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«I. The Utility of Activation Domain Mimics as Targeted Inhibitors of Transcription II. The Design and Implementation of a Peer-Led Module in ...»

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I. The Utility of Activation Domain Mimics as Targeted Inhibitors of Transcription

II. The Design and Implementation of a Peer-Led Module in Practical Research Ethics

by

Christopher Taylor

A dissertation submitted in partial fulfillment

of the requirements for the degree of

Doctor of Philosophy

(Chemistry)

in the University of Michigan

Doctoral Committee:

Professor Anna K. Mapp, Chair

Professor E. Neil G. Marsh

Professor John Montgomery

Assistant Professor Matthew B. Soellner © Christopher Taylor All rights reserved

ACKNOWLEDGEMENTS

There are many people I would like to thank for their help over the years. First, I would like to thank my adviser, Dr. Anna Mapp. Anna been an amazing adviser over the years, giving me the freedom to learn and grow as a scientist, but also providing guidance when I needed it. I would also like to thank the mapp lab for their advice and friendship over the years, in particular Dr. Chinmay Majmudar, Dr. Amberlyn Wands, Amanda Dugan, Ningkun Wang, and Paul Bruno. It has been my privilege to work with them.

I would like to thank the other members of my committee, Dr. John Montgomery, Dr. Neil Marsh, and Dr. Matthew Soellner. In particular, Dr. Montgomery has been an excellent mentor over the years, as both a rotation adviser and a committee member. Dr.

Soellner help in writing up publications has been invaluable, and his comments on the work presented in this thesis have been particularly helpful and insightful.

My interest in the sciences is due in large part to the excellent science professors I had as an undergraduate. Although I had the privilege of learning from many gifted professors, there are three I want to thank in particular. Dr. Rachel Brem and Dr. David McAvity were amazing teachers, whose ability to make science fun and interesting without over-simplifying it is something that I strive to emulate in my own teaching. My undergraduate organic chemistry adviser, Dr. Lydia McKinstry, deserves special thanks.

Her amazing work as a teacher and a mentor have been instrumental to my success in graduate school, and her continued support and advice throughout the years has been invaluable.

I would like to thank my family – Deb and Patricia Conklin, Kim O'neal, Greg Taylor, Izzy Martin, Jake and Yara Pasner, and Steve, Lauran, and Andrew Danowitz for ii their support over the years, even when it meant unreturned phone calls and long gaps between visits. I would also like to thank the friends I have made during my time in graduate school – Nate Bynum, Tim Baran, Nick Gutschow, and Jonas Locke – for the many good times I have spent with them, and for their understanding when I have been too busy or too stressed out to enjoy myself.

Finally, I would like to thank the person who has contributed more to my success and mental well-being during graduate school more than anyone else, my wife Amy Danowitz. Amy has been many things to me over the year – a collaborator, a friend, and a partner – and she has always been there for me when I needed her. The joy and comfort that her love has brought me through the darkest times of graduate school, and I know that with her help I can weather any storm.

iii TABLE OF CONTENTS

ACKNOWLEDGMENTS

LIST OF FIGURES

LIST OF TABLES

LIST OF BOXES

LIST OF APPENDICES

CHAPTER I Introduction A. Project Overview

B. Introduction to Transcription

C. Structure and Function of Transcriptional Activators and Coactivators

D. Small Molecules and Peptidomimetics that Target Transcription

E. The Regulation of the ErbB2 oncogene by ESX and the Role of the ErbB2 Protein

F. Research Misconduct in the Sciences

F. Summary of Thesis

II Inhibition of ErbB2(Her2) Expression with Small Molecule Transcription Factor Mimics A. Chapter Overview

B. Background

C. Design and Synthesis of Isoxazolidine iv Based ESX mimics

D. Evaluation of Isoxazolidine Based ESX Mimics

E. Conclusions

F. Supporting Information

G. References

III A Multi-Pronged Approach to Targeting Oncogenic Transcription A. Chapter Overview

B. Background

C. The Use of II-2 in Combination with Other Agents that Target ErbB2 Lifetime and Activity

D. Conclusions

E. Supporting Information

F. References

IV Design and Implementation of a Peer-Led Discussion Module for Teaching Research Ethics to Incoming Graduate Students A. Chapter Overview

