothiazi n - 10 - yl)methyl)benzoate General Procedure. 5%, 37.7 mg as a white solid. 1 H NMR (500 MHz, CDCl 3 J = 8. 0 Hz, 2 H), 7.40 (d, J = 8.0 Hz, 2 H), 7.16 7.06 (m, 1 H), 7.02 6.93 (m, 2 H), 6.88 (t, J = 7.5 Hz, 1H), 6.59 (d, J = 8.5 Hz, 1 H), 6.45 (dd, J = 8.4, 2.4 Hz, 1H), 6.20 (d, J = 2.4 Hz, 1H), 5.11 (s, 2 H), 3. 90 (s, 3 H) , 3.63 (s, 3 H ) . 13 C NM R (126 MHz, CDCl 3 ) 159.7, 145.7, 144.2, 142.3, 130.2, 129.2, 127.4, 127.2, 127.1, 126.9, 122.9, 115.6, 107 .0, 103.6, 55.5, 52.7, 52.3. HRMS (APCI) m/z : [M+H] + 22 H 20 NO 3 S + 378.1158; Found 378.1161 . ATIR: CH (2957 cm - 1 ), aromatic CH (30 09 cm - 1 ), carbonyl (s, 1713 cm - 1 ) 98 Figure E. 16: Compound 3 - 16 M ethyl 4 - ((2 - (ethylthio) - 10H - phenothiazin - 10 - yl)methyl)benzoate Benzylation achieved with the general procedure to give product ( 95.1 mg, 12% ) as a th ick dark yellow oil. 1 H NMR (500 MHz, CDCl 3 ) J = 8.4 Hz, 2H), 7.44 7.35 (m, 2H), 7.10 (dd, J = 7.6, 1.5 Hz, 1H), 7.00 (d, J = 7.9 Hz, 2 H), 6.91 6.72 (m, 2H), 6.61 (dd, J = 8.2, 1.2 Hz, 1H), 6.56 (d, J = 1.7 Hz, 1H), 5.10 (s, 2H), 3.90 (s, 3H) , 2.67 (q, J = 7.3 Hz, 2 H) , 1.09 (t, J = 7. 3 Hz, 3 H) . 13 C NMR (126 MHz, CDCl 3 ) 166.8, 144.6, 144.1, 14 2.1, 135.5, 130.1, 129.2, 127.4, 127.1, 127.1, 126.8, 123.7, 123.6, 122.9, 121.4, 116.5, 115.5, 52.4, 52.2, 28.0, 14.3. HRMS (APCI) m/z : [M+H] + C 23 H 2 2 NO 2 S 2 408.108 6 ; Found 408.1084 . ATIR: CH (2957 cm - 1 ), aromatic CH (30 12 cm - 1 ), carbonyl (s, 171 4 cm - 1 ) 99 Figure E. 17: Compound 3 - 17 M ethyl 4 - ((3 - chloro - 10,11 - dihydro - 5H - dibenzo[b,f]azepin - 5 - yl)methyl)benzoate Synthesized by g eneral p rocedure to give the title compound as a white solid (400 mg, 34% ) . 1 H NMR (500 MHz , CDCl 3 ) J = 8. 4 Hz, 2 H), 7. 44 ( d, J = 8.4 Hz , 2H), 7.1 0 (d, J = 7.1 Hz, 1H), 7.08 7.06 (m, 2 H), 7.03 (d, J = 2.0 Hz, 1H), 6.95 6.90 (m, 2 H ), 6.84 (dd, J = 8.1, 2.1 Hz, 1H), 4.97 (s, 2H), 3.87 (s, 3H), 3.29 3.09 (m, 4H). 13 C NMR (126 MHz, CDCl 3 166. 9, 148.5, 147.5, 143.2, 134.7, 131.6, 131.5, 131.2, 129.7, 129.6, 129.0, 128.0, 126.6, 123.5, 122.4, 120.4, 120.0, 55.4, 52.0, 32.3, 31.8. HRMS (APCI) m/z : [M+H] + C 23 H 2 1 N Cl O 2 + 378.1261 ; Found 378.1257 ATIR: CH ( w, 295 8 cm - 1 ), aromatic CH ( w, 300 6 cm - 1 ) , carbonyl (s, 171 1 cm - 1 ) 100 Figure E. 18: Compound 3 - 18 M ethyl 4 - (((3 - chlorophenyl)(phenyl)amino)methyl)benzoate General Procedure gave the product as a white solid, which decays over a long period of time to a purple solid, 283 mg, 29.8%. 1 H NMR (500 MHz, CDCl 3 J = 8.4 Hz, 2H), 7.49 (d, J = 8. 5 Hz, 2 H), 7.31 (dd, J = 8.7, 7.4 Hz, 4H), 7.13 (dd, J = 8.8, 1.1 Hz, 4 H), 7.05 6.98 (m, 2H), 5.09 (s, 2H), 3.94 (s, 3 H). 13 C NMR (126 MHz, CDCl 3 16 6.9, 147.9, 144.9, 130.0, 129.5, 128.9, 126.6, 121.8, 120 .7, 56.3, 52.1. HRMS ( ESI+ ) m/z : [M+H] + C 21 H 20 NO 2 + 3 18.1494 ; Found 318.1498 ATIR: CH (2957 cm - 1 ), aromatic CH (30 65, 3045, 3009 cm - 1 ), carbonyl (s, 171 1 cm - 1 ) 101 Figure E. 19: Compound 3 - 19 10 - B enzyl - 2 - chloro - 10H - phenothiazine General Procedure provided the product as a colorless oil (200 mg, 17%). 1 H NMR (500 MHz, CDCl 3 ) 7.26 (m, 5 H), 7.12 (dd, J = 7.6, 1.6 Hz, 1 H), 7.04 6.97 (m, 2 H), 6.94 6.84 (m, 2 H), 6.71 6.64 (m, 2 H), 5.06 (s, 2 H). 13 C NMR (126 MHz, CDCl 3 145.8, 143.9, 136.0, 133.1, 128.9, 127.5, 127.4, 127.3, 127.0, 126.6, 123.1, 123.0, 122.4, 121.9, 115.9, 115.7, 52.7. HRMS ( ESI+ ) m/z : [M+H] + for C 19 H 1 5 ClNS + 3 2 4 .0 608 ; Found 3 2 4 . 0611 ATIR: CH (29 24, 2851 cm - 1 ), aromatic CH (3 112 , 30 60 , 30 21 cm - 1 ) 102 Figure E. 20: Compound 3 - 20 5 - B enzyl - 10, 11 - dihydro - 5H - dibenzo[b,f]azepine Synthesized by the g eneral p rocedure to yield the product as a brown solid ( 2.83 mg, 45% ) . 1 H NMR (500 MHz, CDCl 3 ) J = 8.1, 2H), 7.36 ( t , J = 6.5 Hz, 2H), 7.29 7.19 (m, 5 H), 7.08 7.04 (m, 2H), 6.96 (dd, J = 8.1, 2.1 Hz, 1H), 5.01 (s, 2H), 3.35 3.32 (m, 2H), 3.3 3.2 5 ( m , 2H). 13 C NMR (126 MHz, CDCl 3 131.5, 131.2, 129.5, 128.4, 128.1, 127.1, 126.5, 123.3, 122.2, 120.7, 120.2, 55.7, 32.4, 31.9. HRMS (APCI) m/z : [M+H] + C 21 H 1 9 ClN + 320.1200 ; Found 320.1207 Found 378.1257 ATIR: CH ( w, 295 8 cm - 1 ), aromatic CH ( w, 300 6 cm - 1 ) 103 Figure E. 21: Compound 3 - 21 M ethyl 3 - ((2 - chloro - 10H - phenothiazin - 10 - yl)methyl)benzoate General Procedure generated title compound as a white solid ( 535 mg, 20% ) . 1 H NMR (500 MHz, CDCl 3 ) J = 7.7 Hz, 1H), 7.47 (d, J = 7.7 Hz , 1H), 7.37 (t, J = 7.7 Hz, 1H), 7.09 (dd, J = 7.6, 1.6 Hz, 1H), 7.00 6.98 ( m , 2 H), 6.88 (td, J = 7.5, 1.2 Hz, 1H), 6.84 (dd, J = 8.2, 2.0 Hz, 1H), 6.64 6.53 (m, 2H), 5.06 (s, 2H), 3.91 (s, 3H). 13 C NMR (126 MHz, CDCl 3 136.7, 13 3.1, 131.1, 130.8, 129.0, 128.6, 127.8, 127.5, 127.5, 127.1, 123.4, 123.2, 122.6, 122.3, 115.8, 115.6, 52. 3, 52.3. HRMS (APCI) m/z : [M+H] + 21 H 17 ClNO 2 S + 382.0669 ; Found : 382.0670 ATI R Aromatic CH (3100 cm - 1 ), CH(2949 cm - 1 , 2922 cm - 1 ) , (s, 171 3 cm - 1 ) 104 Figure E. 22: Compound 3 - 22 Methyl 3 - ((3 - chloro - 10,11 - dihydro - 5H - dibenzo[b,f]azepin - 5 - yl)methyl)benzoate General procedure provided the produ ct as a pale, sticky oil (18 mg, 30% ) . 1 H NMR (500 MHz, CDCl 3 s, 1H), 7.84 (dt, J = 7.7, 1.5 Hz, 1H), 7.56 (d, J = 7.7 Hz, 1H), 7.32 (t, J = 7.7 Hz, 1H), 7.18 7.03 (m, 4H), 7.00 (d, J = 8.0 Hz, 1H), 6.97 6.90 (m, 1H), 6.85 (dd, J = 8.1, 2.1 Hz, 1H), 4.97 (s, 2H), 3.91 (s, 3H), 3.29 3.21 (m, 2H), 3.22 3.16 (m, 2H). 13 C NMR (126 MHz, CDCl 3 131.2, 130.3, 129.6, 129.3, 128.5, 128.3, 126.6, 123.4, 122.4, 120.5, 120.0, 55.2, 52.1, 32.3, 31.8. HRMS (APCI) m/z : [M+H] + 23 H 2 1 NClO 2 + 378.12 61 ; Found 378.12 73 ATIR: CH (2 848 cm - 1 ), aromatic CH (30 89, 3062 cm - 1 ), carbonyl ( m , 171 9 cm - 1 ) 105 Figure E. 2 3 : Compound 3 - 2 3 3 - ( M ethylthio) - 1H - indole Indole (585 mg, 5 mmol), iodine (634 mg, 2.5 mmol) and dimethyl disulfide (0.24 mL, 2.75 mL) were combined in a round bottom flask charged with ethanol and refluxed for 12 hours. After which, the solution was cooled to room temperatur e, washed with sodium thiosulfate (reaction volume x 2), extracted into EtOAc (reactio n volume x2), dried over sodium sulfite, and purified via MPLC (132.4 mg, 16.2%). 1 H NMR (500 MHz, CDCl 3 8.12 ( bs , 1 N H), 7.91 7.76 (m, 1H), 7.37 (dd, J = 7.0, 1.5 Hz, 1H), 7.33 7.27 (m, 2H), 7.26 (d, J = 2.6 Hz, 1H), 2.43 (d, J = 0.9 Hz, 3H). 13 C NMR (126 MHz, CDCl 3 128.7, 128.0, 122.8, 120.4, 119.2, 111.7, 107.9, 20.3. NMR spectra of the c ompound matched li terature values 180 . 106 Figure E. 24: Compound 3 - 24 Methyl 4 - ((3 - (methylthio) - 1H - indol - 1 - yl)methyl)benzoate Benzylation via general procedure to give the title compound as a yellow solid ( 59 mg, 30%). 1 H NMR (500 MHz, CDCl 3 J = 8.4 Hz, 2H), 7.8 3 7.76 ( m , 1H), 7.33 7.02 (m, 6 H), 5.33 (s, 2H), 3.90 (s, 3 H), 2.40 (s, 3H). 13 C NMR (126 MHz, CDCl 3 142.1, 136.9, 131.6, 130.2, 129.8, 129.6, 126.8, 122.8, 120.4, 119.7, 110.1, 107.6, 52.3, 50.0, 20.5 . HRMS (APCI) m/z : [M+H] + C 18 H 18 NO 2 S + 31 2.1058 ; Found 312. 0369 . ATIR: CH (29 82, 2915 cm - 1 ), aromatic CH (3 108, 3060 cm - 1 ), carbonyl (s, 171 5 cm - 1 ) 107 Figure E. 25: Compound 3 - 25 4 - ( B romomethyl)phenyl acetate P owdered NBS (3.1 g, 17.5 mmol) and benzoyl peroxide (0.8 g, 3.3 mmol) were added to a stirred solution of p - tolyl - acetate in freshly distilled chlroform (20 mL). Solution was refluxed for 4 hours. After cooling to room temperature, solids were removed by f iltrati on before concentrating in vacuo. Residue was then dissolved in DCM, washed with DI water and dried over sodium sulfate. Compound was applied to an MPLC to give the title compound (2.8 g, 74%) with identical NMR spectra to literature 181 . 1 H NMR (500 MHz, CDCl 3 2 (d, J = 6.8 Hz, 2H), 7. 08 (d, J = 6.8 Hz, 2H) , 4.4 3 (s, 2H), 2.31 (s, 3H). 13 C NMR (126 MHz, CDCl 3 108 Figure E. 26: Compound 3 - 26 E thyl 1 - (4 - acetoxybenzyl) - 1H - indole - 2 - carboxylate Benzylation by general procedure gave the title compound as a clear colorless oil (303 mg, 30%) . 1 H NMR (500 MHz, CDCl 3 ) J = 8.0 Hz, 1H), 7.46 ( s, 1H), 7.42 7.32 (m, 2H), 7.22 (t, J = 7.9 Hz, 1H), 7.12 (d, J = 8.6 Hz, 2H), 7.05 6.99 (d, J = 8.6 Hz, 2H), 5.86 (s, 2H), 4.38 (q, J = 7.1 Hz, 2H), 2.28 (s, 3H), 1.40 (t, J = 7.1 Hz, 3H). 13 C NMR (126 MHz, CDCl 3 169.4, 161.9, 149.7, 139.4, 135.9, 127.6, 127.4, 126.1, 125.4, 122.7, 121.7, 120.9, 111.1, 110.8, 60.6, 47.3, 21.1, 14.3 . HRMS (APCI) m/z : [M+H] + C 20 H 20 NO 4 + 338.1392 ; Found 388.1 38 8 ATIR: CH (29 85, 2930 cm - 1 ), aromatic CH (3 108, 3061 cm - 1 ), ca rbonyl (s, 17 04 cm - 1 ), carbonyl (s, 17 62 cm - 1 ) 109 Figure E. 27: Compound 3 - 27 E thyl 1 - (4 - (methoxycarbonyl)benzyl) - 1H - indole - 2 - carboxylate Benzylation by general procedure gave the title compound as a beige solid (444 mg, 43%). 1 H N MR (500 MHz, CDCl 3 ) 7.93 (m, 2H), 7.76 (d, J = 8.0 Hz, 1H), 7.47 (s, 1H), 7.36 7.25 (m, 2H), 7.23 7.13 (m, 1H), 7.11 (d, J = 8.1 Hz, 2H), 5.87 (s, 2H), 4.35 (q, J = 7.1 Hz, 2H), 3.87 (s, 3H), 1.37 (t, J = 7.1 Hz, 3H). 