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Antimicrob. Agents Chemother. doi:10.1128/AAC.01432-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Comparison of Phosphorylation of 4'-Ethynyl 2',3'-dihydro-3'-deoxythymidine with Other Anti-HIV Thymidine Analogs

Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520, USA; School of Pharmaceutical Sciences, Showa University, Tokyo 142-8555, Japan; Center for Chronic Viral Diseases, Division of Antiviral Chemotherapy, Faculty of Medicine, Kagoshima University, Kagoshima 890-8520, Japan; Department of Oncology, National Taiwan University Hospital, Taipei, Taiwan

^* To whom correspondence should be addressed. Email: yccheng{at}yale.edu.

	Abstract

Thymidine analogs, including 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxy-3'-deoxythymidine (D4T), are important anti-retroviral agents. To exert anti-retroviral activity, these analogs undergo a stepwise phosphorylation intracellularly to the active triphosphate metabolites. We previously reported that 4'-substituted D4T with an ethynyl group (i.e., 4'-ethynyl D4T) increased the anti-HIV activity and was active against multi-drug resistant HIV strains. 4'-Ethynyl D4T is a better substrate for phosphorylation by human thymidine kinase 1 than D4T. In this report, we first studied the enzymes involved in the phosphorylation of 4'-ethynyl D4T from monophosphate (MP) to triphosphate (TP) metabolites. The 4'-ethynyl D4TMP is phosphorylated by recombinant human TMP kinase with a Km 19 ± 4 µM and a kcat 0.007 ± 0.001 sec^-1; the relative efficiency is about 9% and 15% of that of D4TMP and AZTMP, respectively. Several enzymes from crude cellular extracts, including nucleoside diphosphate kinase, pyruvate kinase, creatine kinase and 3-phosphoglycerate kinase, could phosphorylate 4'-ethynyl D4T-diphosphate (DP). The relative phosphorylation efficiencies of 4'-ethynyl D4TDP were 3 25% of those of D4TDP, and were generally similar to those of AZTDP. In T-lymphoid cells lines, there was preponderant accumulation of 4'-ethynyl D4TMP, suggesting that TMP kinase could be the rate-limiting enzyme in the metabolism of 4'-ethynyl D4T. Although the same enzymes are involved in the stepwise phosphorylation of thymidine analogs, their behaviors in phosphorylating metabolites of 4'-ethynylD4T are different from those of D4T and AZT. Qualitatively, the metabolism of 4'-ethynyl D4T is more similar to that of AZT than to its progenitor, D4T.

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