Document Type

Dissertation

Date of Degree

Spring 2016

Degree Name

PhD (Doctor of Philosophy)

Degree In

Pharmaceutical Sciences and Experimental Therapeutics

First Advisor

David L. Roman

Abstract

Regulator of G Protein Signaling 4 (RGS4) mediates motor defects in Parkinson's disease. Small molecule RGS4 inhibitors (e.g. CCG-50014) modify buried cysteine residues, but the structural and dynamic mechanisms underpinning specificity of inhibitors for RGS4 within the RGS family are poorly understood. We used NMR and other biophysical methods to examine ligand-induced structural changes and the dynamics of unliganded RGS4 and RGS8 that allow ligand binding. NMR and fluorescence spectroscopy data reveal details of the hidden, excited conformational state of RGS4 that exposes Cys148, one of the buried cysteines bound by inhibitors. We further show that specificity of RGS4 inhibitors is driven by differential accessibility of the target cysteine compared to its equivalent in RGS8. Cys148 is buried beneath the lid at the center the α4-α7 helix bundle, and this bundle is destabilized in RGS4 compared to RGS8. Notably, helix 6 is highly destabilized in RGS4 compared to RGS8 and is likely the key mediator of access to Cys148. Our findings provide key insight into the mechanism of allosteric RGS4 inhibition and show that dynamics drive inhibitory specificity among RGS proteins.

Public Abstract

Drugs are chemicals we take for a specific, desired biological effect. The discovery and development of new drugs is a long and difficult process that requires a thorough understanding of the processes that keep our bodies and minds working. Once we understand the processes that break down in a disease and the proteins and cells involved in those processes, scientists discover and/or develop chemicals specifically suited to restore their function.

I have been working on a project to understand how certain chemicals interact with a protein called regulator of G protein signaling 4 (RGS4). RGS4 is overactive in the brains of patients with Parkinson’s disease, and we want to develop chemicals into drugs to block RGS4 to help treat these patients. My work on this project has two components: measuring the specific way the chemicals interact with the protein, and identifying factors that contribute to the sensitivity of RGS4 to these chemicals compared to a related protein, RGS8.

I showed that the chemicals destabilized the structure of RGS4 and that RGS4 is more sensitive to these effects because its structure is more malleable than the structure of RGS8. My work will help the next generation of research aimed at creating those drugs and ensuring that they are safe and have minimal side effects. This project is still in its infancy, and it will take years of hard work and good fortune for those drugs to reach patients.

Keywords

publicabstract, dynamic, nmr, regulator of g protein signaling, rgs4, structure

Pages

xiii, 138 pages

Bibliography

Includes bibliographical references (pages 130-138).

Copyright

Copyright 2016 Colin Anthony Higgins

Share

COinS