Computational Studies of Ligand Protein Interactions Part I: The T-Taxol Conformation Part II: Elucidating Interdependent Binding Sites on Tubulin Open Access

Alcaraz, Ana Angelica (2009)

Permanent URL: https://etd.library.emory.edu/concern/etds/9z903039w?locale=en
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Abstract

Part I: T-Taxol is a proposal for the bioactive conformation of paclitaxel (PTX) derived from fitting ligand conformations to the electron crystallography (EC) density of the tubulin-ligand complex. It has been confirmed by independent refinement of the PTX-tubulin structure, the activity of bridged T-Taxol analogs, and mutation studies in the yeast tubulin framework. Nonetheless, some structural ambiguities remain based on an uncritical interpretation of the solid state REDOR measurement of two internuclear 13C---19F distances in a fluorinated derivative of PTX. The issues are evaluated by an analysis of the static and dynamic properties of PTX and the PTX-tubulin complex, torsional force field parameters, and the error assigned to the REDOR distances (± 0.5 Å). In addition, a proposed alternative to T-Taxol (PTX-NY) is shown to be incompatible with both the EC density and the activity of a highly active series of bridged taxanes. The development of series of bridged and simplified taxanes using the T-Taxol conformation resulted in analogs of the former that induce microtubule formation more efficiently. NAMFIS analysis, along with molecular dynamics simulations and docking, are used to analyze the compounds, and we propose that the enhanced ability to assemble microtubules by these taxane derivatives is linked to their ability to effectively shape the conformation of the M-loop of tubulin for cross-protofilament interaction.

Part II: A model of colchicine (COL) and 2-methoxyestardiol (2ME2) binding to tubulin was developed using molecular dynamics and docking. Available experimental data, including a number of labeling studies, supports the two-site hypothesis in which conformational changes in the H7--H8 loop open opposing sites with differing binding properties and affinities. The β-Val236Ile mutation that causes resistance to 2ME2 with no cross-resistance to COL is located in the site designated for the former ligand and not near the site for the later one.

Table of Contents

List of Figures
List of Tables
List of Abbreviations

The T-Taxol Conformation
Chapter One

Drug Discovery 1
Rational Drug Design 3
Computational Techniques 5
Taxol® (Paclitaxel) 6
Microtubules 9
Cancer 12


Chapter Two

Background 15
The Bioactive Conformation of PTX 16
The Polar and Non-polar Conformations 16
Deconvolution of Conformationally Averaged Structures 20
The T-Taxol Conformation 22
The REDOR Conformation 25
Methods 28
Results and Discussion 33
Electron Crystallographic Density Compatibility 33
REDOR Experiment Limitations 38
Conformer Variability 45
Taxane Analogs 51
T-Taxol Inspired REDOR Experiment 62
Summary and Conclusions 68


Chapter Three

Design of Constrained Analogs 73
Methods 77
Results and Discussion 81
Summary 103
Simplified Taxanes 104
Methods 105
Results and Discussion 106
C-2 Sulfur Linked PTX Analogs 109
Methods 111
Results and Discussion 113
Conclusions 116
Elucidating Interdependent Binding Sites on Tubulin
Background 120
Binding Site A 121
Binding Site B 124
Methods 127
Results and Discussion 129
Conclusions 132
References 134

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