Pre-steady state kinetic analysis of cyclobutyl derivatives of 2′-deoxyadenosine 5′-triphosphate as inhibitors of HIV-1 reverse transcriptase

ABSTRACT Recent studies have identified a role for mutations in the connection and RNase H domains of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) resistance to nucleoside analog RT inhibitors (NRTI). To provide insight into the biochemical mechanism(s) involved, we investigated the effect of the G333D mutation in the connection domain of RT on resistance to zidovudine (AZT) and lamivudine (3TC) in enzymes that contain both M184V and thymidine analog mutations (TAMs; M41L, L210W, and T215Y). Our results from steady-state kinetic, pre-steady-state kinetic, and thermodynamic analyses indicate that G333D facilitates dual resistance to AZT and 3TC in two ways. First, in combination with M184V, G333D increased the ability of HIV-1 RT to effectively discriminate between the normal substrate dCTP and 3TC-triphosphate. Second, G333D enhanced the ability of RT containing TAMs and M184V to bind template/primer terminated by AZT-monophosphate (AZT-MP), thereby restoring ATP-mediated excision of AZT-MP under steady-state assay conditions. This study is the first to elucidate a molecular mechanism whereby a mutation in the connection domain of RT can affect NRTI susceptibility at the enzyme level.

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Pre-Steady-State Kinetic Characterization of RNA-Primed Initiation of Transcription by HIV-1 Reverse Transcriptase and Analysis of the Transition to a Processive DNA-Primed Polymerization Mode†

Biochemistry ◽

10.1021/bi981102t ◽

1998 ◽

Vol 37 (38) ◽

pp. 13349-13358 ◽

Cited By ~ 31

Author(s):

Ruth Krebs ◽

Birgitta M. Wöhrl ◽

Luciano Cellai ◽

Roger S. Goody ◽

Tobias Restle

Keyword(s):

Steady State ◽

Reverse Transcriptase ◽

Kinetic Characterization ◽

Steady State Kinetic ◽

Initiation Of Transcription ◽

State Kinetic ◽

Hiv 1

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00083-08 ◽

2008 ◽

Vol 52 (6) ◽

pp. 2035-2042 ◽

Cited By ~ 7

Author(s):

Guangwei Yang ◽

Jimin Wang ◽

Yao Cheng ◽

Ginger E. Dutschman ◽

Hiromichi Tanaka ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Steady State ◽

Reverse Transcriptase ◽

Structural Basis ◽

Mechanism Of Inhibition ◽

Steady State Kinetic ◽

Immunodeficiency Virus ◽

State Kinetic ◽

Hiv 1

ABSTRACT 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 Kd (dissociation constant) values than d4TTP, while d4TTP has up to eightfold-higher Kd 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.

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