4th International Workshop on HIV Drug Resistance & Treatment Strategies Sitges, Spain, 12–16 June 2000

THE EFFECT OF DRUG RESISTANCE MUTATIONS ON KINETICS OF HIV PROTEASE INACTIVATION BY IRREVERSIBLE INHIBITORS

Antivir Ther. 2000 Jun 12-16; 5 (Suppl. 3):6 (Abstract 7

S Gulnik^†, M Pototschnik, T Guerassina^†, M Smrcina, TN Bhat, M Eissenstat^† and JW Erickson^†
SAIC-Frederick, NCI-FCRDC, Frederick, Md., USA; ^†Present address: Tibotec Inc., Rockville, Md., USA

Extensive use of potent HIV protease inhibitors has led to the generation of resistant mutant viruses. Many of these mutants evolved to become highly cross-resistant to most if not all currently approved protease inhibitors. In this connection, irreversible inhibitors are considered to be an attractive alternative to conventional drugs, whose binding is non-covalent and thermodynamically reversible. It has been proposed that irreversible inhibitors may be less sensitive to protease mutations, since complete inactivation of the enzyme can be achieved over time despite a reduction in inhibitor potency. A number of potent and apparently selective irreversible inhibitors based on a cis-epoxide core have been recently described. However, these inhibitors had rather high molecular weight and contained naturally occurring amino acids, which are undesirable for metabolic stability. We have solved a crystal structure of one of such inhibitor, LB71350, complexed with HIV protease, and used structural information to design shorter, non-peptidic, cis-epoxide-based inhibitors. Binding (K_i) and inactivation rate (k_inact) constants for these inhibitors were determined using stopped-flow kinetics techniques. We showed that truncation resulted in an increase in K_i, but little change in k_inact. The most potent inhibitors had K values in the range of 10–100 nM and inactivated HIV protease with a half-life of 15–30 s at inhibitor concentrations equal to K_i. The effect of several drug-resistant mutations, including V82A, V82F/I84V, G48V/V82A and L10F/M46I/ I47V/I50V, on the kinetics of enzyme inactivation has been evaluated. While k_inact did not change significantly, mutant-dependent increases of K_i up to 100-fold were detected, which led to corresponding increase of half-life of enzyme inactivation. Our data suggests that this type of irreversible inhibitor may have an advantage over reversible compounds in treating drug-resistant viruses only if HIV protease is required to be active over a period of at least several minutes during viral maturation.

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2000-06-12
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