Luận án tiến sĩ: A chemical genomic approach towards determining the molecular target of anti-proliferative compounds

BOSTON UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES Dissertation A CHEMICAL GENOMIC APPROACH TOWARDS DETERMINING THE MOLECULAR TARGET OF ANTI-PROLIFERATIVE COMPOUNDS by ERIN L., University of Massachusetts Dartmouth, 1999 Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2007 UMI Number: 3246602 Copyright 2006 by Eastwood, Erin L. All rights reserved. INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction.

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This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P. Box 1346 Ann Arbor, MI 48106-1346 © Copyright by ERIN L. EASTWOOD 2006 Approved by First Reader Soe Scott E.

Assistant Professor of Chemistry and Pharmacology Second Reader ⁄⁄⁄⁄ zZ⁄Z Mark W. Associate Professor of Chemistry and Biomedical Engineering ACKNOWLEDGEMENTS I would like to thank the Boston University Department of Chemistry and the Center for Chemical Methodology and Library Development for this opportunity and for providing financial support. Elliott, and Dr. Pinghua Liu, thank you for serving on my committee and for your guidance.

I would like to thank my research advisor, Dr. Schaus, for giving me the chance and the confidence to grow as a scientist. Thank you for your support over the last five years. I would also like to thank the past and present Schaus group members including Josh Giguere, John Westbrook, Nolan McDougal, Melissa Dominguez, Sha Lou, Stacy Rodgen, Whitney Trevellini, Christiane Bode, Amal Ting, Jen Goss, Josh Bishop, Phil Moquist, Allison Wensley, Elise Birkett, Laura Kliman, Andrew Wojtovich, and Valerie Curtis for providing laughs as well as support in and outside the lab.

You made me smile on my worst days. I could not have asked for a better group of people to work with. I wish you all the best. I would like to thank our collaborators; Sarah Chobot and Mike Hamil! from the Elhott lab in the BU Chemistry Department for their help with the thioredoxin assays; Melissa Landon and Dr.

Sandor Vajda from the BU Bioinformatics Department for the GOLD studies; the Gardner and Collins labs at the Center for BioDynamics and iv Department of Biomedical Engineering at BU for the predictions of the target of PTSB using the MNI; and Dr. John Tullai from the Cooper lab in the Department of Biology at BU for his help with the western blot analysis. I would like to thank the office staff in the chemistry department for all of their hard work. A special thank you goes to Katinka Csigi for her help in my job search as well as her suggestions on my presentations and résumé.

You are a wonderful lady. I am very thankful to the CMLD faculty and staff, especially Paul Ferrari and Aruna Jain. Aaron Beeler, Dayle Acquilano, and Chris Singleton, thank you for the help with my library synthesis and data analysis. To my friends and family, I am so lucky to have such a strong support system.

Stacy, Christiane, Melissa, Jen, and Allison, I am grateful to have such a great set of friends that are also my colleagues. SuzAnn, we have been through a lot together. I do not know how I would have made it through those first years without you. Thank you for being the sister that I never had.

My family has always been an important part of my life. I would not be who I am without their love and support. Mom and Dad, words can not explain how much you both mean to me. I love you both very much.

Thank you for letting me find my own way and making me believe in myself. The support of my family extends beyond my parents. I am lucky to have a wonderful extended family, including my aunts, uncles, cousins, and grandparents who have encouraged and supported me over the years. I love you all very much.

You have showed me what really matters in life. Matt, thank you for being so supportive, and understanding about my work schedule. It really made me happy to share my free time with you. I have found the chemical reaction that I was looking for, and I look forward to what the future may hold.

vi A CHEMICAL GENOMIC APPROACH TOWARDS DETERMINING THE MOLECULAR TARGET OF ANTI-PROLIFERATIVE COMPOUNDS (Order No. EASTWOOD Boston University Graduate School of Arts and Sciences, 2007 Major Professor: Scott E. Schaus, Assistant Professor of Chemistry and Pharmacology ABSTRACT Drug target identification is a time consuming stage of the drug discovery process. Chemical genomics offers a solution to this hurdle.

