High potency of indolyl aryl sulfone nonnucleoside inhibitors towards drug-resistant human immunodeficiency virus type 1 reverse transcriptase mutants is due to selective targeting of different mechanistic forms of the enzyme

Abstract

Indolyl aryl sulfone (IAS) nonnucleoside inhibitors have been shown to potently inhibit the growth of wild-type and drug-resistant human immunodeficiency virus type 1 (HIV-1), but their exact mechanism of action has not been elucidated yet. Here, we describe the mechanism of inhibition of HIV-1 reverse transcriptase (RT) by selected IAS derivatives. Our results showed that, depending on the substitutions introduced in the IAS common pharmacophore, these compounds can be made selective for different enzyme-substrate complexes. Moreover, we showed that the molecular basis for this selectivity was a different association rate of the drug to a particular enzymatic form along the reaction pathway. By comparing the activities of the different compounds against wild-type RT and the nonnucleoside reverse transcriptase inhibitor-resistant mutant Lys103Asn, it was possible to hypothesize, on the basis of their mechanism of action, a rationale for the design of drugs which could overcome the steric barrier imposed by the Lys103Asn mutation.

FIG. 1.

Schematic diagram of the HIV-1 reverse transcriptase RNA-dependent DNA polymerization reaction pathway. (A) Full reaction pathway. (B) The general steady-state kinetic analysis was simplified by varying one of the substrates (either TP or dNTP) while the other was kept constant. When the TP substrate was held constant at saturating concentration and the inhibition at various concentrations of dNTPs was analyzed, at the steady state all of the input RT was in the form of the RT/TP binary complex and only two forms of the enzyme (the binary complex and the ternary complex with dNTP) could react with the inhibitor (left part of the panel). Similarly, when the dNTP concentration was kept constant at saturating levels and the inhibition at various TP concentrations was analyzed, RT was present either as a free enzyme or in the ternary complex with TP and dNTP (right part of the panel). For details, see Materials and Methods.

FIG. 2.

Structures of the compounds used in this study.

FIG. 3.

Mechanisms of inhibition of HIV-1 RT by the IAS derivatives. (A) Increasing concentrations of RS1866 were titrated in the presence of 5 nM RT and either 2 μM, 4 μM, 10 μM, or 20 μM dTTP or 0.04 μM, 0.08 μM, 0.2 μM, and 0.4 μM (as 3′-OH ends) poly(rA)/oligo(dT) under the conditions described in Materials and Methods. Initial velocities of the reaction were plotted as a function of the substrate concentration. The variations of the k_cat(app) values for the reaction derived from these experiments were plotted as a function of the RS1866 concentrations. Data were fitted to equation 6 as described in Materials and Methods. (B) Increasing concentrations of RS1588 were titrated in the presence of 5 nM RT and 2 μM, 4 μM, 10 μM, and 20 μM dTTP or 0.04 μM, 0.08 μM, 0.2 μM, and 0.4 μM (as 3′-OH ends) of poly(rA)/oligo(dT) under the conditions described in Materials and Methods. Initial velocities of the reaction were plotted as a function of the substrate concentration. The variation of the k_cat(app) values for the reaction derived from these experiments was plotted as a function of the RS1588 concentrations. Data were fitted to equation 4 as described in Materials and Methods. (C) Increasing concentrations of RS1202 were titrated in the presence of 5 nM RT and 2 μM, 4 μM, 10 μM, and 20 μM dTTP or 0.04 μM, 0.08 μM, 0.2 μM, and 0.4 μM (as 3′-OH ends) poly(rA)/oligo(dT) under the conditions described in Materials and Methods. Initial velocities of the reaction were plotted as a function of the substrate concentration. The values of the apparent affinity for the nucleic acid substrate (K_s3′-OH) derived from these experiments were plotted as a function of the inhibitor concentration. Data were fitted to both equation 2 and equation 3, as described in Materials and Methods. (D) The variation of the k_cat(app) values for the reaction derived as described for panel C was plotted as a function of the RS1202 concentrations. Data were fitted to equation 4 as described in Materials and Methods. (E) Increasing concentrations of RS1980 were titrated in the presence of 5 nM RT and 2 μM, 4 μM, 10 μM, and 20 μM dTTP or 0.04 μM, 0.08 μM, 0.2 μM, and 0.4 μM (as 3′-OH ends) poly(rA)/oligo(dT) under the conditions described in Materials and Methods. Initial velocities of the reaction were plotted as a function of the substrate concentration. The values of the apparent affinity for the nucleotide (K_sTTP, circles) derived from these experiments were plotted as a function of the inhibitor concentration. Data were fitted to both equation 2 and equation 3, as described in Materials and Methods. (F) The variation of the k_cat(app) values for the reaction derived as described for panel E was plotted as a function of the RS1980 concentrations. Data were fitted to equation 4 as described in Materials and Methods.

FIG. 4.

Structure-activity relationships for the IAS derivatives used in this study. The different substituents on the common IAS scaffold are highlighted with circles. Arrows indicate the structural relationships among the different compounds. For each compound, the mechanism of action is indicated below its structure. For details, see the Discussion.