Structural basis of HIV inhibition by translocation-defective RT inhibitor 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA)

Abstract

4′-Ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) is the most potent nucleoside analog inhibitor of HIV reverse transcriptase (RT). It retains a 3′-OH yet acts as a chain-terminating agent by diminishing translocation from the pretranslocation nucleotide-binding site (N site) to the posttranslocation primer-binding site (P site). Also, facile misincorporation of EFdA-monophosphate (MP) results in difficult-to-extend mismatched primers. To understand the high potency and unusual inhibition mechanism of EFdA, we solved RT crystal structures (resolutions from 2.4 to 2.9 Å) that include inhibition intermediates (i) before inhibitor incorporation (catalytic complex, RT/DNA/EFdA-triphosphate), (ii) after incorporation of EFdA-MP followed by dT-MP (RT/DNA EFdA-MP P •dT-MP N), or (iii) after incorporation of two EFdA-MPs (RT/DNA EFdA-MP P •EFdA-MP N); (iv) the latter was also solved with EFdA-MP mismatched at the N site (RT/DNA EFdA-MP P •EFdA-MP *N). We report that the inhibition mechanism and potency of EFdA stem from interactions of its 4′-ethynyl at a previously unexploited conserved hydrophobic pocket in the polymerase active site. The high resolution of the catalytic complex structure revealed a network of ordered water molecules at the polymerase active site that stabilize enzyme interactions with nucleotide and DNA substrates. Finally, decreased translocation results from favorable interactions of primer-terminating EFdA-MP at the pretranslocation site and unfavorable posttranslocation interactions that lead to observed localized primer distortions.EFdA | HIV-1 reverse transcriptase | inhibitors | NRTIs | X-ray crystallography

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

“…When insertion of the analog was favored, the difference between MK-8591-TP and dATP was attributable to a lower K d (dissociation constant) for the former (26,27), suggesting that MK-8591-TP bound to RT with a higher affinity than dATP. This inference is supported by surface plasmon resonance data (27) and by crystallographic data from the recently solved structure of HIV-1 RT with MK-8591-TP as the incoming nucleotide (i.e., in the N site of the polymerase) (28). In this structure, the 4=-ethynyl moiety of the analog is embedded within a well-defined hydrophobic pocket in the polymerase active site.…”

“…To our knowledge, this remains the only report of an NRTI that, when tested against a diverse panel of HIV-1 and HIV-2 isolates, exhibits morepotent inhibition of HIV-2 replication. Molecular modeling suggests that the ethynyl group of BMS-986001-5=-triphosphate fits within a hydrophobic pocket in HIV-2 RT that corresponds to the aforementioned 4= pocket of the HIV-1 enzyme (28,32). In agreement with this model, other 4=-modified nucleosides with 2=-deoxy (14, 33) and 2=-deoxy-2=-␤-fluoro (34) sugar configurations also inhibit HIV-2 replication, with EC 50 s in the nanomolar range.…”

Structural Insights into HIV Reverse Transcriptase Mutations Q151M and Q151M Complex That Confer Multinucleoside Drug Resistance

“…5). The RT-bound ddATP and dATP have 3=-exo versus 3=-endo conformations for their respective sugar rings; however, the flexible sugar ring of a ddNTP also has a 3=-endo conformation, particularly in high-resolution RT/nucleic acid complexes in which both of the catalytic Mg 2ϩ ions are present at the polymerase active site (41,42). The dNTP-binding pocket would undergo a series of structural transitions in the process of nucleotide incorporation starting from the structurally observed dNTP-bound state.…”

Physiological Mg2+ Conditions Significantly Alter the Inhibition of HIV-1 and HIV-2 Reverse Transcriptases by Nucleoside and Non-Nucleoside Inhibitors in Vitro

“…41 Crystal structures containing RT, the primer–template hybrid, and EFdA mono- and triphosphates corroborate this finding as the ethynyl group is able to access a previously unexploited conserved hydrophobic pocket in the polymerase active site that may lead to stabilization of binding. 67 The major effect of the fluorine group was enhancing resistance to adenosine deaminase. 41 It is possible that these unique structural or chemical properties rather than the presence of a 3′-OH were responsible for EFdATP’s ability to associate more strongly with RT under low-Mg 2+ conditions.…”