Part I: Conformational Analysis of Bioactive Molecules inSolutionPart II: Structure-Based Design of Selective Inhibitors ofCyclin-Dependent Kinase 7 公开

Abstract

Abstract

The biological activity of flexible organic molecules crucially depends on their conformation. Knowledge of binding conformation of druglike molecules can help in understanding the fundamental factors operating in protein-ligand interactions and can pave the way toward improvement of biological properties by synthetic modification. Current methods used for determining conformation of flexible, rapidly interconverting molecules in solution provide average data and are therefore inadequate. NAMFIS is a method that combines average conformational data from Nuclear Magnetic Resonance (NMR) spectroscopy with extensive force field based conformational searching and derives Boltzmann populations for such flexible molecules in solution. We use this method to investigate the solution conformational profiles of Dictyostatin and Discodermolide, two important potential anticancer molecules binding to the protein tubulin. Using known experimental data, we investigate prior hypotheses regarding their conformations and explore the relation between their solid-state, solution-state and protein bound conformations to construct models of these molecules in the tubulin binding site. The analysis reveals dissimilar and unique conformational behavior in both cases. Using the same technique, we also investigate conformational profiles for Noscapine, another tubulin binding molecule. A study of conformations in the crystal structures and in solution leads to the observation of an unusual pseudoaxial conformational preference for a methyl group in a six membered ring. Finally, NAMFIS analysis has been applied to previous data for cyclic depsipeptides called Stevastelins that possess immunosuppressant and phosphatase inhibitory activity. The analysis reveals the presence of several previously unobserved unusual conformations that may contribute to the biological activity of the molecule.

The amyloid Aß (16-22) peptide forms diverse and highly organized structures induced by small changes in physicochemical conditions. The peptide is of interest as a part of the larger amyloid Aß (1-42) peptide implicated in Alzheimer's disease and forms tubes and fibers under only slightly different conditions of pH. Using molecular dynamics (MD) simulations, molecular mechanics calculations, and structural data obtained from a variety of experiments done by Emory researchers, we investigate the factors responsible for self-assembly. Single-point mutants are utilized to study the contribution of cross- strand pairing to amyloid assembly.

Cyclin-dependent kinase 7 (CDK7) is a protein kinase that plays a crucial role in both phosphorylation of key substrate kinases in the cell cycle as well as transcriptional activation, and has been implicated in breast cancer. Using a structure-based design strategy, we have extensively collaborated with synthetic chemists and biologists at Imperial College London and have designed BS-181, the first potent and highly selective inhibitor of CDK7 that demonstrates antitumor activity.

Table of Contents

Chapter 1: Molecules, Motion and Life

1.1: Introduction………………………………………………………………….......1

1.2: NMR and molecular structure………………………………………………2

1.3: Conformation and drug design…….......................…………..4

1.4: NMR and conformation…………………………………....................6

1.5: Augmenting NMR data with computational methods……………..10

1.6: NAMFIS…………………………………………………………………………….12 1.7: Problems with purely theoretical methods

for studying conformation……………………………………………………18

Chapter 2: Dictyostatin and Discodermolide

2.1: Microtubules, tubulin and cell division…………………………………22

2.2: Dictyostatin and Discodermolide…………………………………………28

2.3: Dictyostatin……………………………………………………………………….32

2.4: Dictyostatin conformations…………………………………………………38

2.5: NAMFIS conformations in DMSO-d6

2.6: Dictyostatin conformations in the tubulin binding pocket……..48

2.7: Outlook and conclusions…………………………………………………….52

2.8: Discodermolide………………………………………………………………...53

2.9: Conformations of DDM in DMSO-d6

2.10: Simplified DDM constructs………………………………………………63

2.11: The tubulin-bound conformation of DDM………………………….67

2.12: Conclusions and outlook…………………………………………………..71

2.13: Perspective: Conformations of tubulin-binding agents………..74

Chapter 3: Noscapine

3.1: Introduction………………………………………………………………………77

3.2: NOESY spectra of noscapine……………………………………………….78

3.3: Noscapine x-ray crystal structures……………………………………….80

3.4: Conformer classification……………………………………………………..81

3.5: Noscapine hydrochloride conformations in solution………………82

3.6: Neutral noscapine conformers in solution…………………………….87

3.7: Comparison of solution and solid-state conformers……………….93

3.8: Summary and outlook………………………………………………………...94

Chapter 4: The Stevastelins

4.1: Cyclic peptides…………………………………………………………………….96

4.2: The Stevastelins………………………………………………………………….100

4.3: Stevastelin conformations……………………………………………………103

4.4: Effect of d-Serine on conformations……………………………………..113

4.5: Energetic analysis………………………………………………………………..115

4.6: Conclusions and outlook.......................……………………………118

Chapter 5: Selective Inhibitors of Cyclin-Dependent

Kinase 7

5.1: Cancer…………………………………………………………………………………122

5.2: Phosphorylation…………………………………………………………………..126

5.3: Protein kinases…………………………………………………………………….127

5.4: Kinase inhibitors in medicine………………………………………………..129

5.5: Kinase structure…………………………………………………………...........133

5.6: Cyclin-dependent kinases and CDK7………………………………………136

5.7: Design of selective CDK7 inhibitors………………………………………..141

5.8: Computational modeling of BS-181………………………………………..142

5.9: Biological studies with BS-181……………………………………………….152

5.10: Rationalization of CDK7 selectivity and implications……………..157

5.11: Future directions………………………………………………………………...162

Chapter 6: Self-assembly of Aß (16-22)

6.1: Alzheimer's disease……………………………………………………………….165

6.2: Amyloid Aß and Alzheimer's disease………………………………………167

6.3: Self-assembly of Aß (16-22).…………………………………………………..173

6.4: Cross-strand pairing and ß-sheet assembly……………………………..187

6.5: Summary and future directions………………………………………………195

Chapter 7: A Bird's Eye View of Computer-Aided Drug Design

7.1 Drugs and rational drug discovery…………………………………………200

7.2 Structure-based drug design and CADD…………………………….....203

7.3: Virtual screening…………………………………………………………...…..205

7.4: Pose prediction…………………………………………………………..……...208

7.5: Binding affinity prediction…………………………………………………..209

7.6: Models, computers and drug discovery……………………………………...211

About this Dissertation

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School	Laney Graduate School
Department	Chemistry
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Chemistry, Organic Chemistry, Pharmaceutical Chemistry, Physical
关键词	CDK7 kinase stevastelins amyloid computational chemistry tubulin dictyostatin noscapine molecular modeling conformational analysis discodermolide
Committee Chair / Thesis Advisor	Liotta, Dennis C, Emory University
Committee Members	Snyder, James P, Emory University Menger, Fred M, Emory University Gallivan, Justin, Emory University

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