Author ORCID Identifier

https://orcid.org/0000-0001-7997-6126

Date of Award

8-13-2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Irene Weber

Second Advisor

Robert Harrison

Third Advisor

Richard Plemper

Abstract

HIV affects the lives of about 37 million patients worldwideincluding around one million Americans. Infection is treated via drugs that inhibit several targets in the virus life cycle such as the HIV protease, which is essential for processing viral polyproteins. Antiviral therapy has been successful; however, emergence of drug resistance is a serious problem for the increasing population of HIV patients. It is unquestionable that there is an urgent need to design new inhibitors against drug-resistant targets in the virus, including HIV proteases. To meet this challenge, molecular mechanisms of resistance must be understood to provide suggestions for drug design.

Mutation L76V in the protease is associated with major resistance towards clinical inhibitors darunavir and lopinavir, which are two of the three protease inhibitors that are prescribed in combination therapy. The prevalence of L76V increases from 1.5-3.2% to 5.4% in patients undergoing treatment failure; the mutation causes impaired autoprocessing and decreased protease stability. We tested the effectiveness of antivirals including clinical protease inhibitors (saquinavir, tipranavir and lopinavir) and promising investigational drugs (GRL-519 and GRL-5010) on the single mutant protease bearing L76V. We solved 6 crystal structures at high-resolution and proposed a mechanism of drug resistance at the structural level in which the mutation alters interactions at the interface of the hydrophobic core with the flap base.

L76V as a single mutation is rare in the clinic with a frequency of 0.04% and is often found in combination with other resistance mutations. Compensatory mutations rescue weakened viral fitness and impaired autoprocessing caused by L76V. Structural studies on mutants bearing L76V in addition to other mutations are not available. We applied biochemical and biophysical analysis on a clinical isolate bearing over 20 mutations in addition to L76V. This cross-resistant mutant exhibited greater loss of susceptibility towards a panel of inhibitors and experienced partial compensation of protease stability.We solved the first crystal structures of ligand-free and inhibitor-complex for this multiple mutant. The structures display alterations of intramolecular interactions and extreme conformations. The present work on molecular mechanisms of drug resistance provides clues for structure-guided drug design.

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