Activity against Human Immunodeficiency Virus Type 1, Intracellular Metabolism, and Effects on Human DNA Polymerases of 4′-Ethynyl-2-Fluoro-2′-Deoxyadenosine

Nakata, Hirotomo; Amano, Masayuki; Koh, Yasuhiro; Kodama, Eiichi; Yang, Guangwei; Bailey, Christopher M.; Kohgo, Satoru; Hayakawa, Hisao; Matsuoka, Masao; Anderson, Karen S.; Cheng, Yung Chi; Mitsuya, Hiroaki

doi:10.1128/aac.00277-07

Cited by 97 publications

(113 citation statements)

References 26 publications

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“…We propose this is due to improved binding and catalytic incorporation into the viral DNA (Fig. 4B); also, the 3′-OH of EFdA contributes to its rapid and facile activation by the deoxycytidine kinase (6,18). Finally, regarding 2-F, it contributes to improved inhibitor binding at the polymerase active site (Fig.…”

Section: Resultsmentioning

confidence: 94%

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

Salie

Kirby

Michailidis

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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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

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Section: Resultsmentioning

confidence: 94%

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

Salie

Kirby

Michailidis

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…The lack of a 3Ј-OH on current clinically used NRTIs can reduce recognition by cellular nucleoside/nucleotide kinases that have evolved to interact with the 3Ј-OH present in their natural nucleoside substrates (9). Preliminary studies suggest that EFdA appears to undergo rapid and facile intracellular conversion to the active antiviral EFdA-TP (36), showing that the 4Ј-ethynyl group does not interfere with recognition by cellular nucleoside/nucleotide kinases. Furthermore, the presence of fluorine at position 2 of the adenine base of EFdA helps to stabilize intracellular levels of EFdA and its phosphorylated products by hindering adenosine deaminasecatalyzed degradation of the molecule (13).…”

Section: Discussionmentioning

confidence: 99%

Mechanism of Inhibition of HIV-1 Reverse Transcriptase by 4′-Ethynyl-2-fluoro-2′-deoxyadenosine Triphosphate, a Translocation-defective Reverse Transcriptase Inhibitor

Michailidis

Marchand

Kodama

et al. 2009

Journal of Biological Chemistry

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Nucleoside reverse transcriptase inhibitors (NRTIs) are employed in first line therapies for the treatment of human immunodeficiency virus (HIV) infection. They generally lack a 3-hydroxyl group, and thus when incorporated into the nascent DNA they prevent further elongation. In this report we show that 4-ethynyl-2-fluoro-2-deoxyadenosine (EFdA), a nucleoside analog that retains a 3-hydroxyl moiety, inhibited HIV-1 replication in activated peripheral blood mononuclear cells with an EC 50 of 0.05 nM, a potency several orders of magnitude better than any of the current clinically used NRTIs. This exceptional antiviral activity stems in part from a mechanism of action that is different from approved NRTIs. Reverse transcriptase (RT) can use EFdA-5-triphosphate (EFdA-TP) as a substrate more efficiently than the natural substrate, dATP. Importantly, despite the presence of a 3-hydroxyl, the incorporated EFdA monophosphate (EFdA-MP) acted mainly as a de facto terminator of further RT-catalyzed DNA synthesis because of the difficulty of RT translocation on the nucleic acid primer possessing 3-terminal EFdA-MP. EFdA-TP is thus a translocation-defective RT inhibitor (TDRTI). This diminished translocation kept the primer 3-terminal EFdA-MP ideally located to undergo phosphorolytic excision. However, net phosphorolysis was not substantially increased, because of the apparently facile reincorporation of the newly excised EFdA-TP. Our molecular modeling studies suggest that the 4-ethynyl fits into a hydrophobic pocket defined by RT residues Ala-114, Tyr-115, Phe-160, and Met-184 and the aliphatic chain of Asp-185. These interactions, which contribute to both enhanced RT utilization of EFdA-TP and difficulty in the translocation of 3-terminal EFdA-MP primers, underlie the mechanism of action of this potent antiviral nucleoside.Nucleoside reverse transcriptase inhibitors (NRTIs) 4 are central components of first line regimens for treatment of HIV infections (1-6). Currently, there are eight clinically approved NRTIs: AZT, 3TC, FTC, ABC, ddI, ddC, d4T, and the nucleotide tenofovir (TFV; reviewed in Refs. 7 and 8). A structural hallmark of these NRTIs is the lack of a 3Ј-OH; it has long been considered that the absence of the 3Ј-OH is essential for antiviral activity. However, the absence of the 3Ј-OH in NRTIs also imparts detrimental properties to the inhibitor, including reduced affinity for RT compared with the analogous dNTP substrate, as well as reduced intracellular conversion to the active nucleoside triphosphate (9).Previously we described a series of 4Ј-substituted NRTIs (10) that retain the 3Ј-OH group and have excellent antiviral properties and significantly improved selectivity indices (CC 50 / EC 50 ) compared with the approved NRTIs. Furthermore, these NRTIs efficiently suppress various NRTI-resistant HIV. The most potent of these 4Ј-substituted NRTIs are the adenosine analogs that have an ethynyl group at the 4Ј position of the ribose ring. Despite their high anti-HIV activity, 4Ј-substituted compounds are susce...

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“…EFdA and ECldA showed very high activity against all HIVs and acceptable SIs, however, the activity of ECldA is lower than that of EFdA. These results indicated that the 3'-OH played important roles not only for the phosphorylation of 5'-OH, but also for the activity against drug-resistant HIVs [34].…”

Section: Intravenous Administrationmentioning

confidence: 88%

A New Paradigm for Developing Antiviral Drugs Exemplified by the Development of Supremely High Anti-HIV Active EFdA

Ohrui¹

2014

J Antivir Antiretrovir

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Activity against Human Immunodeficiency Virus Type 1, Intracellular Metabolism, and Effects on Human DNA Polymerases of 4′-Ethynyl-2-Fluoro-2′-Deoxyadenosine

Cited by 97 publications

References 26 publications

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

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

Mechanism of Inhibition of HIV-1 Reverse Transcriptase by 4′-Ethynyl-2-fluoro-2′-deoxyadenosine Triphosphate, a Translocation-defective Reverse Transcriptase Inhibitor

A New Paradigm for Developing Antiviral Drugs Exemplified by the Development of Supremely High Anti-HIV Active EFdA

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