Selective Interaction of the Human Immunodeficiency Virus Type 1 Reverse Transcriptase Nonnucleoside Inhibitor Efavirenz and Its Thio-Substituted Analog with Different Enzyme-Substrate Complexes

Abstract

Accumulating data have brought the nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) into the forefront of antiretroviral therapy. Among the emerging compounds in this class, a particularly attractive one is efavirenz (Sustiva), recently approved for clinical use by the U.S. Food and Drug Administration. In the present study, the equilibrium dissociation constants for efavirenz binding to the different catalytic forms of human immunodeficiency virus type 1 RT as well as the association and dissociation rates have been determined using a steady-state kinetic approach. In addition, the same enzymological analysis has been extended to the thio-substituted analog, sefavirenz, which showed comparable activity in vitro against RT. Both compounds have been found to act as purely uncompetitive inhibitors at low drug concentrations (5 to 50 nM) and as mixed noncompetitive inhibitors at higher doses (50 to 500 nM). This behavior can be interpreted in terms of the relative affinities for the different catalytic forms of the enzyme. Both efavirenz and sefavirenz showed increasing affinities for the different forms of RT in the following order: free enzyme < (i.e., bound with lower affinity) binary RT-template-primer (TP) complex < ternary RT-TP-deoxynucleoside triphosphate (dNTP) complex. The rate of binding of the two inhibitors to the different enzyme-substrate complexes was well below the diffusion limit (on the order of 10 4 M ؊1 s ؊1 ); however, both inhibitors, when bound to the ternary RT-TP-dNTP complex, showed very low dissociation rates, on the order of 10 ؊4 s ؊1 for both compounds, typical of tightly binding inhibitors. Thus, efavirenz and its thio-substituted derivative sefavirenz appear to be peculiar in their mechanism of action, being selective tightly binding inhibitors of the ternary RT-TP-dNTP complex. Efavirenz is the first clinically approved NNRTI to show this property.

The Stereoselective Targeting of a Specific Enzyme-Substrate Complex Is the Molecular Mechanism for the Synergic Inhibition of HIV-1 Reverse Transcriptase by (R)-(−)-PPO464

“…Since NNRTI binding involves a rearrangement of the structural elements surrounding the binding pocket, it is possible that local differences might facilitate the access of (R)-(Ϫ)-PPO464 to the binding site, partially explaining the observed mechanism of inhibition. Other second generation NNRTIs have been shown to bind with variable affinities to different reaction intermediates (26,27); moreover, in a previous work, we have shown that the second generation NNRTI efavirenz displays preferential affinity for either the binary or the ternary complex of RT with its substrates, with respect to the unliganded enzyme (28). Thus, it is conceivable to propose an induced fit mechanism for binding of some classes of NNRTIs, which is triggered by complex formation among RT and the TP and dNTP substrates.…”

“…Because combination therapy using HIV-1 protease inhibitors will be increasingly used but these agents have the potential to interact with drug-metabolizing enzymes, these studies also preliminarily assessed how this interaction affected the pharmacokinetics of the new antiviral agent (R)-(Ϫ)-PPO464. Ritonavir was selected because it is the strongest inhibitor of P450 enzymes in vitro and in vivo, particularly of the CYP3A subfamily (28).…”

Non nucleoside reverse transcriptase inhibitors

“…Two additional tight-binding NNRTI have now been identified. These are 5-chloro-3-phenylsulfonylindole-2-carboxamide (CSIC) (Motakis et al, submitted) and the clinically used efavirenz (10). Our comparison of the tight-binding NNRTI CSIC, EFV, and UC781 with the rapid-equilibrium inhibitors DLV, NVP, and UC84 in a variety of virucidal tests now firmly establishes the tight-binding mode of inhibition as an essential parameter that must be met by NNRTI for use in virucidal applications.…”

Development of a New Methodology for Screening of Human Immunodeficiency Virus Type 1 Microbicides Based on Real-Time PCR Quantification

“…EFV binds rapidly to RT but, once bound, dissociates only very slowly. Thus, RT remains inhibited for prolonged periods of time after being bound, even in the absence of significant levels of unbound inhibitor or the wash procedure to remove the excess inhibitor (14). Therefore, we suggested that tightly binding inhibition might be an important criterion for the NNRTI microbicide screening, and compounds classified as tightly binding, such as EFV, have great potential as microbicides.…”

“…NVP is a rapid-equilibrium inhibitor and requires an excess of the drug over the enzyme concentration (27). On the other hand, EFV is a tightly binding inhibitor of HIV-1 RT (14). Tightly binding inhibitors exhibit unique properties in their interactions with enzymes, which distinguish them from rapid-equilibrium inhibitors (31).…”