title: Cyclosaligenyl-2',3'-didehydro-2',3'-dideoxythymidine monophosphate: efficient intracellular delivery of d4TMP.
abstract: Cyclosaligenyl-2',3'-didehydro-2', 3'-dideoxythymidine-5'-monophosphate (cycloSal-d4TMP) is a potent and selective inhibitor of human immunodeficiency virus replication in cell culture and differs from other nucleotide prodrug approaches in that it is designed to selectively deliver the nucleotide 5'-monophosphate by a controlled, chemically induced hydrolysis. Its antiviral efficacy in cell culture is at least as good as, if not superior to, that of d4T. CycloSal-d4TMP was found to lead to the efficient intracellular release of d4TMP in a variety of cell lines, including both wild-type CEM and thymidine kinase-deficient CEM/TK(-) cells. Under similar experimental conditions, exposure of CEM/TK(-) cells to d4T failed to result in significant d4TTP levels. The intracellular conversion of cycloSal-d4TMP proved to be both time and dose dependent. The half-life of d4TTP generated intracellularly from d4T- or cycloSal-d4TMP-treated CEM cells was approximately 3.5 h, and the intracellular ratios of d4TTP/d4TMP in cells exposed to cycloSal-d4TMP gradually increased from 1 to 3.4 upon prolonged incubation. Radiolabeled cycloSal-d4TMP could be separated as its two R(p) and S(p) diastereomers on high-performance liquid chromatography. The R(p) diastereomer of cycloSal-d4TMP was 3- to 7-fold more efficient in releasing d4TMP and generating d4TTP than the S(p) cycloSal-d4TMP diastereomer. This correlated well with the 5-fold more pronounced antiviral activity of the R(p) diastereomer versus the S(p) diastereomer. d4TMP is a poor substrate for the cytosolic 5'(3')-deoxyribonucleotidase (V(max)/K(m) for d4TMP: 0.08 of V(max)/K(m) for dTMP) and is only slowly hydrolyzed to d4T. This contributes to the efficient conversion of the prodrug of d4TTP.
authors.affiliation: Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium. jan.balzarini@rega.kuleuven.ac.be
authors.forename: J
authors.initials: J
authors.lastname: Balzarini
authors.forename: S
authors.initials: S
authors.lastname: Aquaro
authors.forename: T
authors.initials: T
authors.lastname: Knispel
authors.forename: C
authors.initials: C
authors.lastname: Rampazzo
authors.forename: V
authors.initials: V
authors.lastname: Bianchi
authors.forename: C F
authors.initials: CF
authors.lastname: Perno
authors.forename: E
authors.initials: E
authors.lastname: De Clercq
authors.forename: C
authors.initials: C
authors.lastname: Meier
mesh_terms: D015720:5'-Nucleotidase / Q000378:metabolism; D019380:Anti-HIV Agents / Q000378:metabolism* / Q000494:pharmacology; D003470:Culture Media; D054306:Dideoxynucleotides; D006207:Half-Life; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D009000:Monocytes / Q000378:metabolism; D018119:Stavudine / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D013936:Thymidine / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D013937:Thymidine Kinase / Q000378:metabolism; D013938:Thymidine Monophosphate / Q000378:metabolism; D013942:Thymine Nucleotides / Q000378:metabolism; D013997:Time Factors; D014316:Tritium; D014407:Tumor Cells, Cultured; D015215:Zidovudine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: C113124:2',3'-dideoxy-2',3'-didehydrothymidine monophosphate; D019380:Anti-HIV Agents; D003470:Culture Media; C415254:Cyclosaligenyl-2',3'-didehydro-2',3'-dideoxythymidine monophosphate; D054306:Dideoxynucleotides; D013942:Thymine Nucleotides; D014316:Tritium; C057287:3'-azido-3'-deoxythymidine 5'phosphate; D013938:Thymidine Monophosphate; D015215:Zidovudine; D018119:Stavudine; D013937:Thymidine Kinase; C063766:5'(3')-nucleotidase; D015720:5'-Nucleotidase; D013936:Thymidine