Hypersusceptibility mechanism of Tenofovir-resistant HIV to EFdA

Abstract

BackgroundThe K65R substitution in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is the major resistance mutation selected in patients treated with first-line antiretroviral tenofovir disoproxil fumarate (TDF). 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), is the most potent nucleoside analog RT inhibitor (NRTI) that unlike all approved NRTIs retains a 3'-hydroxyl group and has remarkable potency against wild-type (WT) and drug-resistant HIVs. EFdA acts primarily as a chain terminator by blocking translocation following its incorporation into the nascent DNA chain. EFdA is in preclinical development and its effect on clinically relevant drug resistant HIV strains is critically important for the design of optimal regimens prior to initiation of clinical trials.ResultsHere we report that the K65R RT mutation causes hypersusceptibility to EFdA. Specifically, in single replication cycle experiments we found that EFdA blocks WT HIV ten times more efficiently than TDF. Under the same conditions K65R HIV was inhibited over 70 times more efficiently by EFdA than TDF. We determined the molecular mechanism of this hypersensitivity using enzymatic studies with WT and K65R RT. This substitution causes minor changes in the efficiency of EFdA incorporation with respect to the natural dATP substrate and also in the efficiency of RT translocation following incorporation of the inhibitor into the nascent DNA. However, a significant decrease in the excision efficiency of EFdA-MP from the 3’ primer terminus appears to be the primary cause of increased susceptibility to the inhibitor. Notably, the effects of the mutation are DNA-sequence dependent.ConclusionWe have elucidated the mechanism of K65R HIV hypersusceptibility to EFdA. Our findings highlight the potential of EFdA to improve combination strategies against TDF-resistant HIV-1 strains.

MK-8591 (4′-Ethynyl-2-Fluoro-2′-Deoxyadenosine) Exhibits Potent Activity against HIV-2 Isolates and Drug-Resistant HIV-2 Mutants in Culture

“…In these experiments, the mean EC 50 for HIV-1 NL4-3 was 2.0 Ϯ 0.6 nM. This result agrees with previously published measurements for HIV-1 in single-cycle assays (1.1 nM for HIV-1 BH10 in MAGI cells [14], 3.2 nM for HIV-1 NL101 in TZM-bl cells [15]). In contrast, within each assay run, the EC 50 for HIV-2 ROD9 was lower than the corresponding value for HIV-1 NL4-3 by a factor of 2.3 to 9.9 ( Fig.…”

“…In contrast, in both HIV-1 and HIV-2, the M184V replacement confers Ͼ1,000-fold resistance to FTC and 3TC (50, 51, 55). Overall, the changes in MK-8591 susceptibility that resulted from single amino acid changes K65R, Q151M, and M184V in HIV-2 ROD9 were similar to those reported for HIV-1 (13)(14)(15). We also observed moderate MK-8591 resistance (13-to 19-fold) for HIV-2 mutants that encode M184V in combination with one or more treatment-associated amino acid changes in RT, including those in the patient-derived clone 4.7a (Table 1).…”

“…In addition, the ability of MK-8591 to inhibit HIV-2 mutants that are resistant to other NRTIs is unknown. In HIV-1, the M184V replacement in RT confers low-level resistance to the drug (7.5-to 15-fold increase in EC 50 ) (13,14), whereas the tenofovir resistance-associated change K65R confers 3-to 5-fold hypersusceptibility to MK-8591 (14,15). However, these findings cannot be extended a priori to HIV-2, as important differences exist between the two HIV types with respect to NRTI resistance pathways and mechanisms (29)(30)(31).…”

“…1) is an investigational nucleoside analog that inhibits HIV-1 replication with 50% effective concentrations (EC 50 s) in the low nanomolar-to-picomolar range (12)(13)(14)(15)(16). Direct comparisons show that MK-8591 is 10-fold more potent than tenofovir disoproxil fumarate (TDF) (15), 10 to100 times more potent than zidovudine (AZT) (12,14), and 1,000 times more potent than emtricitabine (FTC) (16) against HIV-1 in culture. MK-8591 also exhibits favorable pharmacokinetic properties, including a long intracellular half-life compared to those of conventional, FDA-approved NRTIs (16)(17)(18).…”

“…Although MK-8591 has been shown to potently inhibit HIV-1 and SIV in culture (12)(13)(14)(15)(16)19) and in experimentally infected nonhuman primates and humanized mouse models (16,(19)(20)(21), efforts to evaluate the activity of the drug against HIV-2 are limited; a single report showed that a group B strain (HIV-2 EHO ) is sensitive to the drug in spreading infections of MT-4 cells (14). In addition, the ability of MK-8591 to inhibit HIV-2 mutants that are resistant to other NRTIs is unknown.…”