13 C NMR (126 MHz, CDCl 3 ) 161.9, 143.7, 139.5, 130.0, 129.1, 127.6, 126.2, 125.5, 122.8, 121.1, 120.5, 111.3, 110.6, 60.7, 52.0, 47.7, 14.3. HRMS (APCI) m/z : [M+H] + C 20 H 20 NO 4 + 338.1392 ; Found 388.1 372 ATIR: CH (29 8 3 , 2853, 29 24 cm - 1 ), aromatic CH (3 309 , 306 2 c m - 1 ) , carbonyl (s, 17 17 cm - 1 ), carbonyl (s, 17 00 cm - 1 ) 110 Figure E. 28: Compound 3 - 28 4 - ((2 - C hloro - 10H - phenothiazin - 10 - yl)methyl)phenyl acetate Benzylation by general procedure to give an off - white solid ( 12% , 127 mg) . 1 H NMR (500 MHz, CDCl 3 ) J = 8.6 Hz, 2H), 7.09 ( m, 3 H), 6.99 (d, J = 8.2 Hz, 2 H), 6.90 (td, J = 7.5, 1.2 Hz, 1H), 6.86 (dd, J = 8.2, 2.0 Hz, 1H), 6.68 6.61 (m, 2H), 5.02 (s, 2H), 2.31 (s, 3H). 13 C NMR (126 MHz, CDCl 3 , 127.5, 127.5, 127.0, 123.1, 123.1, 122.5, 122.0, 122.0, 115.9, 115.6, 52.2, 21 .2 . HRMS (APCI) m/z : [M+H] + 21 H 17 ClNO 2 S + 382.0663; Found 382.0669 . 111 Figure E. 29: Compound 3 - 29 4 - ((2 - C hloro - 10H - pheno thiazin - 10 - yl)methyl)phenol 4 - ((2 - chloro - 10H - phenothiazin - 10 - yl)methyl)phenyl acetate (88 mg, 0.23 mmol) was added to a stirred solution of KOH (excess) in a mixture of ethanol/water (1:1) and refluxed for 4 hours. After cooling, mixture was concentrated to half volume and extracted with ether. Organic layer was discarded, and the aqueous layer acidified to pH 2 and extracted into ether. Crude material was then concentrated and applied to the MPLC for purification to give a white solid that rapidly turns p urple ( 131 mg, 21% ) 1 H NMR (5 00 MHz, CDCl 3 ) 7. 24 (d, J = 7 . 9 Hz, 1 H), 7.16 (d, J = 8.4 Hz, 2 H), 7. 07 (d d , J = 7.8, 1.4 Hz, 1 H), 7.02 6.94 (m, 2 H), 6.91 6. 82 (m, 2H), 7. 70 (d, J = 9.3 Hz, 2 H) , 6. 68 6. 59 (m, 2 H), 4.98 (s, 2H). 13 C NMR (126 MHz, CDCl 3 ) 128.05, 128.01, 127.51, 127.45, 127.03, 1 23.18, 123.07, 122.43, 122.02, 115.99, 115.88, 115.84, 115.34, 52.23. HRMS (APCI) m/z : [M+H] + C 19 H 1 5 ClNOS + 3 40.0536 ; Found 340.0474 ATIR: CH ( 2962 cm - 1 ), ar omatic CH ( 3062 cm - 1 ), broad OH (3321 cm - 1 ) 112 Figure E. 30: Compound 3 - 30 T ert - butyl 4 - ((2 - chloro - 10H - phenothiazin - 10 - yl)methyl)benzoate Benzylation using general procedure gave the desired compound as a white solid ( 64 mg , 7.6%). 1 H NMR (500 MHz, CDCl 3 ) , J = 8.2 Hz, 2H), 7.36 (d, J = 8.1 Hz, 2H), 7.09 (dd, J = 7.6, 1.5 Hz, 1H), 7.04 6.93 (m, 2H), 6. 92 6.88 (m, 2H), 6. 61 6.55 (m, 2H), 5.07 (s, 2H), 1.60 (s, 9H). 13 C NMR (126 MHz, CDCl 3 145.6, 143.6, 140.8, 133.1, 131.2, 130.1, 12 7.5, 127.4 , 127.1, 126.5, 123.3, 123.2, 122.6, 122.1, 115.8, 115.6, 81.1, 52.4, 28.2 . HRMS (APCI) m/z : [M+H] + 24 H 23 ClNO 2 S + 42 4 .1 133 ; Found 42 4 .1 1 68 . ATIR Aromatic CH (3100 cm - 1 ), CH(2949 cm - 1 , 2922 cm - 1 ) , (s, 171 3 cm - 1 ) 113 Figure E. 3 1 : Compound 3 - 3 1 3 - ((2 - C hloro - 10H - phenothiazin - 10 - yl)methyl)benzoic acid Methyl 3 - ((2 - chloro - 10H - phenothiazin - 10 - yl)methyl)benzoate (0.400 g, 1.05 mmol) was added to a solution of 10% K OH and ethanol (1:1) and refluxed for 2 hours in a round bottom flask . The reaction was extracted with ether and the organic layer discarded. The aqueou s layer was acidified with 10% HCl to pH 2 and extracted into ether (50 mL), washed with brine (50 mL) and dried over sodium sulfate. The resul ting solut ion was concentrated in vacuo to give a white solid ( 376 m g, 97%). 1 H NMR (500 MHz, CDCl 3 ) 1H), 8.01 (d, J = 7.7 Hz, 1H), 7.54 (d, J = 1.5 Hz, 1H), 7.42 (t, J = 7.7 Hz, 1 H), 7.10 (d, J = 7.6, 1H), 7.03 6.95 ( m, 2 H), 6.94 6.83 (m, 2 H), 6.69 6.56 (m, 2 H), 5.10 (s, 2 H). 13 C NMR (126 MHz, CDCl 3 , 132.0, 130.0, 129.3, 129.2, 128.4, 127.6, 127.5, 127.1, 123.5, 123.2, 122.6, 122.4, 115.8, 115.6, 52.2. HRMS (ESI) m/z : [M] - Calcd for C 20 H 1 3 ClNO 2 S - 366.0356; Found 366.0356 ATIR CH Aromatic (2995 cm - 1), CH (2912 cm - 1 ), CO2H (w, br, 3433 cm - 1 ), (w, 1610 cm - 1 ) 114 Figure E. 32: Compound 3 - 32 2 - C hloro - 10 - (3 - nitrobenzyl) - 10H - phenothiazine Benzylation followed the general procedure with one addendum. After addition of 3 - (bromomethyl) - nitrobenzene, potassium iodide (0.166g, 1 mmol) was added and the reaction was stirred while covered in foil at room temperature overnight. Workup is as written in the general procedure to give the title compound as a bright yellow solid (1.16 g, 31 % ). 1 H NMR (500 MHz, CDCl 3 1 3 (d, J = 7.2 Hz, 1H) , 7. 66 (d, J = 6.8 Hz, 1H) , 7.50 (t, J = 7.9 Hz, 1 H), 7.13 (dd, J = 7.6, 1.5 Hz, 1 H), 7.08 6.99 (m, 2 H), 6.95 6.86 (m, 2 H), 6.65 6.50 (m, 2 H), 5.14 (s, 2 H). 13 C NMR (126 MHz, CDCl 3 148.7, 145.4, 143.4, 138 .6, 133.2, 132.8, 12 9.9, 127 .8, 127.5, 127.4, 124.0, 123.5, 122.9, 122.8, 122.6, 121.8, 115.7, 115.6, 51.8. HRMS (APCI) m/z : [M+H] + C 19 H 1 4 ClN 2 O 2 S + 369.0464 ; Found 369.0511 ATIR: CH ( weak, 29 24, 2855 cm - 1 ), aromatic CH (3 309, 3062 cm - 1 ), NO (1 459, 1404 cm - 1 ) 115 Figure E. 33: Compound 3 - 33 3 - ((2 - C hloro - 10H - phenothiazin - 10 - yl)methyl)aniline 2 - chloro - 10 - (3 - nitrobenzyl) - 10H - phenothiazine ( 1.16 g, 3. 1 mmol) was dissolved in EtOAc. Tin(II) chloride dihydrate (10 eq) was added and t he mixture refluxed for 2 h. The mixture was cooled to room temperature and poured into ice , and the pH was adjusted to 10. The resulting s lurry was extracted with EtOAc (3 x 50 mL) and the combined organic fractions washed with brine ( 1 x 50 mL), dried ov er sodium sulfite and concentrated in vacuo to give the title compound in quantitative yield as a white solid (1.17 g). 1 H NMR (500 MHz, CDCl 3 12 ( t , J = 7. 6 Hz, 1H), 7.06 (d, J = 7.5 , 1H), 7.00 6.92 (m, 2H), 6.91 6.79 (m, 2H), 6.74 6.61 (m, 3H), 6.5 8 6.53 ( m , 2H), 4.93 (s, 2H), 3.59 ( b s, 2 NH ) . 13 C NMR (126 MHz, CDCl 3 126.8 , 123. 0, 122.6, 122.3, 121.5, 116.6, 115.9, 115.6, 114.0, 112.8, 52.8. HRMS (APCI) m/z : [M+H] + C 19 H 16 ClN 2 S + 339.0723 ; Found 339.0822 ATIR: NH (3446, 3365 cm - 1 ), CH (29 24 , 285 5 cm - 1 ), aromatic CH (3 205 , 30 11 cm - 1 ), (s, 17 17 cm - 1 ) , NH bend (161 6, 1590 cm - 1 ). 116 Figure E. 34: Compound 3 - 3 4 2 - C hloro - 10 - ( 3 - methoxybenzyl) - 10H - phenothiazine Benzylation was done using the general procedure to isolate product as a yellow oil (99 mg, 28%). 1 H NMR (500 MHz, CDCl 3 21 (d, J = 8.5 Hz, 2H), 7.08 (dd, J = 7.6, 1.6 Hz, 1H), 7.04 6.95 (m, 2H), 6.93 6.79 (m, 4H), 6.71 6.59 (m, 2H), 4.99 (s, 2H), 3.79 (s, 3H). 13 C NMR (126 MHz, CDCl 3 129.8, 127.7, 127.7, 127.4, 127.3, 126.9, 1 23.1, 123.0 , 122.3, 121.9, 115.9, 115.7, 114.3, 113.9, 55.3, 52.1. HRMS (APCI) m/z : [M+H] + 20 H 17 ClNOS + 353.0641 ; Found 353.0672 ATIR: CH (29 24, 2851 cm - 1 ), aromatic CH (3 112, 3060, 3021 cm - 1 ) 117 Figure E. 35: Compou nd 4 - 1 ((4 - B romobenzyl)oxy)(tert - butyl)dimethylsilane p - B romo benzyl alcohol (10 g, 53 mmol) was added to a stirring solution of tert - butyl dimethylsilyl chloride (8.8g, 58.3 mmol) and imidazole (7.9 g, 116.6 mmol) in DCM. Solution was stirred at room t emperature for three days and then quenched with aqueous ammonia chloride. The aqueous layer was extracted with DCM (3x 100 mL) and the combined organic layers were washed with brine (1x 300 mL) and dried over sodium sulfite. Solution was concentrate d in v acuo and purified via hexane:ethyl acetate gradient on the MPLC (16.2 g, 99%). Spectroscopic data matches that reported for this compou nd 162 . 1 H NMR (500 MHz, CDCl 3 7.35 (m, 2 H), 7.35 7.11 ( m, 2 H) , 4.72 ( s , 2 H), 0.98 ( s , 9 H), 0.27 ( s , 6 H). 13 C NMR (126 MHz, CDCl 3 18.4, - 5.2. 118 Figure E. 36: Compound 4 - 2 1 - (4 - ((( T ert - butyldimethylsilyl)oxy)methyl)phenyl) - 2,2,2 - tr ifluor oethan - 1 - one ((4 - B romobenzyl)oxy)(tert - butyl)dimethylsilane (16.2 g, 53 mmol) was dissolved in anhydrous THF and stirred for 15 minutes at - 78 °C (acetone dry ice bath). nBuLi (25.4 mL, 63.6 mmol) was added dropwise under constant temperature over 1 hour. Solution was left to stir at - 78 °C for 1.5 hours before dr opwise addition of ethyl trifluoroacetate (8.9 mL, 74.2 mmol). Temperature and stirring was maintained for another 1.5 hours. To quench, saturated aqueous ammonium chloride (reaction volume) was a dded at - 78 °C and reaction was allowed to warm to room temp erature before being diluted with diethyl ether. Organic layer was washed with saturated aqueous ammonium chloride (2x 100 mL), brine (2x 100 mL). Organic layer was dried over sodium sulfate and c oncentrated in vacuo to give the title compound (10.9 g, 64% ) as a colorless liquid . Product was used without further purification and matched literature 162 . 1 H NMR (500 MHz, CDCl 3 J = 8.0 Hz , 2 H), 7.52 (d, J = 8.1 Hz, 2 H), 4.85 (s, 2 H), 0.98 (s, 9 H), 0.14 (s, 6 H). 13 C NMR (126 MHz, CDCl 3 2 (q, 2 J CF = 38.6 Hz, 1C) , 150.1, 130.2 (q, 4 J CF = 1 Hz , 1C) 128.6, 128.2, 126.9, 126.1, 126.0, 117.9, 116.7 (q, 1 J CF = 292.5 Hz, 1C), 115.6, 64.2, 25.9, - 5.4. 