Access Type

Open Access Dissertation

Date of Award

January 2013

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Biochemistry and Molecular Biology

First Advisor

Ladislau C. Kovari

Abstract

Protein point mutations acquired as a mechanism of survival against therapeutics cause structural changes that effect protein function and inhibitor binding. This work investigates the structural mechanisms that lead to multi-drug resistance to HIV-1 protease and integrase inhibitors.

Proper proteolytic processing of the HIV-1 Gag/Pol polyprotein is required for HIV infection and viral replication. This feature has made HIV-1 protease an attractive target for antiretroviral drug design for the treatment of HIV-1 infected patients, thus the development of drug resistance has arisen as a major therapeutic and drug design challenge. To understand the molecular mechanisms leading to drug resistance we selected and characterized three multi-drug resistant HIV-1 protease patient isolates, identifying a previously unreported structural role for V32I, I47V, I54M and L90M in protease dynamics. To examine the role of the P1 and P1' positions of the substrate in inhibitory efficacy of multi-drug resistant HIV-1 protease 769 (MDR 769), we performed a structure-function studies. We designed a series of ligands and evaluated them using a combination of computational and experimental methods. Our results suggest two important strategies for rational drug design of protease inhibitors: (1) the presence of fluorinated P1 or P1' groups enhance the binding affinities in both wild-type and MDR PR variants and (2) non-identical P1/P1' residues play an important role in binding to multi-drug resistant HIV-1 protease.

HIV-1 integrase is an essential enzyme necessary for the replication of the HIV virus as it catalyzes the insertion of the viral genome into the host chromosome. Raltegravir was the first integrase inhibitor approved by the FDA for treatment of HIV-1 infection. HIV patients on raltegravir containing regimens may develop drug resistance mutations at residue 140 and 148 in the catalytic 140's loop resulting in a 5-10 fold decrease in susceptibility to raltegravir. To understand the molecular mechanisms of drug resistance in HIV-1 integrase we performed molecular dynamics studies on the catalytic core domain of raltegravir resistant HIV-1 protease in complex with raltegravir. These experiments suggest a gating function of the catalytic 140's loop in active site accessibility as well as reduced flexibility as a mechanism of raltegravir resistance. In addition, we developed a model for the full length HIV-1 integrase and performed molecular dynamics of our model in complex with raltegravir and the viral DNA ends, identifying unique alterations in non-bonded interactions between the protein, DNA, and raltegravir as a result of the drug resistant mutations.

The results of this work provide detailed structural information on HIV-1 protease and integrase in order to aid in the development of new therapeutics against these targets. Our work has implications not only on HIV-1 therapy but the techniques described here can be used to study other proteins, design new compounds, and help aid in our understanding of protein structure.

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