Novel Mechanism of Inhibition of HIV-1 Reverse Transcriptase by a New Non-nucleoside Analog, KM-1

Louise Z Wang,George L Kenyon,Kenneth A Johnson

doi:10.1074/jbc.m406241200

Abstract

2-Naphthalenesulfonic acid (4-hydroxy-7-[[[[5-hydroxy-6-[(4 cinnamylphenyl)azo]-7-sulfo-2-naphthalenyl]amino]-carbonyl]amino]-3-[(4-cinnamylphenyl)]azo (KM-1)) is a novel non-nucleoside reverse transcriptase inhibitor (NNRTI) that was designed to bind at an unconventional site on human immunodeficiency virus type 1 reverse transcriptase (RT) (Skillman, A. G., Maurer, K. W., Roe, D. C., Stauber, M. J., Eargle, D., Ewing, T. J., Muscate, A., Davioud-Charvet, E., Medaglia, M. V., Fisher, R. J., Arnold, E., Gao, H. Q., Buckheit, R., Boyer, P. L., Hughes, S. H., Kuntz, I. D., and Kenyon, G. L. (2002) Bioorg. Chem. 30, 443-458). We have investigated the mechanism by which KM-1 inhibits wild-type human immunodeficiency virus type 1 RT by using pre-steady state kinetic methods to examine the effect of KM-1 on the parameters governing the single nucleotide incorporation catalyzed by RT. Analysis of the pre-steady-state burst phase of dATP incorporation showed that KM-1 decreased the amplitude of the reaction as previously shown for other NNRTIs, because of the slow equilibration of the inhibitor with RT. In the ternary enzyme-DNA-KM-1 complex (E-DNA-I), incorporation of the next nucleotide onto the primer is blocked. However, unlike conventional NNRTIs, the inhibitory effect was caused primarily by weakening the DNA binding affinity and displacing DNA from the enzyme. Wild-type RT binds a 25/45-mer DNA duplex with an apparent K(d) of 3 nm, which was increased to 400 nm upon saturation with KM-1. Likewise, the apparent K(d) for KM-1 binding to RT increased at higher DNA concentrations. We therefore conclude that KM-1 represents a new class of inhibitor distinct from nevirapine and related NNRTIs. KM-1 can bind to RT in both the absence and presence of DNA but weakens the affinity for DNA 140-fold so that it favors DNA dissociation. The data suggest that KM-1 distorts RT conformation and misaligns DNA at the active site.

Highlights

Since the introduction of antiretroviral therapy, the life span of patients infected with HIV1 or suffering from AIDS has been
non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) bind in a hydrophobic pocket ϳ10 Å away from the RT polymerase active site in the palm domain of the p66 subunit and distort the key residues that comprise the aspartic triad (D110, D185, and D186) so that the carboxyl groups of the side chains are out of alignment leading to slower rates of catalysis [13,14,15]
We propose that KM-1 binds to RT with or without DNA and precludes proper alignment of DNA at the polymerase active site, depleting the active DNA-bound RT (E-D) complex required for nucleotide incorporation

Summary

Introduction

Since the introduction of antiretroviral therapy, the life span of patients infected with HIV1 or suffering from AIDS has been. Nucleoside analog RT inhibitors, such as 3Ј-azido3Ј-deoxythymidine and 2Ј,3Ј-dideoxycytidine, are nucleosides that become phosphorylated by cellular enzymes to their triphosphate form and are incorporated by RT, acting as chain terminators to prevent further polymerization. To varying degrees, they block cellular DNA polymerases, in particular the mitochondrial DNA polymerase (Pol ␥) [10], and their kinetics of incorporation are correlated with the toxic side effects of these drugs (reviewed in Ref. 11). Drug-resistant RT mutants have been proposed to obstruct the binding of the drug [16, 17], reposition the template/primer (18 –20), or facilscriptase; NNRTI, non-nucleoside reverse transcriptase inhibitor; E-D, enzyme-DNA complex; E-D-I, enzyme-DNA-inhibitor complex; Pol ␥, mitochondrial DNA polymerase. It weakens the binding of DNA, whereas conventional NNRTIs are thought to strengthen DNA binding by slowing the DNA dissociation rate from the closed complex formed after the binding of the correct nucleotide [15]

Objectives

Results

Conclusion