Mechanism of inhibition of HIV-1 reverse transcriptase by 4'-Ethynyl-2-fluoro-2'-deoxyadenosine triphosphate, a translocation-defective reverse transcriptase inhibitor.

Abstract

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.

• Authors: Eleftherios Michailidis, Bruno Marchand, Eiichi N Kodama, Kamlendra Singh, Masao Matsuoka, Karen A Kirby, Emily M Ryan, Ali M Sawani, Eva Nagy, Noriyuki Ashida, Hiroaki Mitsuya, Michael A Parniak, Stefan G Sarafianos
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 284 Issue 51 Pg. 35681-91 (Dec 18 2009) ISSN: 1083-351X [Electronic] United States
• PMID: 19837673 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)

Chemical References

• 4'-ethynyl-2-fluoro-2'-deoxyadenosine triphosphate
• DNA, Viral
• Deoxyadenine Nucleotides
• Reverse Transcriptase Inhibitors
• reverse transcriptase, Human immunodeficiency virus 1
• HIV Reverse Transcriptase

Topics

• DNA, Viral (biosynthesis)
• Deoxyadenine Nucleotides (chemistry, pharmacology)
• HIV Infections (drug therapy, enzymology)
• HIV Reverse Transcriptase (antagonists & inhibitors, chemistry, metabolism)
• HIV-1 (enzymology)
• Humans
• Hydrophobic and Hydrophilic Interactions
• Leukocytes, Mononuclear (virology)
• Models, Molecular
• Protein Structure, Secondary
• Reverse Transcriptase Inhibitors (chemistry, pharmacology)
• Reverse Transcription (drug effects)
• Virus Replication (drug effects)