Antiretroviral potency of 4'-ethnyl-2'-fluoro-2'-deoxyadenosine, tenofovir alafenamide and second-generation NNRTIs across diverse HIV-1 subtypes

4'-Ethnyl-2'-fluoro-2'-deoxyadenosine (EFdA) is a novel translocation-defective reverse transcriptase inhibitor. We investigated the virological and biochemical inhibitory potentials of EFdA against a broad spectrum of subtype-specific chimeric viruses and compared it with tenofovir alafenamide, nevirapine, efavirenz, rilpivirine and etravirine. pNL4.3 chimeric viruses encoding gag-pol from treatment-naive patients (n = 24) and therapy-failure patients (n = 3) and a panel of reverse transcriptase inhibitor-resistant strains (n = 7) were used to compare the potency of reverse transcriptase inhibitor drugs. The phenotypic drug susceptibility assay was performed using TZM-bl cells. In vitro inhibition assays were done using patient-derived reverse transcriptase. IC50 values of NNRTIs were calculated using a PicoGreen-based spectrophotometric assay. Steady-state kinetics were used to determine the apparent binding affinity (Km.dNTP) of triphosphate form of EFdA (EFdA-TP) and dATP. Among the chimeric treatment-naive viruses, EFdA had an ex vivo antiretroviral activity [median (IQR) EC50 = 1.4 nM (0.6-2.1 nM)] comparable to that of tenofovir alafenamide [1.6 nM (0.5-3.6 nM)]. Subtype-specific differences were found for etravirine (P = 0.004) and rilpivirine (P = 0.017), where HIV-1C had the highest EC50 values. EFdA had a greater comparative efficiency [calculated by dividing the efficiency of monophosphate form of EFdA (EFdA-MP) incorporation (kcat.EFdA-TP/Km.EFdA-TP) over the efficiency of dATP incorporation (kcat.dATP/Km.dATP)] compared with the natural substrate dATP, with a fold change of between 1.6 and 3.2. Ex vivo analysis on reverse transcriptase inhibitor-resistant strains showed EFdA to have a higher potency. Despite the presence of rilpivirine DRMs, some non-B strains showed hypersusceptibility to rilpivirine. Our combined virological and biochemical data suggest that EFdA inhibits both WT and reverse transcriptase inhibitor-resistant viruses efficiently in a subtype-independent manner. In contrast, HIV-1C is least susceptible to etravirine and rilpivirine.