B. Background

C. Pedagogical Rationale for Module Design

D. Module Layout and Implementation

E. Module Content

F. Response by Students and the Larger Chemical Community

G. Conclusions

H. Course materials

I. References

V Conclusions and Future Directions A. Inhibition of ErbB2(Her2) Expression with Small Molecule Transcription Factor Mimics

B. A Multi-Pronged Approach to Targeting Oncogenic v Transcription

C. Design and Implementation of a Peer-Led Discussion Module for Teaching Research Ethics to Incoming Graduate Students

D. References

Appendices

–  –  –

FIGURES Figure I-1. Initiation of transcription by transcriptional activator proteins





Figure I-2. Possible interacting partners of transcriptional activator proteins

Figure I-3. Anatomy of a transcriptional activator

Figure I-4. Ribbon diagram of the VP16 activation domain

Figure I-5. Small molecule transcriptional inhibitors identified from high throughput screens

Figure I-6. Development of an isoxazolidine based TAD

Figure I-7. ErbB2 is the preferred dimerization partner for erbB signaling.

Figure I-8. The transcriptional activator ESX regulates transcription of erbB2

Figure II-1. The modular nature of activator proteins

Figure II-2. Small molecule transcriptional inhibitors identified from high throughput screens

Figure II-3. Development of an isoxazolidine based TAD

Figure II-4. Effects of ESX knockdown on erbB2+ cells

Figure II-5. ErbB dimerization

Figure II-6. The transcriptional activator ESX regulates transcription of erbB2

Figure II-7. General synthetic scheme for inhibitors

Figure II-8. Synthesis of II-2 and II-3

Figure II-9 Synthesis of II-4

Figure II-10. Isoxazolidines II-1 through II-11

vii Figure II-12. Effects of II-1 and II-2 on erbB2 expression

Figure II-13. Effects of II-2 on erbB2 protein and mRNA

Figure II-14. II-2 reduces the viability of erbB2+ SkBr3 cells

Figure II-15. Comparison of the effects of II-2 (SkBr3 vs MCF-7)

Figure II-16. Comparison of the effects of II-2 (SkBr3 vs. IMR90)

Figure II-17. II-1 1H NMR Spectrum

Figure II-18. II-2 1H NMR Spectrum

Figure II-19. II-3 1H NMR Spectrum

Figure II-20. II-4 1H NMR Spectrum

Figure II-21. II-5 1H NMR Spectrum

Figure II-22. II-6 1H NMR Spectrum

Figure II-23. II-7 1H NMR Spectrum

Figure II-24. II-8 1H NMR Spectrum

Figure II-25. II-9 1H NMR Spectrum

Figure II-26. II-10 1H NMR Spectrum

Figure II-27. II-11 1H NMR Spectrum

Figure III-1. Rationale for synergy

Figure III-2. Bliss definition of synergy

Figure III-3. Loewe definition of synergy

Figure III-4. Rationale for synergy between II-2 and geldanamycin

Figure III-5. II-2/geldanamycin combinations (I)

Figure III-6. II-2/geldanamycin combinations (II)

Figure III-7. Selectivity of II-2/geldanamycin combinations (I)

Figure III-8. Selectivity of II-2/geldanamycin combinations (II)

Figure III-9. Rationale for synergy between II-2 and erlotinib

Figure III-10. II-2/erlotinib combinations (I)

Figure III-11. II-2/erlotinib combinations (II)

Figure III-12. Rationale for synergy between II-2 and lapatinib

Figure III-13. II-2/lapatinib combinations (I)

viii Figure III-14. II-2/lapatinib combinations (II)

Figure III-15. Dose effect curves for II-2/lapatinib combinations

Figure V-1. Rationale for isoxazolidine based inhibitors

Figure V-2. Helical inhibitor of HIF-1α mediated transcription

Figure V-3. Non-biomolecule helical mimics

Figure V-4. Upstream vs. downstream synergy

Figure V-5. Rationale for CH1 binding peptides as inhibitors of p65 mediated transcription

Figure V-6 Complementary approaches to synergy

Figure A1-1. Rationale for inhibition of p65 mediated transcription

Figure A1-2. Major domains of p65

Figure A1-3. Putative interactions between p65 and the coactivator CBP

Figure A1-4. General schematic of the d-peptide inhibitors designed to curb p65 activity

Figure A1-5. Results from initial screen of d-peptides

Figure A1-6. Results from followup evaluation of A1-3 and A1-7

Figure A1-7. Results of fluorescent microscopy experiments

Figure A1-8. Results of luciferase reporter assays

Figure A2-1. Applications for alleneamides

Figure A2-2. Iterative construction of allenamides

Figure A2-3. General scheme for alleneamide synthesis

Figure A2-4. Racemization of A2-14.

