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Antimicrobial Agents and Chemotherapy, June 2008, p. 2035-2042, Vol. 52, No. 6
0066-4804/08/$08.00+0     doi:10.1128/AAC.00083-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Mechanism of Inhibition of Human Immunodeficiency Virus Type 1 Reverse Transcriptase by a Stavudine Analogue, 4'-Ethynyl Stavudine Triphosphate

Department of Pharmacology, School of Medicine, Yale University, New Haven, Connecticut 06520,¹ Department of Molecular Biophysics and Biochemistry, School of Medicine, Yale University, New Haven, Connecticut 06520,² School of Pharmaceutical Sciences, Showa University, Tokyo 142-8555, Japan,³ Division of Antiviral Chemotherapy, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8520, Japan⁴

Received 21 January 2008/ Returned for modification 3 March 2008/ Accepted 28 March 2008

2',3'-Didehydro-3'-deoxy-4'-ethynylthymidine (4'-Ed4T), a recently discovered nucleoside reverse transcriptase (RT) inhibitor, exhibits 5- to 10-fold-higher activity against human immunodeficiency virus type 1 (HIV-1) and less cytotoxicity than does its parental compound d4T (stavudine). Using steady-state kinetic approaches, we have previously shown that (i) 4'-ethynyl-d4T triphosphate (4'-Ed4TTP) inhibits HIV-1 RT more efficiently than d4TTP does and (ii) its inhibition efficiency toward the RT M184V mutant is threefold less than that toward wild-type (wt) RT. In this study we used pre-steady-state kinetic approaches in an attempt to understand its mechanism of inhibition. With wt and the M184V mutant RTs, 4'-Ed4TTP has three- to fivefold-lower K_d (dissociation constant) values than d4TTP, while d4TTP has up to eightfold-higher K_d values than dTTP. Inhibition is more effective in DNA replication with RNA template than with DNA template. In general, the M184V mutant exhibits poorer binding for all three nucleoside triphosphates than does wt RT. The structural basis for the lower binding affinity of d4TTP than of dTTP could be the lack of hydrogen bonds from the missing 3'-hydroxyl group in d4TTP to the backbone amide of Y115 and also to the side chain of Q151. The structural basis for the higher binding affinity of 4'-Ed4TTP than of d4TTP could be the additional binding of the 4'-ethynyl group in a preformed hydrophobic pocket by A114, Y115, M184, F160, and part of D185.

Published ahead of print on 7 April 2008.