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Probing nonnucleoside inhibitor-induced active-site distortion in HIV-1 reverse transcriptase by transient kinetic analyses

¹ Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA
² Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520, USA

(RECEIVED February 14, 2007; FINAL REVISION May 11, 2007; ACCEPTED May 20, 2007)

Nonnucleoside reverse transcriptase inhibitors (NNRTI) are a group of structurally diverse compounds that bind to a single site in HIV-1 reverse transcriptase (RT), termed the NNRTI-binding pocket (NNRTI-BP). NNRTI binding to RT induces conformational changes in the enzyme that affect key elements of the polymerase active site and also the association between the two protein subunits. To determine which conformational changes contribute to the mechanism of inhibition of HIV-1 reverse transcription, we used transient kinetic analyses to probe the catalytic events that occur directly at the enzyme's polymerase active site when the NNRTI-BP was occupied by nevirapine, efavirenz, or delavirdine. Our results demonstrate that all NNRTI–RT–template/primer (NNRTI–RT–T/P) complexes displayed a metal-dependent increase in dNTP binding affinity (K_d ) and a metal-independent decrease in the maximum rate of dNTP incorporation (k _pol). The magnitude of the decrease in k _pol was dependent on the NNRTI used in the assay: Efavirenz caused the largest decrease followed by delavirdine and then nevirapine. Analyses that were designed to probe direct effects on phosphodiester bond formation suggested that the NNRTI mediate their effects on the chemistry step of the DNA polymerization reaction via an indirect manner. Because each of the NNRTI analyzed in this study exerted largely similar phenotypic effects on single nucleotide addition reactions, whereas each of them are known to exert differential effects on RT dimerization, we conclude that the NNRTI effects on subunit association do not directly contribute to the kinetic mechanism of inhibition of DNA polymerization.

Keywords: HIV; reverse transcriptase; nonnucleoside reverse transcriptase inhibitor; presteady-state kinetics; phosphorothioate metal effect

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