Novel nonnucleoside inhibitors that select nucleoside inhibitor resistance mutations in human immunodeficiency virus type 1 reverse transcriptase

Abstract

Mutations in and around the catalytic site of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) are associated with resistance to nucleoside RT inhibitors (NRTIs), whereas changes in the hydrophobic pocket of the RT are attributed to nonnucleoside RT inhibitor (NNRTI) resistance. In this study, we report a novel series of nonnucleoside inhibitors of HIV-1, exemplified by VRX-329747 and VRX-413638, which inhibit both NNRTI- and NRTI-resistant HIV-1 isolates. Enzymatic studies indicated that these compounds are HIV-1 RT inhibitors. Surprisingly, however, following prolonged (6 months) tissue culture selection, this series of nonnucleoside inhibitors did not select NNRTI-resistant mutations in HIV-1 RT. Rather, four mutations (M41L, A62T/V, V118I, and M184V) known to cause resistance to NRTIs and two additional novel mutations (S68N and G112S) adjacent to the catalytic site of the enzyme were selected. Although the M184V mutation appears to be the initial mutation to establish resistance, this mutation alone confers only a two- to fourfold decrease in susceptibility to VRX-329747 and VRX-413638. At least two additional mutations must accumulate for significant resistance. Moreover, while VRX-329747-selected viruses are resistant to lamivudine and emtricitabine due to the M184V mutation, they remain susceptible to zidovudine, stavudine, dideoxyinosine, abacavir, tenofovir, and efavirenz. These results directly demonstrate that VRX-329747 and VRX-413638 are novel nonnucleoside inhibitors of HIV-1 RT with the potential to augment current therapies.

FIG. 1.

Structures of VRX-329747 and VRX-413638.

FIG. 2.

Time course of VRX-329747-resistant mutant selection. NL4-3 virus-infected SupT1 cells were cultured in the presence of VRX-329747. At each virus breakthrough point (massive syncytium formation), the compound concentration was doubled. Proviruses from each breakthrough point were sequenced. RT mutations and their prevalences (subscripts) in recovered pools are shown for 0.8, 1.6, 3.2, and 20 μM VRX-329747.

FIG. 3.

VRX-329747-selected mutations in HIV-1 RT. HIV-1 RT amino acids 1 to 550 are represented on the top line of the figure. Individual proviral clones recovered by PCR and characterized by sequence analysis are listed horizontally for 0.8, 1.6, 3.2, 6.4, and 20 μM VRX-329747. The dominant mutations M41L, A62T/V, S68N, G112S, V118I, and M184V are shaded.

FIG. 4.

VRX-329747-selected mutations map near the HIV-1 RT catalytic complex (front and back views are shown). VRX-329747-selected (pink) and common NRTI (black) mutations are mapped on the complex structure of Huang et al. (16). The template, primer, dTTP, and Mg²⁺ are depicted in green, blue, orange, and yellow, respectively. The six mutations selected by VRX-329747 encompass the RT catalytic site.

FIG. 5.

Steady-state inhibition by VRX-413638 of HIV-1 RT synthesis on poly(A)/oligo(dT). (A) Inhibition with respect to TTP. Reactions were carried out as described in Materials and Methods, using 0 (□), 0.1 (•), 0.2 (⧫), 0.3 (▾), 0.4(▴), or 0.5 (▪) μM VRX-413638. Data were globally fit to a mixed-type inhibition pattern. (B) Inhibition with respect to poly(rA)/oligo(dT)₁₈. Data are shown for 0 (□), 0.1 (•), 0.2 (⧫), 0.3 (▾), 0.4(▴), and 0.5 (▪) μM VRX-413638. Data were globally fit to an uncompetitive inhibition pattern.

FIG. 6.

Kinetic scheme of inhibition by VRX-413638. The inhibitor does not bind to RT but can bind after RT forms a complex with the template/primer (T/P). The inhibitor binds to the RT/T/P complex with a K_i of 0.05 μM or to the RT/T/P/TTP complex with a K_i of 0.9 μM, leading to the formation of an inactive complex.