Dataset Information

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.

SUBMITTER: Takamatsu Y

PROVIDER: S-EPMC6261781 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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

Takamatsu Yuki Y Das Debananda D Kohgo Satoru S Hayashi Hironori H Delino Nicole S NS Sarafianos Stefan G SG Mitsuya Hiroaki H Maeda Kenji K

Cell chemical biology 20180830 10

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- ...[more]

PMID: 30174310

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Project description:We tested three compounds for their ability to inhibit the RNase H (RH) and polymerase activities of HIV-1 reverse transcriptase (RT). A high-resolution crystal structure (2.2 Å) of one of the compounds showed that it chelates the two magnesium ions at the RH active site; this prevents the RH active site from interacting with, and cleaving, the RNA strand of an RNA-DNA heteroduplex. The compounds were tested using a variety of substrates: all three compounds inhibited the polymerase-independent RH activity of HIV-1 RT. Time-of-addition experiments showed that the compounds were more potent if they were bound to RT before the nucleic acid substrate was added. The compounds significantly inhibited the site-specific cleavage required to generate the polypurine tract (PPT) RNA primer that initiates the second strand of viral DNA synthesis. The compounds also reduced the polymerase activity of RT; this ability was a result of the compounds binding to the RH active site. These compounds appear to be relatively specific; they do not inhibit either Escherichia coli RNase HI or human RNase H2. The compounds inhibit the replication of an HIV-1-based vector in a one-round assay, and their potencies were only modestly decreased by mutations that confer resistance to integrase strand transfer inhibitors (INSTIs), nucleoside analogs, or nonnucleoside RT inhibitors (NNRTIs), suggesting that their ability to block HIV replication is related to their ability to block RH cleavage. These compounds appear to be useful leads that can be used to develop more potent and specific compounds.IMPORTANCE Despite advances in HIV-1 treatment, drug resistance is still a problem. Of the four enzymatic activities found in HIV-1 proteins (protease, RT polymerase, RT RNase H, and integrase), only RNase H has no approved therapeutics directed against it. This new target could be used to design and develop new classes of inhibitors that would suppress the replication of the drug-resistant variants that have been selected by the current therapeutics.

Project description:The steric flexibility or rigidity of polymerase active sites may play an important role in their fidelity of nucleic acid synthesis. In this regard, reverse transcriptases offer an unusual opportunity to compare two enzymatic activities that proceed in the same active site. For HIV-1 reverse transcriptase, reverse transcription (RNA-templated synthesis) is known to proceed with lower fidelity than DNA-templated synthesis. Here, we describe the use of a set of variably sized nonpolar thymidine and uracil mimics as molecular rulers to probe the active site steric constraints of HIV-1 RT, and for the first time, we directly compare the functional flexibility of these two activities. Steady-state kinetics of incorporation for natural dNTPs opposite unnatural template bases as well as for unnatural dNTPs opposite natural template bases are reported for the DNA-templated DNA synthesis, and comparison is made with recent data for the RNA-templated activity. Kinetics for extension beyond a base pair containing the analogue template bases are also reported both for RNA and DNA templates. Our results show that the DNA-dependent polymerization by HIV-RT is highly sensitive to size, strongly biasing against both too-small and too-large base pairs, while, by contrast, the RNA-dependent polymerization is only biased against analogues that are too small, and is much more accepting of larger base pairs. In addition, base pair extension with HIV-RT is found to be relatively insensitive to varied base pair size, consistent with its high mutagenicity. Overall, the data show greater rigidity with a DNA template as compared with an RNA template, which correlates directly with the higher fidelity of the DNA-templated synthesis. Possible structural explanations for these differences are discussed. We also report kinetics data for two HIV-1 RT mutants reported to have altered fidelity (F61A and K65R) using DNA templates containing nonpolar base analogues, and find that one of these (F61A) is a high-fidelity enzyme that appears to be sensitive to a loss of hydrogen-bonding groups.

Dataset Information

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

Publications

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

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