119 Figure E. 37: Compound 4 - 3 (E) - 1 - (4 - ((( T ert - butyldimethylsilyl)oxy)methyl)phenyl) - 2,2,2 - trifluoroethan - 1 - one O - tosyl oxime Hydroxylamine hydrochloride salt (7.3 g, 105 mmol) and sodium acetate (11.5 g , 140 mmol) were com bined in ethanol stirred for 15 minutes at room temperature. Solids were filtered and the supernatant was added to a stirring solution of 1 - (4 - (((tert - butyldimethylsilyl)oxy)methyl)phenyl) - 2,2,2 - trifluoroethan - 1 - one (10.9 g, 35 mmol) an d exce ss sodium sulf ate. Mixture was refluxed overnight. After cooling to room temperature, solution is filtered and concentrated in vacuo . The crude material is backfilled with argon and charged with anhydrous DCM (5 mL/2 mmol of product calculated from c rude m ass), pyridine (5.6 mL, 70 mmol ), DMAP (3.8 g, 31.5 mmol) and cooled to zero centigrade. In batches, p - toluene sulfonyl chloride (7.3 g, 38.5 mmol) was added and mixture stirred for 0.5 h at zero then 2 hours at room temperature. Reaction was then qu enched with DI water and extracted with DCM (3x 100 mL). Combined organic layers were dried over sodium sulfate, concentrated in vacuo and purified on silica gel with 10% EtOAc in hexanes (2 g, 13.7%) as a pale green oil . Compound exists as a mixture of E/ Z isom ers and matches spectra from the literature 162 . 120 Figure E. 38: Compound 4 - 5 3 - (4 - ((( T ert - butyldimethylsilyl)oxy)methyl)phenyl) - 3 - (trifluoromethyl) - 3H - diazirine (E) - 1 - ( 4 - (((t ert - butyldimethylsilyl)oxy)methyl )phenyl) - 2,2,2 - trifluoroethan - 1 - one O - tosyl oxime (2 g, 4.7 mmol) was dissolved in diethyl ether (anhydrous 20 mL) and cooled to - 78 °C. Ammonia was bubbled through until ~60 mL had condensed and reaction was stirred for 3 hours at - 78 °C, then allowed to warm to room temperature over 2 hours. After evaporation of ammonia, the reaction mixture was filtered and concentrated to give a translucent paste that was used in subsequent transformations without further purifica tion . The paste was dissolved in anhydr ous MeOH and TEA (excess) in a darkened fume hood. Molecular iodine (0.6 g, 2.4 mmol) was added in portions until a red/orange color persists. After stirring for 20 minutes at room temperature, the reaction was quench ed wit h a few drops of 5% sodium metabi sulphite and neutralized with 10% citric acid. Mixture was poured into ether (150 mL), dried over sodium sulfate, and concentrated in vacuo . Crude material was purified via MPLC in the dark using 2:1 hexane/DCM to giv e the title compound as a light yellow oil (303 mg, 20%). Spectral data matches literature report 162 . 1 H NMR (500 MHz, CDCl 3 7.36 (m, 2H), 7.19 (d, J = 8.1 Hz, 2H), 4.77 (s, 2H), 0.97 (d, J = 1.0 Hz, 9H), 0.13 (d, J = 1.0 Hz, 6H). 13 C NMR (126 MHz, CDCl 3 127.6, 126.4, 126.4, 126.2, 125.5, 123.3, 121.1, 118.9, 64.2, 25.9, 18.4, - 5.4. 121 Figure E. 39: Compound 4 - 6 (4 - (3 - ( T rifluoromethyl) - 3H - diazirin - 3 - yl)pheny l)meth anol 3 - (4 - ((( T ert - butyldimethylsilyl)oxy)methyl)phenyl) - 3 - (trifluoromethyl) - 3H - diazirine (303 mg, 0.9 mmol) was dissolved in 1M TBAF in THF (2 mL) with 5% water (0.1 mL) in a round bottom flask wrapped i n foil. Reaction was stirred for 5 hours at ro om tem perature before being diluted with diethyl ether (40 mL), washed with DI water (3 x12 mL). Organic layers were combined, dried over sodium sulfate, and concentrated in vacuo . Purify on silica gel with 100 % DCM to give the title compound (quant, 0.9 m mol) a s a pale - yellow oil . Spectral data matches literature report 162 . 1 H NMR (500 MHz, CDCl 3 ) 7.35 (d, J = 8.0 Hz, 2H), 7.17 (d, J = 8.0 Hz, 2H), 4.66 (d, J = 5.5 Hz, 2H) , 2.3 (bs, 1H). 13 C NMR (126 MHz, CDCl 3 , 126.6, 122.1 (q, 1 J CF = 275.4 Hz, 1C), 64.3. 122 Figure E. 40 : Compound 4 - 7 2,5 - D ioxopyrrolidin - 1 - yl 3 - ((2 - chloro - 10H - phenothiazin - 10 - yl)methyl)benzoate Compound 3 - 31 (460 mg, 1.3 mmol) , EDCI (255 mg, 2 mmol), and DIPEA (0.7 mL, 2 mmol) were combined in anhydrous DMF and stirred at room temperature. After 1 hour, N - hydroxysuc cinamide w as added and allowed to stir for 4 hours at room temperature. Solution was then diluted with EtOAc (~ 100 mL), washed with saturated bicarbonate solution (2x 100 mL), DI water (3x 100 mL), and brine (1x 100 mL). Organic layer was dried over sodiu m sulfite and concentrated in vacuo before being purified via standard MPLC conditions to give the title compound (22 mg, 3.6%) as a white solid . 1 H NMR (500 MHz, CDCl 3 s, 1H), 8 .05 ( d, J = 8 . 7 Hz, 1 H ), 7. 59 ( d, J = 7.6 Hz , 1H ), 7.45 ( t , J = 7.8 H z, 1H), 7.10 (d, J = 7.6 Hz, 1H), 7.05 6.97 (m, 2H), 6.95 6.80 (m, 2H), 6.65 6.52 (m, 2H), 5.11 (s, 2H), 2.91 (s, 4H). 13 C NMR (126 MHz, CDCl 3 143.4, 137.4, 133.3, 133.1, 129.6, 129.5, 128.8, 127.7, 127.5, 127.2, 125.8 , 123.6, 123.3, 122.7, 122.4, 115.8, 115.5, 52.0, 25.7. HRMS (APCI) m/z : [M+H] + C 24 H 1 8 ClN 2 O 2 S + 465.0676 ; Found 465.0 6 93 123 Figure E. 41 : Compound 5 - 4 1 - ( B ut - 3 - yn - 2 - yl) - 1 - hydroxyurea But - 3 - yn - 2 - ol (5 mL, 63 mmol) was d issolved in dry DCM (ca. 110 mL) to give a colorless solution which was cooled to 0°C before freshly distilled TEA (11.67 mL, 83 mmol) was added as a single portion. This solution was allowed to stir for 10 min at 0° C before methane sulfonyl ch loride (5.9 6 mL, 76.7 mmol) was added dropwise while keeping the temperature below 10°C. The reaction was stirred below 10°C until TLC showed complete conversion of starting material. An equal volume of 0.5 M HCl was added and the layers were separated. Th e aqueous l ayer was further extracted with DCM (2x30 mL). Organic layers were combined, washed with brine (2x70 mL), dried over MgSO 4 , filtered and dried in vacuo before dissolving the residue in MeOH and cooling to 0°C. Hydroxylamine (50% aq. solution, 31.3 mmol) wa s added and the mixture was allowed to warm to room temperature with stirring. After stirring for 16 hr, the solution was concentrated in vacuo and pH adjusted to 9 with sodium hydroxide pellets. The mixture was then extracted with ethyl acetate (4x 100 mL ) and concentrated to ca. 30 mL. To this pale yellow solution, potassium cyanate (13.8g, 170 mmol) in ca. 100 mL water was added in a single portion and allowed to sitr for 20 minutes. Afterwards, fuming HCl (15.45 mL, 502 mmol) was added dropwise via addi tion funnel while keeping the temperature below 10°C with an ice bath. After addition was complete, the solution was warme d to room temperature and stirred for an additional 14 hr. The layers were then separated and the aqueous layer further extracted with EtOAc (5 x 200 mL). The organic layers were combined into two fractions 124 [due to limitation of available glassware size], both washed with brine (3x 100mL) and dried over MgSO4. The solvent was removed under reduced pressure to give ca. 30mL. This was dilu ted under vigorous stirring with heptane (100 mL, white precipitate forms) and concentrated under reduced pressure. The re sidue was recrystallized from EtOAc/diethyl ether 1:1 and the resulting white solid was filtered off and the mother liquour concentrat ed under reduced pressure without heat to precipitate out additional product. The flask was placed into a fridge overnight and the resulting white solid was also filtered and combined with the previous fraction and dried under reduced pressure to yield the title compound (2.8 g, 22 %) as a white free flowing powder. Spectral data match literature report 151 . 1 HNMR (500 MHz, DMSO - d 6 9.23 (s, 1H), 6 .50 (s, 2H), 4.84 (q, J = 7.0 Hz, 1H), 3.03 (d, J = 2.2 Hz, 1H), 1.24 (d, J = 7.1 Hz, 3H). 13 CNMR (126 MHz, DMSO - d 6 162.0, 84.3, 73.3, 45.6, 18.9. 125 Figure E. 42: Compound 5 - 5 3 - (3 - I odophen oxy)propan - 1 - ol A round bottom flask was charged with THF and sodium hydride (1.05 g, 26.25 mmol) under an inert atmosphere . M - iodophenol (6g, 27.5 mmol) was carefully added in 3 portions and stirred for 15 minutes at room temperature. 3 - Bromo - propa nol (2. 3 mL, 25 mmol) was then added dropwise and the solution was stirred for 3 days. Solution was then concentrated to dryness in vacuo then diluted with DI water before being extracted into diethyl ether. Organic layer was washed with Brine (2 x 150 mL) , dried over sodium sulfite and concentrated in vacuo . Crude material was purified on a large silica gel column eluted with 100% hexanes to give the title compound (6.58 g, 94%). Spectral data matched literature 182 . 1 H NMR (500 MHz, CDCl 3 7.22 (m, 2H), 6.99 (t, J = 7.8 Hz, 1H), 6.87 (ddd, J = 8.4, 2.5, 1.0 Hz, 1H), 4.09 (t, J = 6.0 Hz, 2H), 3.85 (t, J = 5.9 Hz, 2H), 2.03 (p, J = 6.0 Hz, 2H). 13 C NMR (126 MHz, CDCl 3 .0, 123.6, 114.2, 94.4, 65.7, 60.2, 31.9. 126 Figure E. 43: Compound 5 - 6 3 - (3 - I odophenoxy)propanal 3 - (3 - I odophenoxy)propan - 1 - ol (2g, 7.2 mmol) and IBX (4.03 g, 14.4 mmol) were combined in a round bottom flask charged with EtOAc and brought to reflux for 9 hours. A fter cooling, the solution was plac ed in a refrigerator for 1 hour and solids filtered off. Organic liquid was concentrated in vacuo to give the title compound as an oil which was used without further purification ( 2.1g, 99%). 1 H NMR (500 MHz, CDCl 3 5 (t, J = 1.5 Hz, 1H), 7.27 7.16 (m, 2H), 6.96 6.85 (m, 1H), 6.80 (ddd, J = 8.4, 2.5, 1.0 Hz, 1H), 4.16 (t, J = 6.1 Hz, 2H), 2.80 (td, J = 6.0, 1.6 Hz, 2H). 13 C NMR (126 MHz, CDCl 3 159.0, 131.0, 130.2, 123.6, 114.2, 94.6, 61.7, 43.0. 