Template-competitive HIV-1 reverse transcriptase photoprobes and inhibitors: Characterization of the inhibitor binding site via photoaffinity labeling

HIV-1 reverse transcriptase (RT) plays a significant role in the replication of HIV-1. Therefore HIV-1 RT has been an attractive target for AIDS treatment. A series of template-competitive RT inhibitors and photoprobes have successfully been designed and synthesized. These novel adenosine triphosphate derivatives exhibited inhibitory activity against RT at low micromolar concentrations. Inhibition kinetics studies demonstrate that tetrafluoroazido nucleotide 1 is competitive to the template/primer and non-competitive to TTP, suggesting that nucleotide 1 and its analogues have a unique binding site on RT. Nucleotide 1 was further chosen as a photoprobe to label RT due to its excellent photochemical properties. The photo inactivation and photoincorporation results suggest that these processes are light, time and concentration dependent and protected by the template/primer. The labeling site of photoprobe 1 was identified as 77F, which is located in the base of the fingers subdomain. The conformation of photoprobe 1 was studied by molecular mechanic calculations and NMR spectroscopy. Finally, a model of RT bound to photoprobe 1 was proposed. The binding model indicates that the side chain of photoprobe 1 extends into a lipophilic template pocket as designed, and that the nucleoside triphosphate moiety of the photoprobe binds in the dNTP-binding site of RT. This research should have important implications for the design of a novel class of RT for the treatment of AIDS.