title: Mechanism of action of (-)-(2R,4R)-1-(2-hydroxymethyl-1,3-dioxolan-4-yl) thymine as an anti-HIV agent.
abstract: (-)-(2R,4R)-1-(2-Hydroxymethyl-1,3-dioxolan-4yl)thymine (DOT) is a thymidine analogue that has potent in vitro activity against wild-type and nucleoside reverse transcriptase inhibitor (NRTI)-resistant HIV. For nucleoside analogues to inhibit viral replication, they must be metabolized to the active triphosphate, which inhibits the viral reverse transcriptase (RT). Using purified enzymes, the kinetics of DOT phosphorylation, inhibition of wild-type and drug-resistant HIV-1 reverse transcriptase activity, and excision of DOT-5'-monophosphate (DOT-MP) from a chain-terminated primer were examined. DOT was phosphorylated by human thymidine kinase-1 (TK-1) but not by other pyrimidine nucleoside kinases, including the mitochondrial thymidine kinase (TK-2). Resistance to NRTIs involves decreased binding/incorporation and/or increased excision of the chain-terminating NRTI. RTs containing the D67N/K70R/T215Y/K219Q or T695-SS/T215Y mutations show enhanced removal of DOT-MP from terminated primer as well as approximately four-fold decreased binding/incorporation. The Q151M and K65R mutations appear to cause decreased inhibition by DOT-TP. However, both the K65R and Q151M mutations show decreased excision, which would confer greater stability on the terminated primer. These opposing mechanisms could offset the overall resistance profile and susceptibility. Little or no resistance was observed with the enzymes harbouring mutations resistant to lamivudine (M184V) and non-nucleoside RT inhibitors (K103N).
authors.affiliation: Pharmasset Inc., Princeton, NJ, USA. eisuke.murakami@pharmasset.com
authors.forename: Eisuke
authors.initials: E
authors.lastname: Murakami
authors.forename: Haiying
authors.initials: H
authors.lastname: Bao
authors.forename: Aravind
authors.initials: A
authors.lastname: Basavapathruni
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Bailey
authors.forename: Jinfa
authors.initials: J
authors.lastname: Du
authors.forename: Holly M Micolochick
authors.initials: HM
authors.lastname: Steuer
authors.forename: Congrong
authors.initials: C
authors.lastname: Niu
authors.forename: Tony
authors.initials: T
authors.lastname: Whitaker
authors.forename: Karen S
authors.initials: KS
authors.lastname: Anderson
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Otto
authors.forename: Phillip A
authors.initials: PA
authors.lastname: Furman
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D019380:Anti-HIV Agents / Q000494:pharmacology*; D024882:Drug Resistance, Viral; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D013941:Thymine / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: C521073:1-(2-hydroxymethyl-1,3-dioxolan-4-yl)thymine; D019380:Anti-HIV Agents; D018894:Reverse Transcriptase Inhibitors; D000255:Adenosine Triphosphate; D054303:HIV Reverse Transcriptase; D013941:Thymine