The High Genetic Barrier of EFdA/MK-8591 Stems from Strong Interactions with the Active Site of Drug-Resistant HIV-1 Reverse Transcriptase

Abstract

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA/MK-8591), a nucleoside reverse transcriptase inhibitor (NRTI) under clinical trials, is a potent and promising long-acting anti-HIV type 1 (HIV-1) agent. EFdA and its derivatives possess a modified 4'-moiety and potently inhibit the replication of a wide spectrum of HIV-1 strains resistant to existing NRTIs. Here, we report that EFdA and NRTIs with a 4'-ethynyl- or 4'-cyano-moiety exerted activity against HIV-1 with an M184V mutation and multiple NRTI-resistant HIV-1s, whereas NRTIs with other moieties (e.g., 4'-methyl) did not show this activity. Structural analysis indicated that EFdA and 4'-ethynyl-NRTIs (but not other 4'-modified NRTIs), formed strong van der Waals interactions with critical amino acid residues of reverse transcriptase. Such interactions were maintained even in the presence of a broad resistance-endowing M184V substitution, thus potently inhibiting drug-resistant HIV-1 strains. These findings also explain the mechanism for the potency of EFdA and provide insights for further design of anti-HIV-1 therapeutics.

Structural features in common of HBV and HIV-1 resistance against chirally-distinct nucleoside analogues entecavir and lamivudine

“…However, ISL's potency that we observed against HIV Q151M and HIV 3MB is not quite consistent with ISL's moderate activity against HBV (IC 50 = 160 nM) 30 . Previous structural studies of HIV-1 RT WT in complex with ISL-triphosphate showed that a 4′-ethynyl of ISL is inserted into the hydrophobic hollow at the N-site, and Phe160 at the bottom of the hollow is likely a key amino acid to accommodate the 4′-ethynyl 31 . Because HIV 3MB/F160M/M184V remains susceptible to ISL, we suspect that Met160 does not interfere with the binding of 4′-ethynyl.…”

Long-Acting Anti-HIV Drugs Targeting HIV-1 Reverse Transcriptase and Integrase

“…EFdA has a high genetic barrier to resistance in culture, which has been attributed to the strong and generally conserved interactions between EFdA and the HIV-1 RT polymerase active site [23,24]. In vitro drug susceptibility assays showed that EFdA had significantly greater efficacy compared to other NRTIs [23]. Moreover, EFdA inhibits both WT and RTI-resistant viruses in a subtype-independent manner [25].…”

Novel Protease Inhibitors Containing C-5-Modifiedbis-Tetrahydrofuranylurethane and Aminobenzothiazole as P2 and P2′ Ligands That Exert Potent Antiviral Activity against Highly Multidrug-Resistant HIV-1 with a High Genetic Barrier against the Emergence of Drug Resistance

“…Viral replication assays. Viral replication assays were performed using recombinant HIV-1 mutants harboring single amino acid substitutions, as previously described (59,66). In brief, recombinant HIV-1 NL4-3 -based infectious clones were generated using the QuikChange II XL site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA) and transfected into HEK293T cells by using Attractene transfection reagent (Qiagen).…”

“…The viruses were harvested at 72 h after the transfection, and viral p24 antigen was measured using a Lumipulse G1200 instrument. Replication of HIV-1 mutants was determined as described previously (59,66), with minor modifications. Briefly, MT-4 cells (density, 1.5 ϫ 10 6 cells in 20 ml RPMI medium) were exposed to the HIV-1 mutants with 20 ng/ml of the p24 antigen and were cultured without the antiretroviral agents for 10 days.…”