127 Figure E. 44: Compound 5 - 7 D iethyl (2,5 - dioxoimidazolidin - 4 - yl)phosphonate Imidazolidine - 2,4 - dione (hydantoin) (20 g, 200 mmol) and acetic acid (80 mL) was heated to 85 °C in an oil bath. An addition funnel was cha rged with bromine (11.2 mL, 220 mmol) and a small amount of bromine (~2 mL ) was introduced into the reaction mixture with vigorous stirring. Once the orange color had disappeared , the remainder of the bromine was added rapidly dropwise to afford a clear so lution. After being stirred at 85 °C for 30 min, the reaction mixture was cooled to 30 °C in an ice bath and triethyl phosphite (47.9 mL, 280 mmol) introduced at such a rate that the internal temperature was maintained between 40 - 45 °C. After the additi on was completed, the mixture was stirred at room temperature for 90 min. The solvent was removed in vacuo and the residue diluted with diethyl ether (80 mL) with stirring to induce precipitation of a white solid. The mixture was poured onto diethyl ether (20 0 mL) with vigorous stirring. After 30 min, filtration afforded the title compound (25.6 g, 54.2%) as a white solid , which matched literature reports 183 and wa s u sed without further purification. 128 Figure E. 44: Compound 5 - 11 5 - (2 - (3 - I odophenoxy)ethyl)imidazolidine - 2,4 - dione To a solution of 1 - iodo - 3 - (prop - 2 - yn - 1 - yloxy)benzene (1 g, 3.9 mmol) in dry DCM ( ca. 35 mL) was adde d borane dimethyl sulfide solution (2.75 mL, 4.5 mmol) at room temperature in the dark under inert atmosphere in one portion. The reaction was stirred vigorously for 0.5 hr, after which 1 mL of MeOH was added carefully, followed by 10% NaOH (4 mL) and 30% peroxide solution (4 mL). This solution was left to stir for 20 hours before extraction with DCM. The organic layers were combined and washed with 10% HCl, brine, and dried over MgSO 4 before being concentrated in vacuo. The crude is combine d with potassium cyanide (0.507 g, 7.8 mmol), and ammonium carbonante (1.5 g, 15.6 mmol) in a round bottom flask in ethanol/water (1:1) mixture and sealed with a rubber septum . The slurry is heated to 70°C and stirred 24 hours. After cooling to room temperature, the solut ion was concentrated to half volume and acidified to pH <6 with concentrated hydrochloric acid. Solution is extracted with diethyl ether and concentrated in va cuo . DCM is added to the crude yellow oil and filtered to collect the white precipitate. The prec ipitate is dried in vacuo to give a white solid (0.030 g, 2.4%). 1 HNMR (DMSO - d 6 10.65 (s, 1 N - - - 1.90 (m, 1H) . 13 CNMR (DMSO - d 6 176.4, 159.5, 157.9, 131.8, 130.0, 123. 6, 114.8, 95.5, 64.3, 55.2, 31.3. MS (ESI+): m/z calculated [M+H] + C 11 H 12 IN 2 O 3 + 346.99, found 347.00 129 Figure E. 45: Compound 5 - 1 2 ( E/ Z) - 5 - (3 - (3 - I odophenoxy)propylidene)imidazolidine - 2,4 - dione D iethyl (2,5 - dioxoimidazolidi n - 4 - yl)phosphonate (2.13g, 9 mmol) is dissolved in ethanol under an inert atmosphere. Sodium metal (216 mg, 9 mmol) is added and mixture stirred for 20 minutes at room temperature. Then, 3 - (3 - iodophenoxy)propanal (2 g, 7.2 mmol) is added as a solution in ethanol and reacti on stirred for 20 minutes. After which, the mixture is poured into 10% HCl solution to precipitate the title compound as a mixture of E/Z isomers (1.18g, 45.7%). 1 H NMR (500 MHz, DMSO - d 6 N H), 10.27 (s, 1 N H), 7.28 ( bs , 2H), 7.17 7.00 (m, 1H ), 7.00 6.89 (m, 1H), 5.55 (t, J = 7.7 Hz, 1H), 4.03 (dt, J = 13.8, 6.5 Hz, 2H), 2.98 (q, J = 6.8 Hz, 1H), 2.58 (q, J = 6.8 Hz, 1H). 13 C NMR (126 MHz, DMSO - d 6 165.1, 164.7, 159.6, 159.6, 155.3, 154.4, 132.2, 131.8, 131.0, 130.0, 128 .1, 124.4, 123.4, 123.4, 115.4, 114.9, 114.9, 112.5, 107.7, 95.5, 67.5, 66.8, 26.7, 25.8. 130 B. Proteomic Methods Determination of the proteasome binding site was performed as described. Compound 4 - 7 was incubated with human proteasome in HEPES Buffer (pH 7.4, 100 mM NaCl) with a drop of DMSO (to solubilize 4 - 7 in the aqueous media) for 1 hour at 37 °C before being frozen in a - 80 °C freezer for an additional hour. This was taken to the metabolomics core where it was thawed, digested with trypsin and subjec ted to mass spectrometry and computational analysis. Analysis constraints were as follows: DATABASE SEARCHING -- Tandem mass spectra were extracted by [unknown] version [unknown]. Charge state deconvolution and deisotoping were not performed. All MS/MS samp les were an alyzed using Mascot (Matrix Science, London, UK; version 2.6.0) and X! Tandem (The GPM, thegpm.org; version X! Tandem Alanine (2017.2.1.4)). Mascot was set up to search the UP_human_crap_20171102 database (unknown version, 160685 entries) assumi ng the dige stion enzyme stricttrypsin. X! Tandem was set up to search a reverse concatenated subset of the UP_human_crap_20171102 database (unknown version, 1404 entries) also assuming strict trypsin. Mascot and X! Tandem were searched with a fragment ion mass tolera nce of 0.020 Da and a parent ion tolerance of 10.0 PPM. Carbamidomethyl of cysteine was specified in Mascot and X! Tandem as a fixed modification. Glu - >pyro - Glu of the n - terminus, ammonia - loss of the n - terminus, gln - >pyro - Glu of the n - terminus, oxidation o f methionine and CJones_mod of cysteine and lysine were specified in X! Tandem as variable modifications. Oxidation of methionine and CJones_mod of cysteine and lysine were specified in Mascot as variable modifications. 131 CRITERIA FOR PROTEIN ID ENTIFICATIO N -- Scaffold (version Scaffold_4.8.9, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 17.0% probabilit y by the Sc affold Local FDR algorithm. Protein identifications were accepted if they could be established at greater than 80.0% probability to achieve an FDR less than 1.0% and contained at least 2 identified peptides. Protein probabilities were assigned by the Prot ein Prophet algorithm (Nesvizhskii, Al et al Anal. Chem. 2003;75(17):4646 - 58). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Proteins s haring sign ificant peptide evidence were grouped into clusters. Tepe - Jones Mod Data 20190122, Publication report created on 04/12/2019" Experiment: Tepe - Jones Mod Data 20190122 Peak List Generator: unknown Version: unknown Charge Sta tes Calculated: unknown Deisotoped: unknown Textual Annotation: unknown Database Set: 2 Databases Database Name: a reverse concatenated subset of the UP_human_crap_20171102 database Version: unknown Taxonomy: All Entries Number of Proteins: 1404 Database Name: the UP_human_crap_20171102 database Version: unknown Taxonomy: All Entries Number of Proteins: 160685 Does database contain common contaminants?: unknown Search Engine Set: 2 Search Engines Searc h En gine: Mascot Version: 2.6.0 Samples: All Samples Fragment Tolerance: 0.020 Da (Monoisotopic) 132 Parent Tolerance: 10.0 PPM (Monoisotopic) Fixed Modifications: +57 on C (Carbamidomethyl) "Variable Modifications: +16 on M (Oxidation ), + 349 on CK (CJones_mod)" "Database: the UP_human_crap_20171102 database (unknown version, 160685 entries)" Digestion Enzyme: stricttrypsin Max Missed Cleavages: 2 Probability Model: "Jones .temp (F012624): LFDR Model, Classifier da ta: Bayes, Good (78%) m:22.9/s:24.9 m:56.1/s:25.3 m:NA m:NA m:NA, Bad (22%) m: - 26.1/s:7.66 m:6.64/s:7.02 m:NA m:NA m:NA [all charge states]" Search Engine: X! Tandem Version: X! Tandem Alanine (2017.2.1.4) Samples: All Samples F ragment Toler ance: 0.020 Da (Monoisotopic) Parent Tolerance: 10.0 PPM (Monoisotopic) Fixed Modifications: +57 on C (Carbamidomethyl) "Variable Modifications: - 18 on Peptide N - Terminal (Glu - >pyro - Glu), - 17 on Peptide N - Terminal (Ammonia - loss), - 17 on Pepti de N - Terminal (Gln - >pyro - Glu), +16 on M (Oxidation), +349 on CK (CJones_mod)" "Database: a reverse concatenated subset of the UP_human_crap_20171102 database (unknown version, 1404 entries)" Digestion Enzyme: stricttrypsin Max Mis sed Cleavages : 2 Probability Model: "Jones .temp (F012624): LFDR Model, No Classifier [all charge states]" Scaffold: Version: Scaffold_4.8.9 Modification Metadata Set: 2334 modifications Source: C: \ Program Files \ Scaffold 4 \ parameters \ u nimod.xml Comment: Protein Grouping Strategy: Experiment - wide grouping with protein cluster analysis Peptide Thresholds: 17.0% minimum Protein Thresholds: 80.0% minimum and 2 peptides minimum Peptide FDR: 0.0% (Decoy) Protein FDR: 0.0% (Decoy) GO Annotation Source(s): Alternate ID Source(s): 133 C. Proteasome Activity Assays The a ctivity assays were carried out in a 1 concentrations of test compounds were added to a black flat/clear bottom 96 - well plate con taining 1 nM of human constitutive 20S proteasome, in 50 mM Tris - HCl at pH 7 .8 and allowed to sit for 1 5 min at 37 °C . Fluorogenic substrates were then added and the enzymatic activity measured at 37 °C on a SpectraMax M5e spectrome ter by measuring the cha nge in fluorescence unit per 5 minutes units for the vehicle control were set at a 100%, and the ratio of drug - treated sample set to that of vehicle control was used to calculate the fold chan ge in enzymatic activity. Fold activity was plotted as a funct ion of drug concentration, using Origin Pro 9 . The fluorogenic substrates used were Suc - LLVY - AMC (CT - - LLE - AMC (Casp - - LRR - AMC (T - L activity, 1 . 134 Figur e E . 46 : Dose Response of Combination Se t 1 135 Figur e E . 4 7 : Dose Response of Combination Set 2 136 Figur e E . 4 8 : Dose Response of Combination Set 3 137 Figur e E . 4 9 : Dose Response of B1 Set 1 138 Figur e E . 