DOI

10.17077/etd.ppygeuv8

Document Type

Thesis

Date of Degree

Fall 2016

Degree Name

MS (Master of Science)

Degree In

Pharmaceutical Sciences and Experimental Therapeutics

First Advisor

Spies, M. Ashley

First Committee Member

Doorn, Jonathan A.

Second Committee Member

Duffel, Michael W.

Abstract

Flexible enzymes are notoriously a bane to structure-based drug design and discovery efforts. This is because no single structure can accurately capture the vast array of conformations that exist in solution and many are subject to ligand-associated structural changes that are difficult to predict. Glutamate racemase (GR) – an antibiotic drug discovery target involved in cell wall biosynthesis – is one such enzyme that has eluded basic structure-based drug design and discovery efforts due to these flexibility issues. In this study, our focus is on overcoming the impediment of unpredictable ligand-associated structural changes in GR drug discovery campaigns. The flexibility of the GR active site is such that it is capable of accommodating ligands with very different structures. Though these ligands may bind to the same pocket, they may associate with quite dissimilar conformations where some are more favorable for complexation than others. Knowledge of these changes is invaluable in guiding drug discovery efforts, indicating which compounds selectively associate with more favorable conformations and are therefore better suited for optimization and providing starting structures to guide structure-based drug design optimization efforts. In this study, we develop a mutant GR possessing a genetically encoded non-natural fluorescent amino acid in a region remote from the active site whose movement has been previously observed to correlate with active site changes. With this mutant GR, we observe a differential fluorescence pattern upon binding of two structurally distinct competitive inhibitors known to associate with unique GR conformations – one to a favorable conformation with a smaller, less solvated active site and the other to an unfavorable conformation with a larger, more solvated active site. A concomitant computational study ascribes the source of this differential fluorescence pattern to ligand-associated conformational changes resulting in changes to the local environment of the fluorescent residue. Therefore, this mutant permits the elucidation of valuable structural information with relative ease by simply monitoring the fluorescence pattern resulting from ligand binding, which indicates whether the ligand has bound to a favorable or unfavorable conformation and offers insight into the general structure of this conformation.

Public Abstract

Drug discovery efforts often rely on detailed structural information of proteins that play an important role in some biological process whose disruption or alteration could result in a desired outcome for improved health (i.e., proteins that make for promising drug targets). However, this structural information can be difficult to attain in some cases due to the fact that some proteins are quite flexible such that they adopt an array of structural variants that are difficult to capture. This is problematic because structure-based drug development efforts rely on accurate and precise structural information to examine (a) whether a compound will bind and (b) what a protein–compound structure looks like. This information may be examined virtually but must be validated experimentally. Experimental assessment of binding is often a rather facile process, but experimental assessment of the structure of the protein–compound complex can be quite difficult to determine. In this study, we have developed a method of assessing both binding and quality of binding of compounds to a flexible protein that is a promising antibiotic drug target. The protein of interest has been mutated such that it fluoresces. Different fluorescence patterns are observed upon complexation with two compounds known to associate with distinct structural variants, indicating its capacity to identify these structural variants, one of which is more favorable for compound binding than the other. The described method may be applied to high-throughput screening campaigns to identify favorable protein-compound complexes and guide optimization efforts following the discovery of an initial hit.

Keywords

allostery, flexible enzymes, glutamate racemase, structure based drug design and discovery

Pages

xii, 47 pages

Bibliography

Includes bibliographical references (pages 44-47).

Copyright

Copyright © 2016 Sondra Faye Dean

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