Biosensor-Based Kinetic Characterization of the Interaction between HIV-1 Reverse Transcriptase and Non-nucleoside Inhibitors

Abstract

Details of the interaction between HIV-1 reverse transcriptase and non-nucleoside inhibitors (NNRTIs) have been elucidated using a biosensor-based approach. This initial study was performed with HIV-1 reverse transcriptase mutant K103N, the phenethylthioazolylthiourea compound (PETT) MIV-150, and the three NNRTIs licensed for clinical use: nevirapine, delavirdine, and efavirenz. Mathematical evaluation of the experimental data with several interaction models revealed that the four inhibitors interacted with HIV-1 RT with varying degrees of complexity. The simplest adequate model accounted for two different conformations of the free enzyme, of which only one can bind the inhibitor, consistent with a previously hypothesized population-shift model including a preformation of the NNRTI binding site. In addition, a heterogeneous binding was observed for delavirdine, efavirenz, and MIV-150, indicating that two noncompetitive and kinetically distinct enzyme-inhibitor complexes could be formed. Furthermore, for these compounds, there were indications for ligand-induced conformational changes.

The paradoxical effects of using antiretroviral-based microbicides to control HIV epidemics

“…This will be closely followed by a microbicide containing dapivirine, which belongs to the nonnucleoside reverse transcriptase inhibitor (NNRTI) class of ARVs (6); trial results will be available in 2010. Three other ARV-based microbicides, containing NNRTIs, are in earlier-phase trials (3,5,(7)(8)(9)(10). ARV-based microbicides are anticipated to be highly effective; however, there is concern that, if used by HIV-positive women, class-specific drug resistance may emerge.…”

Inhibition of HIV-1 by non-nucleoside reverse transcriptase inhibitors via an induced fit mechanism—Importance of slow dissociation and relaxation rates for antiviral efficacy

“…14 The choice of biosensor-based interaction analysis for this study was based on our experience that it can provide detailed information on the interaction between NNRTIs and the enzyme [5][6][7], although not as mechanistically revealing as the pre-steady state kinetic analysis of the enzyme activity [14] previously used to determine the mechanism of NNRTI inhibition. However, there were several experimental challenges to overcome in the present study.…”

“…The wild type and three drug resistant enzyme variants (Y181C, K103N and L100I) of reverse transcriptase (EC 2.7.7.49) of HIV-1 (BH10 isolate) were obtained as previously [5,11] described. All enzymes contained the additional substitution E478Q to abolish the RNase H activity.…”

“…In order to establish the kinetic mechanism of the interaction and qualitatively determine the difference in the kinetics for the different inhibitors, sensorgrams were simulated with a set of mechanistic models representing steps of the complex mechanism previously found to describe the interactions of NNRTIs with the K103N variant of HIV-1 RT 10 [5]. An induced fit mechanism was the simplest model that captured the basic features of the experimental data (Fig.…”

“…We have previously used this technology to characterize the interaction mechanism for NNRTIs with their target and to determine the resistance profiles of NNRTIs [5][6][7]. Due to the tight-binding interaction of the clinically used NNRTIs, the initial characterization was performed with a drug resistant variant from which the compounds dissociated.…”