50 : Dose Response of B 1 Set 2 139 Figur e E . 51 : Dose Response of B1 Set 3 140 Figur e E . 52 : Dose Response of B 2 Set 1 141 Figur e E . 5 3 : Dose Response of B 2 Set 2 142 Figur e E . 5 4 : Dose Response of B 2 Set 3 143 Figur e E . 5 5 : Dose Response of B 5 Set 1 144 Figur e E . 5 6 : Dose Resp onse of B 5 Set 2 145 Figur e E . 5 7 : Dose Response of B 5 Set 3 146 A PPENDIX 147 Figure A.1: 1 H and 13 C NMR Spectra of Compound 3 - 1 148 Figure A.2: 1 H and 13 C NMR Spectra of Compoun d 3 - 2 149 Figure A.3: 1 H and 13 C NMR Spectra of Compound 3 - 3 150 Figure A. 4 : 1 H and 13 C NMR Spectra of Compound 3 - 4 151 Figure A.5: 1 H and 13 C NMR Spectra of Compound 3 - 5 152 Figure A.6: 1 H and 13 C NMR Spectra of Compound 3 - 6 153 Figure A.7: 1 H and 13 C NMR Spectra of Compound 3 - 7 154 Figure A.8: 1 H and 13 C NMR Spectra of Compound 3 - 8 155 Figure A.9: 1 H and 13 C NMR Spectra of Compound 3 - 9 156 Figure A.10: 1 H and 13 C NMR Spectra of Compound 3 - 10 157 Figure A.11: 1 H and 13 C NMR Spectra of Compound 3 - 11 158 Figure A.1 2 : 1 H and 13 C NMR Spectra of Compound 3 - 1 2 159 Figure A.1 3: 1 H and 13 C NMR Spectra of Compound 3 - 13 160 Figure A.1 4 : 1 H and 13 C NMR Spectra of Compound 3 - 1 4 161 Figure A.15: 1 H and 13 C NMR Spectra of Compound 3 - 15 162 Figure A.1 6 : 1 H and 13 C NMR Spectra of Compound 3 - 1 6 163 Figure A.1 7 : 1 H and 13 C NMR Spectr a of Compound 3 - 1 7 164 Figure A.1 8 : 1 H and 13 C NMR Spectra of Compound 3 - 1 8 165 Figure A.1 9 : 1 H and 13 C NMR Spectra of Compoun d 3 - 1 9 166 Figure A. 20 : 1 H and 13 C NMR Spectra of Compound 3 - 20 167 Figure A. 21 : 1 H and 13 C NMR Spectra of Compound 3 - 21 168 Figure A. 22 : 1 H and 13 C NMR Spectra of Compound 3 - 22 169 Figure A. 23 : 1 H and 13 C NMR Spectra of Compound 3 - 23 170 Figure A. 24 : 1 H and 13 C NMR Spectra of Compound 3 - 24 171 Figure A. 25 : 1 H and 13 C NMR Spectra of Compound 3 - 25 172 Figure A. 26 : 1 H and 13 C NMR Spectra of Compound 3 - 26 173 Figure A. 27 : 1 H and 13 C NMR Spectra of Compound 3 - 27 174 Figure A. 2 8 : 1 H and 13 C NMR Spectra of Compound 3 - 2 8 175 Figure A. 29 : 1 H and 13 C NMR Spectra of Compound 3 - 29 176 Figure A. 30 : 1 H and 13 C NMR Spectra of Compound 3 - 30 177 Figure A. 31 : 1 H and 13 C NMR Spectra of Compound 3 - 31 178 Figure A. 32 : 1 H and 13 C NMR Spectra of Compound 3 - 32 179 Figure A. 33 : 1 H and 13 C NMR Spectra of Compound 3 - 33 180 Figure A . 34 : 1 H and 13 C NMR Spectra of Compound 3 - 34 181 Figure A. 35 : 1 H and 13 C NMR Spectra of Compound 4 - 1 182 Figure A. 36 : 1 H and 13 C NMR Spectra of Compound 4 - 2 183 Figure A. 37 : 1 H and 13 C NMR Spectra of Compound 4 - 3 184 Figure A. 38 : 1 H and 13 C NMR Spectr a of Compound 4 - 4 185 Figure A. 39 : 1 H and 13 C NMR Spectra of Compound 4 - 5 186 Figure A. 40 : 1 H and 13 C NMR Spectra of Compound 4 - 7 187 Figure A. 41 : 1 H and 13 C NMR Spectra of Compound 5 - 4 188 Figure A. 42 : 1 H and 13 C NMR Spectra of Compound 5 - 5 189 Figure A. 43 : 1 H and 13 C NMR Spectra of Compound 5 - 6 190 Figure A. 44 : 1 H and 13 C NMR Spectra of Compound 5 - 7 191 Figure A. 45 : 1 H and 13 C NMR Spectra of Compound 5 - 11 192 Figure A. 46 : 1 H and 13 C NMR Spectra of Compound 5 - 12 193 REFERENCES 194 REFERENCES 1. Kulak, N. A.; Geyer, P. E.; Mann, M., Loss - less Nano - fractionator for High Sensitivity, High Coverage Proteomics. Molecular & Cellular Proteomics 2017, 16 (4), 694 - 705. 2. Hetz, C.; Glimcher, L. H., Protein homeostasis networks in physiology and diseas e. Current opinion in cell biology 2011, 23 (2), 123 - 125. 3. Rouillard, A. D.; Gundersen, G. W.; Fernandez, N. F.; Wang, Z.; Monteiro, C. D.; gathere d to serve and mine know ledge about genes and proteins. Database 2016, 2016 , baw100 - baw100. 4. Brehme, M.; Voisine, C.; Rolland, T.; Wachi, S.; Soper, James H.; Zhu, Y.; Orton, K.; Villella, A.; Garza, D.; Vidal, M.; Ge, H.; Morimoto, Richard I., A Chapero me Subnetwork Safeguards Proteostasis in Aging and Neurodegenerative Disease. Cell Reports 2014, 9 (3), 1135 - 1150. 5. García - Prat, L.; Martínez - Vicente, M.; Perdiguero, E.; Ortet, L.; Rodríguez - Ubreva, J.; Rebollo, E.; Ruiz - Bonilla, V.; Gutarra, S.; Balles tar, E.; Serrano, A. L.; Sandri, M.; Muñoz - Cánoves, P., Autophagy maintains stemness by preventing senescence. Nature 2016, 529 , 37. 6. Bartlett, A. I.; Radford, S. E., An expanding arsenal of experimental methods yields an explosion of insights into prote in folding mechanisms. N ature Structural &Amp; Molecular Biology 2009, 16 , 582. 7. Ellis, R. J.; Minton Allen, P., Protein aggregation in crowded environments. In Biological Chemistry , 2006; Vol. 387, p 485. 8. Klaips, C. L.; Jayaraj, G. G.; Hartl, F. U., Pathways of cellular pro teostasis in aging and disease. The Journal of Cell Biology 2017 . 9. Kaushik, S.; Cuervo, A. M., Proteostasis and aging. Nature Medicine 2015, 21 , 1406. 10. Henning, R. H.; Brundel, B. J. J. M., Proteostasis in cardiac health and di sease. Nature Reviews Ca rdiology 2017, 14 , 637. 11. Tanaka, K.; Matsuda, N., Proteostasis and neurodegeneration: The roles of proteasomal degradation and autophagy. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 2014, 1843 (1), 197 - 204. 195 12. Labbadia, J.; Morimoto, R. I., The Biology of Proteostasis in Aging and Disease. Annual Review of Biochemistry 2015, 84 (1), 435 - 464. 13. Medinas, D. B.; Valenzuela, V.; Hetz, C., Proteostasis disturbance in amyotrophic lateral sclerosis. Human Molecular G enetics 2017, 26 (R2), R 91 - R104. 14. 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. 15. Korovila, I.; H ugo, M.; Castro, José P. ; Weber, D.; Höhn, A.; Grune, T.; Jung, T., Proteostasis, oxidative stress and aging. Redox Biology 2017, 13 , 550 - 567. 16. Morley, J. F.; Brignull, H. R.; Weyers, J. J.; Morimoto, R. I., The threshold for polyglut amine - expansion pro tein aggregation and cel lular toxicity is dynamic and influenced by aging in Caenorhabditis elegans. Proc Natl Acad Sci U S A 2002, 99 (16), 10417 - 22. 17. David, D. C.; Ollikainen, N.; Trinidad, J. C.; Cary, M. P.; Burlingame, A. L.; Ken yon, C., Widespread protein aggregation as an inherent part of aging in C. elegans. PLoS biology 2010, 8 (8), e1000450. 18. Ben - Zvi, A.; Miller, E. A.; Morimoto, R. I., Collapse of proteostasis represents an early molecular event in Caenorhabditis elegans aging. Proc Natl Ac ad Sci U S A 2009, 106 (35), 14914 - 9. 19. Taylor, R. C.; Berendzen, K. M.; Dillin, A., Systemic stress signalling: understanding the cell non - autonomous control of proteostasis. Nature reviews. Molecular cell biology 2014, 15 (3), 211 - 21 7. 20. Hipp, M. S.; Park, S. - H.; Hartl , F. U., Proteostasis impairment in protein - misfolding and - aggregation diseases. Trends in Cell Biology 2014, 24 (9), 506 - 514. 21. Halliwell, B.; Hu, M. - L.; Louie, S.; Duvall, T. R.; Tarkington, B. K.; Motchnik, P.; C ross, C. E., Intera ction of nitrogen d ioxide with human plasma Antioxidant depletion and oxidative damage. FEBS Letters 1992, 313 (1), 62 - 66. 22. Menzel, D. B., The toxicity of air pollution in experimental animals and humans: the role of oxidative stress. Toxicology Letters 1994, 72 (1), 269 - 277. 23. Hu, M. - L.; Tappel, A. L., POTENTIATION OF OXIDATIVE DAMAGE TO PROTEINS BY ULTRAVIOLET - A AND PROTECTION BY ANTIOXIDANTS. Photochemistry and Photobiology 1992, 56 (3), 357 - 363. 24. Kappus, H., Oxidative stress i n chemical toxicity. Archives of Toxic ology 1987, 60 (1), 144 - 149. 196 25. Davies, K. J. A., Oxidative stress: the paradox of aerobic life. Biochemical Society Symposium 1995, 61 , 1 - 31. 26. Appelqvist, H.; Kågedal, K.; Wäster, P.; Öllinger, K., The lysosome: f r om waste bag to potential therapeutic target. Journal of Molecular Cell Biology 2013, 5 (4), 214 - 226. 27. Glick, D.; Barth, S.; Macleod, K. F., Autophagy: cellular and molecular mechanisms. The Journal of pathology 2010, 221 (1), 3 - 12. 28. Hayat, M. A., C h apter 1 - Overview of Autophagy. In A utophagy: Cancer, Other Pathologies, Inflammation, Immunity, Infection, and Aging , Hayat, M. A., Ed. Academic Press: 2017; pp 3 - 90. 29. Feng, Y.; He, D.; Yao, Z.; Klionsky, D. J., The machinery of macroautophagy. Cell R esearch 2013, 24 , 24. 30. Levine, B.; Mizushima, N.; Virgin, H. W., Autophagy in immunity and inflammation. Nature 2011, 469 , 323. 31. Shang, L.; Chen, S.; Du, F.; Li, S.; Z hao, L.; Wang, X., Nutrient starvation elicits an acute autophagic response mediat e d by Ulk1 dephosphorylation and its subsequent dissociation from AMPK. Proceedings of the National Academy of Sciences of the United States of America 2011, 108 (12), 4788 - 4 793. 32. Li, W. - w.; Li, J.; Bao, J. - k., Microautophagy: lesser - known self - eating. C ellular and Molecular Life Sciences 2012, 69 (7), 1125 - 1136. 33. Bandyopadhyay, U.; Kaushik, S.; Varticovski, L.; Cuervo, A. M., The Chaperone - Mediated Autophagy Receptor Or ganizes in Dynamic Protein Complexes at the Lysosomal Membrane. Molecular and Cell u lar Biology 2008, 28 (18), 5747 - 5763. 34. Youle, R. J.; Narendra, D. P., Mechanisms of mitophagy. Nature Reviews Molecular Cell Biology 2010, 12 , 9. 35. Liu, K.; Czaja, M. J ., Regulation of lipid stores and metabolism by lipophagy. Cell Death And Differen t iation 2012, 20 , 3. 