A binding site for TSAO-m(3)T at the interface between the p66 and p51 subunits of HIV-1 reverse transcriptase (RT) and distinct from that of "classical" HIV-1 non-nucleoside inhibitors is proposed. The feasibility of the binding mode was assessed by carrying out nanosecond molecular dynamics simulations for the complexes of TSAO-m(3)T with reduced models of both the wild-type enzyme and a more sensitive R172A mutant. The molecular model is in agreement with a previous proposal, with known structure-activity and mutagenesis data for this unique class of inhibitors, and also with recent biochemical evidence indicating that TSAO analogues can affect enzyme dimerization. The relative importance of residues involved in dimer formation and TSAO-RT complex stabilization was assessed by a combination of surface area accessibility, molecular mechanics, and continuum electrostatics calculations. A structure-based modification introduced into the lead compound yielded a new derivative with improved antiviral activity.
The highly conserved Tyr-318 is part of the non-nucleoside reverse transcriptase inhibitor (NNRTI)-specific lipophilic pocket of human immunodeficiency virus type I reverse transcriptase (RT) and makes contact within 4 Å with the NNRTIs in all reported RT/NNRTI complexes. Using site-directed mutagenesis, six mutant RTs were constructed bearing the mutations Y318H, Y318K, Y318L, Y318C, Y318W, and Y318F. We found that only the Y318W and Y318F mutant RTs retained substantial RT activity, whereas the catalytic activities of the Y318K, Y318C, Y318H, and Y318L RT mutants were less than 5% of the wild-type activity. The Y318F mutant RT retained substantial sensitivity to the majority of NNRTIs tested, whereas the Y318W mutant RT showed varying degrees of resistance to NNRTIs. Subunit-specific site-directed mutagenesis revealed that there was no difference in the catalytic activity or resistance/sensitivity spectrum toward NNRTIs regardless of whether the Tyr-318 mutation was introduced in both subunits or only in the p66 subunit of RT. Recombinant viruses harboring the Y318F or Y318W mutation in the RT showed a similar resistance/sensitivity pattern to NNRTIs as their corresponding 318 mutant recombinant RTs. Our findings stress a functional or structural role for Tyr-318 in wild-type RT and argue for the design of novel NNRTIs that interact more closely with this amino acid in the NNRTI-specific pocket of human immunodeficiency virus type I RT.
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