Computational Studies of Ligand Protein Interactions Part I: TheT-Taxol Conformation Part II: Elucidating Interdependent BindingSites on Tubulin Pubblico
Alcaraz, Ana Angelica (2009)
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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