36. Dikic, I., Proteasomal and Autophagic Degradation Systems. Annual Review of Biochemistry 2017, 86 (1), 193 - 224. 37. Lilienbaum, A., Relationship betw een the proteasomal system and autophagy. International Journal of Biochemistry an d Molecular Biology 2013, 4 (1), 1 - 26. 38. Goldberg, A. L., Functions of the proteasome: from protein degradation and immune surveillance to cancer therapy. Biochemical Society Transactions 2007, 35 (1), 12 - 17. 197 39. Smith, D. M.; Benaroudj, N.; Goldberg, A. , Proteasomes and their associated ATPases: A destructive combination. Journal of Structural Biology 2006, 156 (1), 72 - 83. 40. Finley, D.; Chen, X.; Walters, K. J., Gates, Channels, and Switches: Elements of the Proteasome Machine. Trends in Biochemical Sc i ences 2016, 41 (1), 77 - 93. 41. Budenholzer , L.; Cheng, C. L.; Li, Y.; Hochstrasser, M., Proteasome Structure and Assembly. J Mol Biol 2017 . 42. Schmidt, M.; Finley, D., Regulation of proteasome activity in health and disease. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 2014, 1843 (1), 13 - 25. 43. Collins, G. A.; Goldberg, A. L., The Logic of the 26S Proteasome. Cell 2017, 169 (5), 792 - 806. 44. Pickering, A. M.; Davies, K. J. A., Chapter 6 - Degradation of Damaged Proteins: The Main Functio n of the 20S Proteasome. In Progress in Mol ecular Biology and Translational Science , Grune, T., Ed. Academic Press: 2012; Vol. 109, pp 227 - 248. 45. Ronald Hough, G. P., and Martin Rechsteiner, Purification of Two High Molecular Weight Proteasomes from Rabb i t Reticulocyte Lysate. The Journal of Biol ogical Chemistry 1987, (262). 46. Lowe, J.; Stock, D.; Jap, B.; Zwickl, P.; Baumeister, W.; Huber, R., Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 A resolution. Science 1995, 2 6 8 (5210), 533 - 539. 47. Glickman, M. H.; Ci echanover, A., The Ubiquitin - Proteasome Proteolytic Pathway: Destruction for the Sake of Construction. Physiological Reviews 2002, 82 (2), 373 - 428. 48. Ciechanover, A., Proteolysis: from the lysosome to ubiquitin a nd the proteasome. Nature Reviews Molecula r Cell Biology 2005, 6 , 79. 49. Tanaka, K., The proteasome: overview of structure and functions. Proceedings of the Japan Academy. Series B, Physical and biological sciences 2009, 85 (1), 12 - 36. 50. Bard, J. A. M. ; Goodall, E. A.; Greene, E. R.; Jonsson, E .; Dong, K. C.; Martin, A., Structure and Function of the 26S Proteasome. Annual Review of Biochemistry 2018, 87 (1), 697 - 724. 51. Choi, W. H.; de Poot, S. A. H.; Lee, J. H.; Kim, J. H.; Han, D. H.; Kim, Y. K.; Fi n ley, D.; Lee, M. J., Open - gate mutants of the mammalian proteasome show enhanced ubiquitin - conjugate degradation. Nature Communications 2016, 7 , 10963. 52. Pickart, C. M.; Eddins, M. J., Ubiquitin: structures, functions, mechanisms. Biochimica et Biophysi c a Acta (BBA) - Molecular Cell Research 200 4, 1695 (1), 55 - 72. 198 53. Mukhopadhyay, D.; Riezman, H., Proteasome - Independent Functions of Ubiquitin in Endocytosis and Signaling. Science 2007, 315 (5809), 201 - 205. 54. Schnell, J. D.; Hicke, L., Non - traditional F unctions of Ubiquitin and Ubiquitin - bindin g Proteins. Journal of Biological Chemistry 2003, 278 (38), 35857 - 35860. 55. Finley, D., Recognition and Processing of Ubiquitin - Protein Conjugates by the Proteasome. Annual Review of Biochemistry 2009, 78 (1), 47 7 - 513. 56. Pickart, C. M., Mechanisms Under lying Ubiquitination. Annual Review of Biochemistry 2001, 70 (1), 503 - 533. 57. Groettrup, M.; Pelzer, C.; Schmidtke, G.; Hofmann, K., Activating the ubiquitin family: UBA6 challenges the field. Trends in Biochemic a l Sciences 2008, 33 (5), 230 - 237. 58. Wijk, S. J. L. v.; Timmers, H. T. M., The family of ubiquitin - conjugating enzymes (E2s): deciding between life and death of proteins. The FASEB Journal 2010, 24 (4), 981 - 993. 59. Nakayama, K. I.; Nakayama, K., Ubiquit i n ligases: cell - cycle control and canc er. Nature Reviews Cancer 2006, 6 , 369. 60. Kwon, Y. T.; Ciechanover, A., The Ubiquitin Code in the Ubiquitin - Proteasome System and Autophagy. Trends in Biochemical Sciences 2017, 42 (11), 873 - 886. 61. Komander, D.; R a pe, M., The Ubiquitin Code. Annual Rev iew of Biochemistry 2012, 81 (1), 203 - 229. 62. Strickland, E.; Hakala, K.; Thomas, P. J.; DeMartino, G. N., Recognition of misfolding proteins by PA700, the regulatory subcomplex of the 26 S proteasome. J Biol Chem 20 0 0, 275 (8), 5565 - 72. 63. Livneh, I.; C ohen - Kaplan, V.; Cohen - Rosenzweig, C.; Avni, N.; Ciechanover, A., The life cycle of the 26S proteasome: from birth, through regulation and function, and onto its death. Cell research 2016, 26 (8), 869 - 885. 64. Zhu, Y. ; Wang, W. L.; Yu, D.; Ouyang, Q.; Lu, Y.; Mao, Y., Structural mechanism for nucleotide - driven remodeling of the AAA - ATPase unfoldase in the activated human 26S proteasome. Nature Communications 2018, 9 (1), 1360. 65. Eisele, M. R.; Reed, R. G.; Rudack, T. ; Schweitzer, A.; Beck, F.; Nagy, I.; P feifer, G.; Plitzko, J. M.; Baumeister, W.; Tomko, R. J.; Sakata, E., Expanded Coverage of the 26S Proteasome Conformational Landscape Reveals Mechanisms of Peptidase Gating. Cell Reports 2018, 24 (5), 1301 - 1315.e5. 6 6 . Wehmer, M.; Rudack, T.; Beck, F.; Au fderheide, A.; Pfeifer, G.; Plitzko, J. M.; Förster, F.; Schulten, K.; Baumeister, W.; Sakata, E., Structural insights into the functional 199 cycle of the ATPase module of the 26S proteasome. Proceedings of the National A cademy of Sciences 2017, 114 (6), 1305 - 1310. 67. Enenkel, C., Proteasome dynamics. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 2014, 1843 (1), 39 - 46. 68. Nandi, D.; Tahiliani, P.; Kumar, A.; Chandu, D., The ubiquitin - proteasome system. J o urnal of Biosciences 2006, 31 (1), 137 - 155. 69. 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. 7 0. Kusmierczyk, A. R.; Kunjappu, M. J. ; Kim, R. Y.; Hochstrasser, M., A conserved 20S proteasome assembly factor requires a C - terminal HbYX motif for proteasomal precursor binding. Nature structural & molecular biology 2011, 18 (5), 622 - 9. 71. Smith, D. M . ; Chang, S. - C.; Park, S.; Finley, D.; Cheng, Y.; Goldberg, A. L., Opens the Gate for Substrate Entry. Molecular Cell 2007, 27 (5), 731 - 744. 72. Förster, A.; Masters, E. I. ; Whitby, F. G.; Robinson, H.; Hill, C. P., The 1.9 Å Structure of a Proteasome - 11S Activator Complex and Implications for Proteasome - PAN/PA700 Interactions. Molecular Cell 2005, 18 (5), 589 - 599. 73. Rabl, J.; Smith, D. M.; Yu, Y.; Chang, S. - C.; Goldberg, A . L.; Cheng, Y., Mechanism of Gate Ope ning in the 20S Proteasome by the Proteasomal ATPases. Molecular Cell 2008, 30 (3), 360 - 368. 74. Chen, S.; Wu, J.; Lu, Y.; Ma, Y. - B.; Lee, B. - H.; Yu, Z.; Ouyang, Q.; Finley, D. J.; Kirschner, M. W.; Mao, Y., Structura l basis for dynamic regulation of the h uman 26S proteasome. Proceedings of the National Academy of Sciences 2016, 113 (46), 12991 - 12996. 75. Dong, Y.; Zhang, S.; Wu, Z.; Li, X.; Wang, W. L.; Zhu, Y.; Stoilova - McPhie, S.; Lu, Y.; Finley, D.; Mao, Y., Cryo - E M structures and dynamics of substrate - engaged human 26S proteasome. Nature 2019, 565 (7737), 49 - 55. 76. Njomen, E.; Osmulski, P. A.; Jones, C. L.; Gaczynska, M.; Tepe, J. J., Small Molecule Modulation of Proteasome Assembly. Biochemistry 2018, 57 (28), 42 1 4 - 4224. 77. Wright, P. E.; Dyson, H. J., Intrinsically unstructured proteins: re - assessing the protein s tructure - function paradigm. Journal of Molecular Biology 1999, 293 (2), 321 - 331. 78. Dunker, A. K.; Brown, C. J.; Lawson, J. D.; Iakoucheva, L. M.; Obr a Intrinsic Disorder and Protein Function. Biochemistry 2002, 41 (21), 6573 - 6582. 200 79. Oates, M. E.; Romero, P.; Ishida, T.; Ghalwash, M.; Mizianty, M. J.; Xue, B.; Dosztányi, Z.; Uversky, V. N.; Obradovic, Z.; Kurgan, L.; Dunker, A. K.; Gough, J. , D(2)P(2): database of disordered protein predictions. Nucleic Acids Research 2013, 41 (Database issue), D508 - D516. 80. Intrinsic Disorder in Cell - signaling and Cancer - associate d Proteins. Journal of Molecular Biology 2002, 323 (3), 573 - 584. 81. Galea, C. A.; Wang, Y.; Sivakolundu, S. G.; Kriwacki, R. W., Regulation of Cell Division by Intrinsically Unstructured Proteins: Intrinsic Flexibility, Modularity, and Signaling Conduits. Biochemistry 2008, 47 (29), 7598 - 7609. 82. Perkins, J. R.; Diboun, I.; Dessailly, B. H.; Lees, J. G.; Or engo, C., Transient Protein - Protein Interactions: Structural, Functional, and Network Properties. Structure 2010, 18 (10), 1233 - 1243. 83. Tompa, P., In t rinsically disordered proteins: a 10 - year recap. Trends in Biochemical Sciences 2012, 37 (12), 509 - 516. 84. Tompa, P., Intrinsically unstructured proteins. Trends in Biochemical Sciences 2002, 27 (10), 527 - 533. 85. Uversky, V. N., Introduction to Intrinsi c ally Disordered Proteins (IDPs). Chemical Reviews 2014, 114 (13), 6557 - 6560. 86. Dunker, A. K.; Babu, M. M.; Barbar, E.; Blackledge, M.; Bondos, S. E.; Dosztányi, Z.; Dyson, H. J.; Forman - Kay, J.; Fuxreiter, M.; Gsp oner, J.; Han, K. - H.; Jones, D. T.; Long h i, S.; Metallo, S. J.; Nishikawa, K.; Nussinov, R.; Obradovic, Z.; Pappu, R. V.; Rost, a name? Why these proteins are intrinsica lly disordered: Why these proteins are i n trinsically disordered. Intrinsically Disordered Proteins 2013, 1 (1), e24157. 