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Proc Natl Acad Sci U S A. 2016 Aug 16;113(33):9274-9. doi: 10.1073/pnas.1605223113. Epub 2016 Aug 3.

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

Author information

1
C. S. Bond Life Sciences Center, Department of Biochemistry, University of Missouri, Columbia, MO 65211;
2
C. S. Bond Life Sciences Center, Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, MO 65211;
3
Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, PA 15261; Magee-Womens Research Institute, University of Pittsburgh, Pittsburgh, PA 15213;
4
Division of Infectious Diseases, Tohoku University and Tohoku Medical Megabank Organization, Sendai 980-8575, Japan;
5
Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; Department of Infectious Diseases, Kumamoto University Graduate School of Medical Sciences, Kumamoto 860-8556, Japan; Department of Hematology, Kumamoto University Graduate School of Medical Sciences, Kumamoto 860-8556, Japan; Research Institute, National Center for Global Health and Medicine, Tokyo 162-8655, Japan; Center for Clinical Sciences, National Center for Global Health and Medicine, Tokyo 162-8655, Japan;
6
Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219.
7
C. S. Bond Life Sciences Center, Department of Biochemistry, University of Missouri, Columbia, MO 65211; C. S. Bond Life Sciences Center, Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, MO 65211; sarafianoss@missouri.edu.

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/DNAEFdA-MP(P)• dT-MP(N) ), or (iii) after incorporation of two EFdA-MPs (RT/DNAEFdA-MP(P)• EFdA-MP(N) ); (iv) the latter was also solved with EFdA-MP mismatched at the N site (RT/DNAEFdA-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.

KEYWORDS:

EFdA; HIV-1 reverse transcriptase; NRTIs; X-ray crystallography; inhibitors

PMID:
27489345
PMCID:
PMC4995989
DOI:
10.1073/pnas.1605223113
[Indexed for MEDLINE]
Free PMC Article

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