In chemical genomics, a target specific chemical ligand is applied on a genomic scale. This technique was used to identify the molecular target of anti-proliferative agents using changes in mRNA transcript levels upon treatment. Whole-genome transcription profiling experiments employed the eukaryotic model organism Saccharomyces cerevisiae for small-molecule perturbation experiments in addition to traditional genetics. Chemical genomics was used to examine the molecular target of borrelidin, a macrolide with conflicting published biological activities.

The initial transcription profiles showed an increase in the transcript ratios of genes involved in amino acid biosynthesis upon treatment with borrelidin. In yeast, the GCN4 pathway regulates general amino acid control. The accumulation of uncharged tRNA activates Gen2p which vil prevents the formation of the eIF-2 complex. In turn, this simulates the translation of Gen4p, which results in the transcription of over 30 genes involved in amino acid biosynthesis.

Experiments using GCN2 and GCN4 gene deletions determined that borrelidin targets the amino acid biosynthetic pathway through GCN4p. The profiling data indicates that an alternative mechanism exists for the translational regulation of Gen4p other than through Gen2p, which was confirmed using immunoblot analysis with elF2 « and phosphorylated elF2 o antibodies. In the second application of chemical genomics, a diverse collection of synthetic compounds was evaluated in a cell-based toxicity assay. The screen revealed a subset of cyclic sulfones that inhibited growth of A549, human small lung carcinoma, cells.

Within this subset, 4-(1-phenyl-1H-tetrazole-5-sulfonyl)-butyronitrile (PTSB) was the most active compound. PTSB was shown to inhibit growth of both wild-type S. Whole-genome transcription profiling experiments in S. cerevisiae indicated that PTSB is involved in the cellular response to oxidative stress.

Analysis of the profiling data using systems biology predicted the thioredoxin pathway as the target. Biochemical assays with thioredoxin (Trx) and thioredoxin reductase (TrxR) validated that PTSB inhibits TrxR. The structure of PTSB suggests a novel mechanism of inhibition. This research illustrates the significance of applying chemical genomics to the target validation stage of drug discovery.