87. van der Lee, R.; Buljan, M.; Lang, B.; Weatheritt, R. J.; Daughdrill, G. W.; Dunker, A. K.; Fuxreiter, M.; Gough, J.; Gsponer, J.; J ones, D. T.; Kim, P. M.; Kriwacki, R. W. ; Oldfield, C. J.; Pappu, R. V.; Tompa, P.; Uversky, V. N.; Wright, P. E.; Babu, M. M., Classification of Intrinsically Disordered Regions and Proteins. Chemical Reviews 2014, 114 (13), 6589 - 6631. 88. Habchi, J.; Tom pa, P.; Longhi, S.; Uversky, V. N., Intr o ducing Protein Intrinsic Disorder. Chemical Reviews 2014, 114 (13), 6561 - 6588. 89. Dyson, H J., Making Sense of Intrinsically Disordered Proteins. Biophysical Journal 2016, 110 (5), 1013 - 1016. 90. Drake, J. A.; Pett itt, B. M., Thermodynamics of Conformati o nal Transitions in a Disordered Protein Backbone Model. Biophysical Journal 2018, 114 (12), 2799 - 2810. 201 91. Dyson, H. J.; Wright, P. E., Intrinsically unstructured proteins and their functions. Nature Reviews Molecul ar Cell Biology 2005, 6 , 197. 92. Uversk y , V. N., Functional roles of transiently and intrinsically disordered regions within proteins. The FEBS Journal 2015, 282 (7), 1182 - 1189. 93. Wright, P. E.; Dyson, H. J., Intrinsically disordered proteins in cellula r signalling and regulation. Nature Revi e ws Molecular Cell Biology 2014, 16 , 18. 94. Uversky, V. N.; Oldfield, C. J.; Dunker, A. K., Showing your ID: intrinsic disorder as an ID for recognition, regulation and cell signaling. Journal of Molecular Recognition 2005, 18 (5), 343 - 384. 95. Uversky, V . N., Flexible Nets of Malleable Guardians: I ntrinsically Disordered Chaperones in Neurodegenerative Diseases. Chemical Reviews 2011, 111 (2), 1134 - 1166. 96. Babu, M. M.; van der Lee, R.; de Groot, N. S.; Gsponer, J., Intrinsically disordered proteins: reg u lation and disease. Current Opinion in Struc tural Biology 2011, 21 (3), 432 - 440. 97. Uversky, V. N., Wrecked regulation of intrinsically disordered proteins in diseases: pathogenicity of deregulated regulators. Frontiers in Molecular Biosciences 2014, 1 , 6 . 98. Uversky, V. N.; Oldfield, C. J.; Dunke r, A. K., Intrinsically Disordered Proteins in Human Diseases: Introducing the D2 Concept. Annual Review of Biophysics 2008, 37 (1), 215 - 246. 99. Darling, A. L.; Uversky, V. N., Intrinsic Disorder and Posttransl a tional Modifications: The Darker Side of the Biological Dark Matter. Frontiers in Genetics 2018, 9 , 158. 100. Brucale, M.; Schuler, B.; Samorì, B., Single - Molecule Studies of Intrinsically Disordered Proteins. Chemical Reviews 2014, 114 (6), 3281 - 3317. 10 1 . Jensen, M. R.; Zweckstetter, M.; Huang, J. - r.; Blackledge, M., Exploring Free - Energy Landscapes of Intrinsically Disordered Proteins at Atomic Resolution Using NMR Spectroscopy. Chemical Reviews 2014, 114 (13), 6632 - 6660. 102. Wei, G.; Xi, W.; Nussinov, R.; Ma, B., Protein Ensembles: How Does Natu re Harness Thermodynamic Fluctuations for Life? The Diverse Functional Roles of Conformational Ensembles in the Cell. Chemical Reviews 2016, 116 (11), 6516 - 6551. 103. Uversky, V. N., Targeting intrinsically diso r dered proteins in neurodegenerative and prot ein dysfunction diseases: another illustration of the D(2) concept. Expert Review of Proteomics 2010, 7 (4), 543 - 564. 202 104. Csizmok, V.; Follis, A. V.; Kriwacki, R. W.; Forman - Kay, J. D., Dynamic Protein Interact i on Networks and New Structural Paradigms in Signaling. Chemical Reviews 2016, 116 (11), 6424 - 6462. 105. 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., Inh i bition of the Human Proteasome by Imidazolin e Scaffolds. Journal of Medicinal Chemistry 2013, 56 (14), 5974 - 5978. 106. Crick, F., The packing of [alpha] - helices: simple coiled - coils. Acta Crystallographica 1953, 6 (8 - 9), 689 - 697. 107. Lee, B.; Richards, F . M., The interpretation of protein structure s: Estimation of static accessibility. Journal of Molecular Biology 1971, 55 (3), 379 - IN4. 108. Connolly, M., Analytical molecular surface calculation. Journal of Applied Crystallography 1983, 16 (5), 548 - 558. 1 0 9. Kuntz, I. D.; Blaney, J. M.; Oatley, S. J .; Langridge, R.; Ferrin, T. E., A geometric approach to macromolecule - ligand interactions. J Mol Biol 1982, 161 (2), 269 - 88. 110. Sliwoski, G.; Kothiwale, S.; Meiler, J.; Lowe, E. W., Jr., Computational methods in drug discovery. Pharmacological reviews 2 014, 66 (1), 334 - 395. 111. Macalino, S. J.; Gosu, V.; Hong, S.; Choi, S., Role of computer - aided drug design in modern drug discovery. Archives of pharmacal research 2015, 38 (9), 1686 - 701. 112. Adcock, S. A.; M c f Methods for Simulating the Activity of Proteins. Chemical Reviews 2006, 106 (5), 1589 - 1615. 113. Usha, T.; Shanmugarajan, D.; Goyal, A. K.; Kumar, C. S.; Middha, S. K., Recent Updates on Computer - aided Drug Di s covery: Time for a Paradigm Shift. Current topics in medicinal chemistry 2017, 17 (30), 3296 - 3307. 114. Pagadala, N. S.; Syed, K.; Tuszynski, J ., Software for molecular docking: a review. Biophysical reviews 2017, 9 (2), 91 - 102. 115. Chen, Y. C., Beware o f docking! Trends in pharmacological sciences 2015, 36 (2), 78 - 95. 116. Brooijmans, N.; Kuntz, I. D., Molecular Recognition and Docking Algorith ms. Annual Review of Biophysics and Biomolecular Structure 2003, 32 (1), 335 - 373. 117. Barril, X., Ligand discov e ry: Docking points. Nat Chem 2014, 6 (7), 560 - 561. 118. Meng, X., Zhang, H., Mezei, M., Cui, M., Molecular Docking: A powerful approach for str ucture - based drug discovery. Curr Comput Aided Drug Des. 2011, 7 (2), 146 - 157. 203 119. Kitchen, D. B.; Decornez, H. ; Furr, J. R.; Bajorath, J., Docking and scoring in virtual screening for drug discovery: methods and applications. Nature Reviews Drug Discover y 2004, 3 , 935. 120. Halperin, I.; Ma, B.; Wolfson, H.; Nussinov, R., Principles of docking: An overview of sear c h algorithms and a guide to scoring functions. Proteins: Structure, Function, and Bioinformatics 2002, 47 (4), 409 - 443. 121. Huang, L.; Ho, P.; Chen, C. - H., Activation and inhibition of the proteasome by betulinic acid and its derivatives. FEBS letters 20 0 7, 581 (25), 4955 - 4959. 122. Huang, X.; Luan, B.; Wu, J.; Shi, Y., An atomic structure of the human 26S proteasome. Nature Structural &Amp; Mol ecular Biology 2016, 23 , 778. 123. Trott, O.; Olson, A. J., AutoDock Vina: improving the speed and accuracy of d o cking with a new scoring function, efficient optimization and multithreading. Journal of computational chemistry 2010, 31 (2), 455 - 461. 124. Wa ng, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case, D. A., Development and testing of a general amber fo r ce field. J Comput Chem 2004, 25 (9), 1157 - 74. 125. Chang, C. - e. A.; Chen, W.; Gilson, M. K., Ligand configurational entropy and protein bindin g. Proceedings of the National Academy of Sciences 2007, 104 (5), 1534 - 1539. 126. Haelterman, R.; Van Eester, D. ; Verleyen, D., Accelerating the solution of a physics model inside a tokamak using the (Inverse) Column Updating Method. Journal of Computation al and Applied Mathematics 2015, 279 , 133 - 144. 127. Kristiansen, J. E.; Mortensen, I., Antibacterial effect of f o ur phenothiazines. Pharmacology & toxicology 1987, 60 (2), 100 - 3. 128. Dastidar, S. G.; Debnath, S.; Mazumdar, K.; Ganguly, K.; Chakrabarty, A. N., Triflupromazine: a microbicide non - antibiotic compound. Acta microbiologica et immunologica Hungarica 2004, 51 (1 - 2), 75 - 83. 129. Mazumder, R.; Ganguly, K.; Dastidar, S. G.; Chakrabarty, A. N., Trifluoperazine: a broad spectrum bactericide especially active on staphylococci and vibrios. International journal of antimicrobial agents 2001, 18 (4), 403 - 6. 130. VAR G A, B.; CSONKA, Á.; CSONKA, A.; MOLNÁR, J.; AMARAL, L.; SPENGLER, G., Possible Biological and Clinical Applications of Phenothiazines. Anticance r Research 2017, 37 (11), 5983 - 5993. 131. Dronca, R. S.; Loprinzi, C., Chapter 18 - Nausea and Vomiting. In Mana g ement of Cancer in the Older Patient , Naeim, A.; Reuben, D. B.; Ganz, P. A., Eds. W.B. Saunders: Philadelphia, 2012; pp 171 - 181. 204 132. Massie, S. P., The Chemistry of Phenothiazine. Chemical Reviews 1954, 54 (5), 797 - 833. 133. Lester, P. A.; Moore, R. M.; S huster, K. A.; Myers, D. D., Chapter 2 - Anesthesia and Analgesia. In The Laboratory Rabbit, Guinea Pig, Hamster, and Other Rodents , S uckow, M. A.; Stevens, K. A.; Wilson, R. P., Eds. Academic Press: Boston, 2012; pp 33 - 56. 134. Mocko, J. B.; Kern, A.; Mo o smann, B.; Behl, C.; Hajieva, P., Phenothiazines interfere with dopaminergic neurodegeneration in Caenorhabditis elegans models of Par kinson's disease. Neurobiol Dis 2010, 40 (1), 120 - 9. 135. Liemburg, E. J.; Knegtering, H.; Klein, H. C.; Kortekaas, R.; A l eman, A., Antipsychotic medication and prefrontal cortex activation: a review of neuroimaging findings. European neuropsychopharmacolo gy : the journal of the European College of Neuropsychopharmacology 2012, 22 (6), 387 - 400. 136. Pickar, D.; Litman, R. E. ; Konicki, P. E.; Wolkowitz, O. M.; Breier, A., Neurochemical and neural mechanisms of positive and negative symptoms in schizophrenia. Modern problems of pharmacopsychiatry 1990, 24 , 124 - 51. 