Mechanism of inhibition of human immunodeficiency virus type 1 reverse transcriptase by a stavudine analogue, 4'-ethynyl stavudine triphosphate.
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.
Yang
Guangwei
G
Department of Pharmacology, School of Medicine, Yale University, 333 Cedar Street, New Haven, CT 06520, USA.
Wang
Jimin
J
Cheng
Yao
Y
Dutschman
Ginger E
GE
Tanaka
Hiromichi
H
Baba
Masanori
M
Cheng
Yung-Chi
YC
D019380:Anti-HIV Agents / Q000737:chemistry / Q000494:pharmacology*; D001483:Base Sequence; D003460:Crystallization; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D015497:HIV-1 / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011122:Polyphosphates; D018894:Reverse Transcriptase Inhibitors / Q000737:chemistry / Q000494:pharmacology; D018119:Stavudine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship
C505960:4'-ethynylstavudine; D019380:Anti-HIV Agents; D011122:Polyphosphates; D018894:Reverse Transcriptase Inhibitors; D018119:Stavudine; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; C005692:triphosphoric acid