Cyclosaligenyl-2′,3′-didehydro-2′,3′-dideoxythymidine Monophosphate: Efficient Intracellular Delivery of d4TMP
- Jan Balzarini1,
- Stefano Aquaro2,
- Tina Knispel3,
- Chiara Rampazzo4,
- Vera Bianchi4,
- Carlo-Federico Perno2,5,
- Erik De Clercq1 and
- Chris Meier3
- 1Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium (J.B., E.D.C.); 2Department of Experimental Medicine, University of Rome “Tor Vergata,” Rome, Italy (S.A., C.-F.P.); 3Institut für Organische Chemie, Universität Hamburg, Hamburg, Germany (T.K., C.M.); 4Department of Biology, University of Padova, Padova, Italy (C.R., V.B.); and 5Istituto di Ricovero e Cura a Carattere Scientifico Lazzaro Spallanzani, Rome, Italy (C.-F.P.)
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 ∼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 Rp and Spdiastereomers on high-performance liquid chromatography. TheRp diastereomer of cycloSal-d4TMP was 3- to 7-fold more efficient in releasing d4TMP and generating d4TTP than theSp cycloSal-d4TMP diastereomer. This correlated well with the 5-fold more pronounced antiviral activity of the Rp diastereomer versus theSp diastereomer. d4TMP is a poor substrate for the cytosolic 5′(3′)-deoxyribonucleotidase (Vmax/Km for d4TMP: 0.08 ofVmax/Km for dTMP) and is only slowly hydrolyzed to d4T. This contributes to the efficient conversion of the prodrug of d4TTP.
Footnotes
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Send reprint requests to: Jan Balzarini, Rega Institute for Medical Research, Minderbroedersstraat 10, B-3000 Leuven, Belgium. E-mail:jan.balzarini{at}rega.kuleuven.ac.be
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This work was supported by the Biomedical Research Program of the European Commission, the Belgian Geconcerteerde Onderzoeksacties (project 00/12), and the Fonds voor Wetenschappelijk Onderzoek—Vlaanderen (project G.0104.98). Work in V.B.'s laboratory was supported by the Instituto Superiore di Sanitá, AIDS Project, and by the Italian Association for Cancer Research.
- Abbreviations:
- HIV
- human immunodeficiency virus
- d4T
- 2′,3′-didehydro-2′,3′-dideoxythymidine
- TK
- thymidine kinase
- d4TMP
- 2′,3′-didehydro-2′,3′-dideoxythymidine-5′-monophosphate
- cycloSal-d4TMP
- cyclosaligenyl-2′,3′-didehydro-2′,3′-dideoxythymidine-5′-monophosphate
- PBL
- peripheral blood lymphocyte
- HPLC
- high-performance liquid chromatography
- AZTMP
- 3′-azido-2′,3′-dideoxythymidine 5′-monophosphate
- d4TTP
- 2′,3′-didehydro-2′,3′-dideoxythymidine-5′-triphosphate
- ddAMP
- 2′,3′-dideoxyadenosine-5′-monophosphate
- d4AMP
- 2′,3′-didehydro-2′,3′-dideoxyadenosine-5′-monophosphate
- FddAMP
- 2′-fluoro-ddAMP
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- Received February 28, 2000.
- Accepted July 6, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
