title: Relationship between antiviral activity and host toxicity: comparison of the incorporation efficiencies of 2',3'-dideoxy-5-fluoro-3'-thiacytidine-triphosphate analogs by human immunodeficiency virus type 1 reverse transcriptase and human mitochondrial DNA polymerase.
abstract: Emtricitabine [(-)FTC; (-)-beta-L-2'-3'-dideoxy-5-fluoro-3'-thiacytidine] is an oxathiolane nucleoside analog recently approved by the Food and Drug Administration for the treatment of human immunodeficiency virus (HIV). Structurally, (-)FTC closely resembles lamivudine [(-)3TC] except that the former is 5-fluorinated on the cytosine ring. In HIV-1 reverse transcriptase (RT) enzymatic assays, the triphosphate of (-)FTC [(-)FTC-TP] was incorporated into both DNA-DNA and DNA-RNA primer-templates nearly 3- and 10-fold more efficiently than (-)3TC-TP. Animal studies and clinical trial studies have demonstrated a favorable safety profile for (-)FTC. However, a detailed study of the incorporation of (-)FTC-TP by human mitochondrial DNA polymerase gamma, a host enzyme associated with nucleoside toxicity, is required for complete understanding of the molecular mechanisms of inhibition and toxicity. We studied the incorporation of (-)FTC-TP and its enantiomer (+)FTC-TP into a DNA-DNA primer-template by recombinant human mitochondrial DNA polymerase in a pre-steady-state kinetic analysis. (-)FTC-TP was incorporated 2.9 x 10(5)-, 1.1 x 10(5)-, 1.6 x 10(3)-, 7.9 x 10(3)-, and 100-fold less efficiently than dCTP, ddCTP, (+)3TC-TP, (+)FTC-TP, and (-)3TC-TP, respectively. The rate of removal of (-)FTC-MP from the corresponding chain-terminated 24-mer DNA by polymerase gamma's 3'-->5' exonuclease activity was equal to the removal of (+)FTC-MP, 2-fold slower than the removal of (-)3TC-MP and (+)3TC-MP, and 4.6-fold slower than the excision of dCMP. These results demonstrate that there are clear differences between HIV-1 RT and polymerase gamma in terms of preferences for substrate structure.
authors.affiliation: Gilead Sciences, Durham, North Carolina 27707, USA.
authors.forename: Joy Y
authors.initials: JY
authors.lastname: Feng
authors.forename: Eisuke
authors.initials: E
authors.lastname: Murakami
authors.forename: Suzana M
authors.initials: SM
authors.lastname: Zorca
authors.forename: Allison A
authors.initials: AA
authors.lastname: Johnson
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Johnson
authors.forename: Raymond F
authors.initials: RF
authors.lastname: Schinazi
authors.forename: Phillip A
authors.initials: PA
authors.lastname: Furman
authors.forename: Karen S
authors.initials: KS
authors.lastname: Anderson
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology* / Q000633:toxicity*; D004247:DNA / Q000378:metabolism; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003845:Deoxycytosine Nucleotides / Q000494:pharmacology* / Q000633:toxicity*; D054306:Dideoxynucleotides; D054303:HIV Reverse Transcriptase / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology* / Q000633:toxicity*; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: C485382:2',3'-dideoxy-5-fluoro-3'-thiacytidine triphosphate; D000998:Antiviral Agents; D003845:Deoxycytosine Nucleotides; D054306:Dideoxynucleotides; D007527:Isoenzymes; D018894:Reverse Transcriptase Inhibitors; D004247:DNA; D054303:HIV Reverse Transcriptase; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase