Template-competitive inhibitors of HIV-1 reverse transcriptase: design, synthesis and inhibitory activity Original Research Article

We report the design, synthesis and activity studies on a novel class of template-competitive reverse transcriptase inhibitors (TCRTIs). The TCRTIs are 1,N6-etheno analogues of a series of dATP-based template-competitive DNA polymerase inhibitors synthesized in our laboratory (Moore, B. M.; Jalluri, R.; Doughty, M.B. Biochemistry 1996, 35, 11634). Thus, nucleotides 2-(4-azidophenacyl)thio-1,N6-etheno-2′-deoxyadenosine 5′-triphosphate 1, the tetrafluoro analogue 2-(4-azido-2,3,5,6-tetrafluorophenacyl)thio-1,N6-etheno-2′-deoxyadenosine 5′-triphosphate 2 and its analogues were synthesized by alkylation of 2-thio-1,N6-etheno-2′-deoxyadenosine 5′-monophosphate with the corresponding chloro- or bromo-alkyl halides and converted to the triphosphate. Kinetically, nucleotides 1 and 2 are both competitive inhibitors of reverse transcriptase versus template/primer with Kiʹs of 8.0 and 7.4 μM, respectively, and non-competitive inhibitors versus TTP with Kiʹs of 15 and 10 μM, respectively. Nucleotide 3, which differs from 1 only in that it lacks the etheno group, non-complementary nucleotide triphosphates, and related monophosphates and nucleosides, are completely inactive as inhibitors of reverse transcriptase at concentrations up to 1 mM. Photoinactivation of RT by 1 was both time- and concentration-dependent, and protected by template/primer but not by dNTPs. The concentration-dependent inactivation data gave a KD,app of 17.2 μM and maximum inactivation of 90%, and radiolabeled [β,γ-32P]-1 photoincorporated specifically and covalently into the p66 subunit of RT. Thus the photoinactivation data support our main conclusion from the kinetic data that this class of RT inhibitors are non-substrate and template-competitive.