title: 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.affiliation: Christopher Bond Life Sciences Center, Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, Missouri 65211, USA.
authors.forename: Eleftherios
authors.initials: E
authors.lastname: Michailidis
authors.forename: Bruno
authors.initials: B
authors.lastname: Marchand
authors.forename: Eiichi N
authors.initials: EN
authors.lastname: Kodama
authors.forename: Kamlendra
authors.initials: K
authors.lastname: Singh
authors.forename: Masao
authors.initials: M
authors.lastname: Matsuoka
authors.forename: Karen A
authors.initials: KA
authors.lastname: Kirby
authors.forename: Emily M
authors.initials: EM
authors.lastname: Ryan
authors.forename: Ali M
authors.initials: AM
authors.lastname: Sawani
authors.forename: Eva
authors.initials: E
authors.lastname: Nagy
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Ashida
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Mitsuya
authors.forename: Michael A
authors.initials: MA
authors.lastname: Parniak
authors.forename: Stefan G
authors.initials: SG
authors.lastname: Sarafianos
mesh_terms: D004279:DNA, Viral / Q000096:biosynthesis*; D003838:Deoxyadenine Nucleotides / Q000737:chemistry / Q000494:pharmacology*; D015658:HIV Infections / Q000188:drug therapy / Q000201:enzymology; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007963:Leukocytes, Mononuclear / Q000821:virology; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D018894:Reverse Transcriptase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D048348:Reverse Transcription / Q000187:drug effects; D014779:Virus Replication / Q000187:drug effects*
chemical_list: C546077:4'-ethynyl-2-fluoro-2'-deoxyadenosine triphosphate; D004279:DNA, Viral; D003838:Deoxyadenine Nucleotides; D018894:Reverse Transcriptase Inhibitors; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase