The phenylmethylthiazolylthiourea nonnucleoside reverse transcriptase (RT) inhibitor MSK-076 selects for a resistance mutation in the active site of human immunodeficiency virus type 2 RT

Abstract

The phenylmethylthiazolylthiourea (PETT) derivative MSK-076 shows, besides high potency against human immunodeficiency virus type 1 (HIV-1), marked activity against HIV-2 (50% effective concentration, 0.63 microM) in cell culture. Time-of-addition experiments pointed to HIV-2 reverse transcriptase (RT) as the target of action of MSK-076. Recombinant HIV-2 RT was inhibited by MSK-076 at 23 microM. As was also found for HIV-1 RT, MSK-076 inhibited HIV-2 RT in a noncompetitive manner with respect to dGTP and poly(rC).oligo(dG) as the substrate and template-primer, respectively. MSK-076 selected for A101P and G112E mutations in HIV-2 RT and for K101E, Y181C, and G190R mutations in HIV-1 RT. The selected mutated strains of HIV-2 were fully resistant to MSK-076, and the mutant HIV-2 RT enzymes into which the A101P and/or G112E mutation was introduced by site-directed mutagenesis showed more than 50-fold resistance to MSK-076. Mapping of the resistance mutations to the HIV-2 RT structure ascertained that A101P is located at a position equivalent to the nonnucleoside RT inhibitor (NNRTI)-binding site of HIV-1 RT. G112E, however, is distal to the putative NNRTI-binding site in HIV-2 RT but close to the active site, implying a novel molecular mode of action and mechanism of resistance. Our findings have important implications for the development of new NNRTIs with pronounced activity against a wider range of lentiviruses.

FIG. 1.

Alignment of important amino acid stretches in the NNRTI-binding pocket of HIV-1 RT with the corresponding amino acids in other lentivirus RTs. Amino acids instrumental in the susceptibility of HIV-1 RT to NNRTIs are shaded and numbered. Amino acid mutations in HIV-2 and HIV-1 RT that are related to MSK-076 resistance are highlighted. The underlined sequence is highly conserved among lentivirus RTs and includes residues D185 and D186, which are critical for polymerase activity.

FIG. 2.

Structures of the PETT derivatives MSK-076 and PETT-2.

FIG. 3.

Time-of-addition experiment. CEM cells were infected with HIV-2(ROD) at approximately 100 times the CCID₅₀ per ml. Test compounds were added at different times postinfection. Viral p24 antigen production was determined at 72 h postinfection. Solid squares, AMD3100 at 10 μM; small solid rectangles, lamivudine at 90 μM; multipliers, tenofovir at 700 μM; solid triangles, MSK-076 at 50 μM; asterisks, ritonavir at 15 μM; plus signs, K-37 at 5 μM; solid diamonds, untreated control.

FIG. 4.

Double-reciprocal plots for inhibition of wild-type HIV-2 RT (A and B) and HIV-1 RT (C and D) by MSK-076. MSK-076 concentrations were as follows: ▪, 52 μM; ▴, 26 μM; ×, 0.052 μM; ○, 0.026 μM; +, 0.013 μM; ♦, 0 μM (control). In panels B and D, 0.1 mM template-primer [poly(rC)·oligo(dG)] and variable concentrations of [³H]dGTP were used. In panels A and C, 1.4 μM [2.8-³H]dGTP and variable concentrations of the template-primer poly(rC)·oligo(dG) were used.

FIG. 5.

Positions of the MSK-076 resistance mutations A101P and G112E in HIV RT. (a) A101P and G112E mutations relative to the polymerase active site and the putative NNRTI site in HIV-2 RT. The protein backbone is shown as blue ribbons and coils, and protein side chains are shown as ball-and-stick structures with carbon, oxygen, and nitrogen atoms represented as orange, red and blue, respectively. The thicker ball-and-stick structure with grey carbon atoms represents the PETT-2 molecule, marking the putative NNRTI site. The possible orientations of 101P and 112E are shown. (b) Position of residue G112 (green sphere) relative to the polymerase active site of HIV-1 RT. Blue ribbons and coils, protein backbones. The protein side chains and the substrate dTTP are shown as thinner and thicker ball-and-stick structures, respectively. The two purple spheres represent manganese ions, and the yellow and green ladder shows the bound oligonucleotide, with the template and primer strands labeled T and P, respectively.