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

• Published in: The Journal of biological chemistry Vol. 284; no. 51; pp. 35681 - 35691
• Main Authors: Michailidis, Eleftherios, Marchand, Bruno, Kodama, Eiichi N., Singh, Kamlendra, Matsuoka, Masao, Kirby, Karen A., Ryan, Emily M., Sawani, Ali M., Nagy, Eva, Ashida, Noriyuki, Mitsuya, Hiroaki, Parniak, Michael A., Sarafianos, Stefan G.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 18.12.2009; American Society for Biochemistry and Molecular Biology
• Subjects: Antiviral activity; Deoxyadenine Nucleotides - chemistry; Deoxyadenine Nucleotides - pharmacology; DNA biosynthesis; DNA, Viral - biosynthesis; DNA: , Repair, Recombination, and Chromosome Dynamics; Elongation; HIV Infections - drug therapy; HIV Infections - enzymology; HIV Reverse Transcriptase - antagonists & inhibitors; HIV Reverse Transcriptase - chemistry; HIV Reverse Transcriptase - metabolism; HIV-1 - enzymology; Human immunodeficiency virus 1; Humans; Hydrophobic and Hydrophilic Interactions; Hydrophobicity; Infection; Leukocytes, Mononuclear - virology; Models, Molecular; Molecular modelling; nucleic acids; nucleoside analogs; nucleoside reverse transcriptase inhibitors; Peripheral blood mononuclear cells; Primers; Protein Structure, Secondary; Replication; Reverse Transcriptase Inhibitors - chemistry; Reverse Transcriptase Inhibitors - pharmacology; Reverse Transcription - drug effects; RNA-directed DNA polymerase; Translocation; Virus Replication - drug effects
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M109.036616

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 EC50 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.