The goal of this project is to understand the molecular basis by which mutations alter the activity and inhibition of the HIV-1 Protease and lead to resistance of human immunodeficiency virus against protease inhibitor drugs.
The Aims of the project are as follows:
Aim 1 : to determine the catalytic activities and inhibitor sensitivities of resistant mutants from 14 major mutation positions of HIV-1 PR. The mutations at these 14 positions in the HIV-1 PR polypeptide chain account for the resistance observed in clinical trials of 3 marketed HIV-1 PR inhibitor drugs (Saquanivir, Idinavir, Ritonavir); the mutations also lead to cross-resistance to other major inhibitors under development for clinical use. The kcat/Km and Ki values will be determined for a panel of substrates for each single-residue mutant form of the HIV-1 PR, and for selected multiple-residue mutants. The data will be analyzed quantitatively by a previously established kinetic model.
Aim 2 : To determine the crystallographic structure of the complexes between inhibitors and resistance mutants of the HIV-1 PR. Crystallographic structures will be determined for chosen single and multiple-site resistant mutants complexed to selected clinically-relevant inhibitors. The differences in the crystal structures between resistant mutants, wild-type, and non-resistant mutants will be critically analyzed and compared to the change in catalytic activities a other kinetic properties.
Aim 3 : To test hypotheses on the mechanisms of HIV-1 PR catalysis, inhibition, and resistance to inhibitors. Kinetic and structural data will set the basis for testing several hypotheses: (1) Catalytic activities of the resistant mutant PR's determine the order of appearance of the mutants and the steady-state population ratios of the mutants during clinical therapy; (2)A major function of the flaps of the HIV-1 PR is the capturing of the sidechains of substrates and inhibitors at the major specificity positions; (3) Most of the clinically selected resistant mutations of HIV-1 PR worsen the binding of the transition state template (in the enzyme molecule) to the isostere of the inhibitors; (4) Inhibitor drugs bind strongest to HIV-1 PR with a transition state conformation that differs from that of the free enzyme.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI038189-08
Application #
6532712
Study Section
AIDS and Related Research Study Section 4 (ARRD)
Program Officer
Miller, Roger H
Project Start
1995-08-01
Project End
2004-06-30
Budget Start
2002-08-01
Budget End
2004-06-30
Support Year
8
Fiscal Year
2002
Total Cost
$356,991
Indirect Cost
Name
Oklahoma Medical Research Foundation
Department
Type
DUNS #
937727907
City
Oklahoma City
State
OK
Country
United States
Zip Code
73104