Figure A2-6. Applications of A2-1

–  –  –

TABLES Table II-1. Effectiveness of isoxazolidines II-1 through II-11

Table II-2. Comparison of the IC50s of II-2

Table A1-1. D-peptides designed to inhibit p65 mediated transcription

Table A2-1. Scope of the rearrangement

–  –  –

BOXES Box IV-1. A Format for Ethical Decision Making

Box IV-2. Sample data interpretation exercise

Box IV-3. Case Study - “Key Result”

Box IV-4 Case Study - “Continuing a Project”

–  –  –

APPENDICES I The Design and Evaluation of d-Peptide Inhibitors of p65 Mediated Transcription A. Overview

B. Background

C. Design of d-peptide Inihibitors of p65 Transcriptional Activity

D. Activity of d-peptide Inhibitors of p65 Mediated Transcription

E. Conclusions

F. Supplementary Information

G. References

II Applications of a Novel Synthesis of Substituted Alleneamides via a [3,3] Sigmatropic Rearrangement A. Overview

B. Background

C. [3,3] Rearrangement of Phosphorimidates

D. Applications of Alleneamides in Organic Synthesis

E. Conclusions

F. Supplementary Information

G. References

–  –  –

A. Project Overview Transcription, in which information stored in the coding regions of the cells DNA is converted into RNA is a fundamental cellular process.1 As such, it is highly regulated in healthy cells. Aberrant transcription is a hallmark of cancer,2,3,4 and is also associated with a host of other pathologies such as metabolic,5,6 inflammatory7,8 and neurological disorders.9,10 The proteins responsible for recruiting the transcriptional machinery to the promoter, transcriptional activator proteins, contact their target proteins, coactivators, through transcriptional activation domains that are often relatively short contiguous groups of amino acids.11,12 Because of the central role that transcription plays in regulating cell function and fate, the ability to cause a targeted increase or decrease in particular transcription programs has tremendous potential as both a therapeutic approach, and as a tool for biochemical investigations.

The design or discovery of ligands, particularly small molecule ligands, that target transcription has proven difficult and only a handful of such molecules are known.12,13 Prominant obstacles include the promiscuous binding profile of many transcriptional activation domains, the low to moderate affinities of activatorcoactivator interactions, and the lack of structural data, or even the identity of, the biologically relevent coactivators in most circumstances. High throughput screening against the well characterized coactivators CBP and p300,14,15 or against particular phenotypes, such as the death of cells which depend on overexpression of a particular gene,16 have seen isolated successes. However, at the time this work was initiated, a general design rationale for small molecule transcriptional inhibitors had not emerged.

The project described in this dissertation grew out of the hypothesis that small molecules which mimic activation domains can be used as scaffolds for the development of transcriptional inhibitors. In this work, I will discuss the potential of small molecules that mimic the activation domain of the transcriptional activator ESX to inhibit the transcription of erbB2, an oncogene whose expression is mediated by ESX.17,18,19 This will be followed by the description of a generalizable strategy for overcoming some of the limitations of small molecule transcriptional inhibitors.

The final portion of this thesis concerns the important issue of education in research practices. A growing body of work and suggests the widespread use of questionable practices by research scientists.20,21, This is supported by the accounts of outright fraud and sabotage involving graduate students and post-doctoral researchers have appeared in the scientific press.22,23,24 Data from the National Research Council, and interviews with those involved indicates that these incidents are connected to percieved pressure on those involved. As part of a collaboration with another graduate student – Dr. Amy Danowitz – I designed, wrote, and lead a discussion module for incoming graduate students about research ethics, data interpretation, and conflict management.



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