vin TABLE OF CONTENTS ACKNOWLEDGEMENTS iv ABSTRACT VI TABLE OF CONTENTS 1X LIST OF TABLES LIST OF FIGURES XI LIST OF SCHEMES XV LIST OF ABBREVIATIONS xvi CHAPTER 1: An introduction to chemical genetics and drug target identification CHAPTER 2: The application of chemical genomics to examine the molecular target of an anti-proliferative compound, borrelidin 14 Introduction 14 Results and Discussion 18 Conclusions 32 Experimental Methods 35 CHAPTER 3: A chemical genomics approach towards the target identification of a novel anti-proliferative compound, PTSB 53 Introduction 53 Results and Discussion 55 Conclusions 82 Experimental Methods 84 Characterization 109 REFERENCES 122 CURRICULUM VITAE 134 ix LIST OF TABLES Table 2.1: Published and experimental ICs values for borrelidin 16 Table 2.2: Fold-change of several over-expressed genes upon treatment with borrelidin 20 Table 2.3: Fold-change of several repressed genes upon treatment with borrelidin 22 Table 2. Fold changes of the transcript ratios for genes involved in amino acid biosynthesis in the drug sensitive (at 30, 60, and 90 minutes), gcn4A, and gcn2A strains upon treatment with borrelidin 50 Table 3.1: ICso values of lead compounds in growth inhibition assays in A549 and HeLa-S3 cell lines 57 Table 3.2: The top five gene ontology predictions for PTSB using the MNI algorithm 67 Table 3.3: Glso values for gene deletion and heterozygous strains of Trx and TrxR in yeast 68 LIST OF FIGURES Figure 1.1: Chemical structures of saframycin A, bishydroquinone derivatives of saframycin A, FK506, rapamycin, and cyclosporin A Figure 1.2: Drug target identification methods Figure 1.3: An overview of chemical genetics Figure 1.4: A chemical genomic approach towards drug target identification using whole-genome transcription profiling experiments 12 Figure 2.1: The chemical structures of borrelidin, 3-amino-1,2,4- triazole (3AT), and several anti-cancer agents 15 Figure 2.2: Color display plot of the expression ratios of genes involved in amino acid biosynthesis in the wild-type, gcn4A, and gcn2A strains as a result of treatment with 100 uM borrelidin 23 Figure 2.3: Color display plot of the expression ratios of genes involved in amino acid biosynthesis in the wild-type, gcen4A, and gen2A strains as a result of treatment with 100 uM borrelidin and 100 mM 3-aminotriazole (3AT), and wt treated with borrelidin and threonine 27 Figure 2.4: Color display plot of the expression ratios of genes involved in amino acid biosynthesis in the wild-type, gcen4A, gcen2A, gcn1A, and gcn20A strains as a result of treatment with 100 uM borrelidin 29 Figure 2.5: Phosphorylation of eIF2o in the presence of borrelidin and 3ATin wild-type and gcn2A yeast strains 31 Figure 2.6: Borrelidin growth inhibition of BY4741 37 Figure 2.7: Borrelidin growth inhibition of BY4743 38 XI Figure 2.8: Borrelidin growth inhibition of CCY333, the drug sensitive strain 39 Figure 2.9: Borrelidin growth inhibition of erg6A strain 40 Figure 2.10: Borrelidin growth inhibition of CDC28::cdc28 heterozygous strain 41 Figure 2.11: Borrelidin growth inhibition of the gcn4A strain 42 Figure 2.12: Borrelidin growth inhibition of gen2A strain 43 Figure 2.13: Borrelidin growth inhibition of the GCD1::gcd1 heterozygous strain 44 Figure 2.14: Borrelidin growth inhibition of hom3A strain 45 Figure 2.15: Growth of CCY333, DS’strain, in the presence of 400 nM borrelidin at various concentrations of threonine 47 Figure 3.1: Representative members from a diverse library 56 Figure 3.2: Representative members of the cyclic sulfone library 60 Figure 3.3: Color display plot of expression ratios of genes involved in oxidative stress, ribosome biogenesis, and rRNA processing upon treatment with PTSB 62 Figure 3.4: (A) DTNB assay for Trx/TrxR activity. (B) DTNB assay for TrxR activity 69 Figure 3.5: DTNB assay for thioredoxin/thioredoxin reductase activity in the presence of 0, 5, and 50 uM PTSB 70 Figure 3.6: DTNB assay for thioredoxin reductase activity in the presence of 0, 12.5, and 25 uM PTSB 72 Figure 3.7: Apparent K,, versus [I] for PTSB 73 Figure 3.8: High throughput screen for thioredoxin reductase activity 74 xil Figure 3.9: Known inhibitors of the Trx/TrxR system 76 Figure 3.10: Docking of AADP” and PTSB in the NADPH binding domain of TrxR from E.11: DTNB assay for thiorexoxin reductase activity in the presence of 12.54M PTSB varying the concentration of NADPH from 50 uM and 200 uM 81 Figure 3. DTNB assay for thioredoxin reductase activity in the presence of 0, 12.5, 25 and 50 uM PTSB 89 Figure 3.

Apparent K,, versus [I] for PTSB 90 Figure 3. Percent growth inhibition of A549 by PTSB 94 Figure 3. Percent growth inhibition of HeLa-S3 cells by PTSB 95 Figure 3. Percent growth inhibition of A549 by 4-[1-(2-Bromo-4-fluoro- phenyl)-1H-tetrazole-5-sulfonyl]-butyronitrile 96 Figure 3.

Percent growth inhibition of HeLa-S3 cells by 4-[1-(2-Bromo- 4-fluoro-phenyl)-1H-tetrazole-5-sulfonyl]-butyronitrile 97 Figure 3. Percent growth inhibition of A549 by 4-[1-(4-Bromo-phenyl)- 1H-tetrazole-5-sulfonyl]-butyronitrile 98 Figure 3. Percent growth inhibition of HeLa-S3 cells by 4-[1-(4-Bromo- phenyl)-1H-tetrazole-5-sulfonyl]-butyronitrile 99 Figure 3. Percent growth inhibition of A549 by 3-(benzothiazole-2- sulfonylmethyl)-benzonitrile 100 Figure 3.

Percent growth inhibition of HeLa-S3 cells by 3- (benzothiazole-2-sulfonylmethyl)-benzonitrile 101 Figure 3. Percent growth inhibition of A549 cells by 4-(1-Phenyl-1H- tetrazole-5-sulfonylmethyl)-benzonitrile 102 Xili Figure 3.