Mechanism-based suppression of dideoxynucleotide resistance by K65R human immunodeficiency virus reverse transcriptase using an alpha-boranophosphate nucleoside analogue.
The amino acid change K65R in human immunodeficiency virus type 1-reverse transcriptase (RT) confers viral resistance to various 2',3'-dideoxynucleoside drugs in vivo. Using pre-steady state kinetic methods, we found that K65R-reverse transcriptase is 3.2-14-fold resistant to 2',3'-dideoxynucleotides in vitro relative to wild-type reverse transcriptase, in agreement with resistance levels observed in vivo. A decreased catalytic rate constant k(pol) mostly accounts for the lower incorporation efficiency observed for 2',3'-dideoxynucleotides. Examination of the crystal structure of the RT.DNA.dNTP complex suggested that both the charge at position 65 and the 3'-OH of the incoming nucleotide act in synergy during the creation of the phosphodiester bond, resulting in a more pronounced decreased catalytic rate constant for 2',3'-dideoxynucleotides than for dNTPs. This type of intramolecular activation of the leaving phosphate by the 3'-OH group appears to be conserved in several nucleotide phosphotransferases. These data were used to design dideoxynucleotide analogues targeting K65R RT specifically. alpha-Boranophosphate ddATP was found to be a 2-fold better substrate than dATP and inhibited DNA synthesis by K65R RT 153-fold better than ddATP. This complete suppression of drug resistance at the nucleotide level could serve for other reverse transcriptases for which drug resistance is achieved at the catalytic step.
Selmi
B
B
Centre National de la Recherche Scientifique and Universités d'Aix-Marseille I et II, UMR 6098, Architecture et Fonction des Macromolécules Biologiques, ESIL-Case 925, 163 avenue de Luminy, 13288 Marseille cedex 9, France.
Boretto
J
J
Sarfati
S R
SR
Guerreiro
C
C
Canard
B
B
D019943:Amino Acid Substitution; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D015224:Dideoxynucleosides / Q000494:pharmacology*; D004352:Drug Resistance, Microbial / Q000235:genetics*; D054303:HIV Reverse Transcriptase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000187:drug effects* / Q000201:enzymology; D007700:Kinetics; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*
D017931:DNA Primers; D015224:Dideoxynucleosides; D018894:Reverse Transcriptase Inhibitors; D054303:HIV Reverse Transcriptase