137. Silberg, I. A.; Cormos, G.; Oniciu, D. C., Retrosynthetic A p proach to the Synthesis of Phenothiazines. In Advances in Heterocyclic Chemistry , Katritzky, A. R., Ed. Academic Press: 2006; Vol. 90, pp 205 - 237. 138. Mitchell, S. C., Phenothiazine: the parent molecule. Current drug targets 2006, 7 (9), 1181 - 9. 139. Jon e s, C. L.; Njomen, E.; Sjögren, B.; Dexheimer, T. S.; Tepe, J. J., Small Molecule Enhancement of 20S Proteasome Activity Targets Intrin sically Disordered Proteins. ACS Chemical Biology 2017, 12 (9), 2240 - 2247. 140. Di, L.; Kerns, E. H.; Carter, G. T., Drug - like property concepts in pharmaceutical design. Curr Pharm Des 2009, 15 (19), 2184 - 94. 141. Shibatani, T.; Ward, W. F., Sodium Dodecyl Sulfate (SDS) Activation of the 20S Proteasome in Rat Liver. Archives of Biochemistry and Biophysics 1995, 321 (1), 160 - 166. 142. Mobley, D. L.; Dill, K. A., Binding of small - molecule ligands to proteins: "what yo u see" is not always "what you get". Structure (London, England : 1993) 2009, 17 (4), 489 - 498. 143. Yale, H. L., The Trifluoromethyl Group in Medical Chemistry. J o urnal of Medicinal and Pharmaceutical Chemistry 1959, 1 (2), 121 - 133. 144. Betageri, R.; Zhan g, Y.; Zindell, R. M.; Kuzmich, D.; Kirrane, T. M.; Bentzien, J.; Cardozo, M.; Capolino, A. J.; Fadra, T. N.; Nelson, R. M.; Paw, Z.; Shih, D. T.; Shih, C. K.; Zu v ela - Jelaska, L.; Nabozny, G.; Thomson, D. S., Trifluoromethyl group as a 205 pharmacophore: effec t of replacing a CF3 group on binding and agonist activity of a glucocorticoid receptor ligand. Bioorg Med Chem Lett 2005, 15 (21), 4761 - 9. 145. Lipinski, C. A.; L ombardo, F.; Dominy, B. W.; Feeney, P. J., Experimental and computational approaches to estim ate solubility and permeability in drug discovery and development settings. Advanced drug delivery reviews 2001, 46 (1 - 3), 3 - 26. 146. Ghose, A. K.; Viswanadhan, V . N.; Wendoloski, J. J., A knowledge - based approach in designing combinatorial or medicinal ch emistry libraries for drug discovery. 1. A qualitative and quantitative characterization of known drug databases. Journal of combinatorial chemistry 1999, 1 (1), 5 5 - 68. 147. 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. 148. Coleman, R. A.; Trader, D. J., Development and Application of a Sensitive Peptide Reporter to Discover 20S Pr oteasome Stimulators. ACS Combinatorial Science 2018, 20 (5), 269 - 276. 149. Kumar, B.; Kim, Y. - C.; DeMartino, G. N., The C Terminus of Rpt3, an ATPase Subunit of P A700 (19 S) Regulatory Complex, Is Essential for 26 S Proteasome Assembly but Not for Activat ion. Journal of Biological Chemistry 2010, 285 (50), 39523 - 39535. 150. Kisselev, Alexei F.; van der Linden, W. A.; Overkleeft, Herman S., Proteasome Inhibitors: A n Expanding Army Attacking a Unique Target. Chemistry & Biology 2012, 19 (1), 99 - 115. 151. Gallastegui, N.; Beck, P.; Arciniega, M.; Huber, R.; Hillebrand, S.; Groll, M., Hydroxyureas as Noncovalent Proteasome Inhibitors. Angewandte Chemie International Ed i tion 2012, 51 (1), 247 - 249. 152. Manasanch, E. E.; Orlowski, R. Z., Proteasome inhibitors in cancer therapy. Nature Reviews Clinical Oncology 2017, 14 , 417. 153. Park, J. E.; Miller, Z.; Jun, Y.; Lee, W.; Kim, K. B., Next - generation proteasome inhibitors f or cancer therapy. Translational Research 2018, 198 , 1 - 16. 154. 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 s timulators. Bioorganic & Medicinal Chemistry Letters 2019, 29 (3), 420 - 423. 155. Kisselev, A. F.; Garcia - Calvo, M.; Overkleeft, H. S.; Peterson, E.; Pennington, M. W.; Ploegh, H. L.; Thornberry, N. A.; Goldberg, A. L., The caspase - like sites of proteasome s , their substrate specificity, new inhibitors and substrates, and allosteric interactions with the trypsin - like sites. J Biol Chem 2003, 278 (38), 35869 - 77. 206 156. Huber, E. M.; Heinemeyer, W.; Li, X.; Arendt, C. S.; Hochstrasser, M.; Groll, M., A unified m e chanism for proteolysis and autocatalytic activation in the 20S proteasome. Nature Communi cations 2016, 7 , 10900. 157. Harshbarger, W.; Miller, C.; Diedrich, C.; Sacchettini, J., Crystal structure of the human 20S proteasome in complex with carfilzomib. S t ructure 2015, 23 (2), 418 - 24. 158. Fabre, B.; Lambour, T.; Garrigues, L.; Ducoux - Petit, M. ; Amalric, F.; Monsarrat, B.; Burlet - Schiltz, O.; Bousquet - Dubouch, M. - P., Label - Free Quantitative Proteomics Reveals the Dynamics of Proteasome Complexes Compositio n and Stoichiometry in a Wide Range of Human Cell Lines. Journal of Proteome Research 2014, 13 (6), 3027 - 3037. 159. Dubinsky, L.; Krom, B. P.; Meijler, M. M., Diazirine based photoaffinity labeling. Bioorganic & Medicinal Chemistry 2012, 20 (2), 554 - 570. 1 6 0. Hill, J. R.; Robertson, A. A. B., Fishing for Drug Targets: A Focus on Diazirine Photoa ffinity Probe Synthesis. Journal of Medicinal Chemistry 2018, 61 (16), 6945 - 6963. 161. Kumar, A. B.; Tipton, J. D.; Manetsch, R., 3 - Trifluoromethyl - 3 - aryldiazirine p h otolabels with enhanced ambient light stability. Chemical Communications 2016, 52 (13), 27 29 - 2732. 162. Smith, D. P.; Anderson, J.; Plante, J.; Ashcroft, A. E.; Radford, S. E.; Wilson, A. J.; Parker, M. J., Trifluoromethyldiazirine: an effective photo - ind u ced cross - linking probe for exploring amyloid formation. Chemical Communications 2008, (4 4), 5728 - 5730. 163. deGruyter, J. N.; Malins, L. R.; Baran, P. S., Residue - Specific Peptide Modification: Biochemistry 2017, 56 (30), 3863 - 3873. 1 6 4. Chen, X.; Wu, Y. - W., Selective chemical labeling of proteins. Organic & Biomolecular Ch emistry 2016, 14 (24), 5417 - 5439. 165. Murata, S.; Yashiroda, H.; Tanaka, K., Molecular mechanisms of proteasome assembly. Nat Rev Mol Cell Biol 2009, 10 (2), 104 - 11 5 . 166. Schrader, J.; Henneberg, F.; Mata, R. A.; Tittmann, K.; Schneider, T. R.; Stark, H. ; Bourenkov, G.; Chari, A., The inhibition mechanism of human 20S proteasomes enables next - generation inhibitor design. Science 2016, 353 (6299), 594 - 8. 167. Gerecke , C.; Fuhrmann, S.; Strifler, S.; Schmidt - Hieber, M.; Einsele, H.; Knop, S., The Diagnosis and Treatment of Multiple Myeloma. Deutsches Arzteblatt international 2016, 113 (27 - 28), 470 - 476. 168. Hewlett, N. M.; Tepe, J. J., Total Synthesis of the Natural Pr o duct (±) - Dibromophakellin and Analogues. Organic Letters 2011, 13 (17), 4550 - 4553. 169. Lansdell, T. A.; Hewlett, N. M.; Skoumbourdis, A. P.; Fodor, M. D.; Seiple, I. B.; l kaloids 207 Dibromophakellin and Dibromophakellstatin Inhibit the Human 20S Proteasome. Journal of Natural Products 2012, 75 (5), 980 - 985. 170. Beck, P.; Lansdell, T. A.; Hewlett, N. M.; Tepe, J. J.; Groll, M., Indolo - Phakellins - Specific Noncovalent Pro teasome Inhibitors. Angewandte Chemie International Edition 2015, 54 (9), 2830 - 2833. 171. Gallastegui, N.; al., e., Angew. Chem., Int. Ed. En gl. 2012, (51), 247 - 249. 172. Kisselev, A. F.; van der Linden, W. A.; Overkleeft, H. S., Chem. Bio. 2012, 19 , 99 - 115. 173. Beck, P.; Lansdell, T. A.; Hewlett, N. M.; Tepe, J. J.; Groll, M., Angew. Chem., Int. Ed. Engl. 2015, (54), 2830 - 2833. 174. Lansdel l, T. A.; al, e., J. Nat. Prod. 2012, 75 , 980 - 985. 175. Frigerio, M.; Santagostino, M.; Sputore, S., A User - Friendly Entry to 2 - Iodoxybenzoic Acid (IBX). The Journal of Organic Chemistry 1999, 64 (12), 4537 - 4538. 176. Gaczynska, M.; Osmulski, P. A.; Gao, Y .; Post, M. J.; Simons, M., Proline - and arginine - rich peptides constitute a novel class of allosteric inhibitors of proteasome activity. Biochemistry 2003, 42 (29), 8663 - 70. 177. Giletto, M. B.; Osmulski, P. A.; Jones, C. L.; Gaczynska, M. E.; Tepe, J. J. , Pipecolic esters as minimized templates for proteasome inhibition. Organic & Biomolecular Chemistry 2019 . 178. Nguyen, L. A.; He, H.; Pham - Huy, C., Chiral drugs: an overview. International journal of biomedical science : IJBS 2006, 2 (2), 85 - 100. 179. Go ttlieb, H. E.; Kotlyar, V.; Nudelman, A., NMR Chemical Shifts of Common Laboratory Solvents as Trace Impurities. The Journal of Organic Chemistry 1997, 62 (21), 7512 - 7515. 180. Chen, S. - Q.; Wang, Q. - M.; Xu, P. - C.; Ge, S. - P.; Zhong, P.; Zhang, X. - H., Iodine - promoted selective 3 - selanylation and 3 - sulfenylation of indoles with dichalcogenides under mild conditions. Phosphorus, Sulfur, and Silicon and the Related Elements 2016, 191 (1), 100 - 103. 181. Nakata, E.; Yukimachi, Y.; Nazumi, Y.; Uto, Y.; Maezawa, H.; Hashimoto, T.; Okamoto, Y.; Hori, H., A newly designed cell - permeable SNARF derivative as an effective intracellular pH indicator. Chemical Communications 2010, 46 (20), 3526 - 3528. 182. Kim, C.; Wallace, J. U.; Chen, S. H.; Merkel, P. B., Effects of Dilut ion, Polarization Ratio, and Energy Transfer on Photoalignment of Liquid Crystals Using Coumarin - Containing Polymer Films. Macromolecules 2008, 41 (9), 3075 - 3080. 208 183. Meanwell, N. A.; Roth, H. R.; Smith, E. C. R.; Wedding, D. L.; Wright, J. J. K., Diethyl 2,4 - dioxoimidazolidine - 5 - phosphonates: Horner - Wadsworth - Emmons reagents for the mild and efficient preparation of C - 5 unsaturated hydantoin derivatives. The Journal of Organic Chemistry 1991, 56 (24), 6897 - 6904.