Mechanistic studies comparing the incorporation of (+) and (-) isomers of 3TCTP by HIV-1 reverse transcriptase.
Among the nucleoside inhibitors used clinically as anti-HIV drugs which target HIV-1 reverse transcriptase (RT), (-)-2', 3'-dideoxy-3'-thiacytidine [(-)SddC or 3TC] is the only analogue with the unnatural L(-) nucleoside configuration. 3TC has been shown to be more potent and less toxic than the D(+) isomer, (+)SddC, which has the natural nucleoside configuration. The mechanistic basis for the stereochemical selectivity and differential toxicity of the isomeric SddC compounds is not completely understood although a number of factors may clearly come into play including differences in uptake, metabolic activation, degradation, and transport. We used a pre-steady-state kinetic analysis to determine the maximum rate of incorporation, kpol, nucleotide-binding affinity, Kd, and efficiency of incorporation, kpol/Kd, for the (-) and (+) isomeric SddCTP compounds as well as the corresponding dideoxy and natural nucleoside triphosphates into a primer-template complex using HIV-1 reverse transcriptase. The affinity (Kd) of the dNTP was much tighter and the efficiency (kpol/Kd) of incorporation by enzyme into the primer-template complex was much higher for the DNA/RNA primer-template compared to DNA/DNA. The maximum rate of incorporation, kpol, followed the trend of dCTP > ddCTP > (+)SddCTP > (-)SddCTP while the Kd values determined for the DNA/RNA primer-template followed the order (-)SddCTP congruent with (+)SddCTP congruent with ddCTP > dCTP. The corresponding efficiency of incorporation followed the trend dCTP > ddCTP > (+)SddCTP > (-)SddCTP. These data suggest that perturbations on the ribose ring of cytidine analogues (C --> S) decrease the rate and efficiency of incorporation but enhance the binding affinity. These results are discussed in the context of a computer modeled structure of the ternary complexes of RT, DNA/RNA primer-template, and SddCTP analogues as well as implications for structure-activity relationships and further drug design. This information provides a mechanistic basis for understanding the inhibition of HIV-1 reverse transcriptase by 3TC.
Feng
J Y
JY
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520-8066, USA.
Anderson
K S
KS
D019380:Anti-HIV Agents / Q000737:chemistry*; D001665:Binding Sites; D017931:DNA Primers / Q000737:chemistry; D004279:DNA, Viral / Q000737:chemistry; D003843:Deoxycytidine Monophosphate / Q000737:chemistry; D003845:Deoxycytosine Nucleotides / Q000737:chemistry*; D003853:Deoxyribonucleosides / Q000737:chemistry; D054306:Dideoxynucleotides; D054303:HIV Reverse Transcriptase / Q000737:chemistry*; D006868:Hydrolysis; D007700:Kinetics; D019259:Lamivudine / Q000737:chemistry*; D008958:Models, Molecular; D012367:RNA, Viral / Q000737:chemistry; D016914:Ribonuclease H / Q000737:chemistry; D013237:Stereoisomerism; D013698:Templates, Genetic
D019380:Anti-HIV Agents; D017931:DNA Primers; D004279:DNA, Viral; D003845:Deoxycytosine Nucleotides; D003853:Deoxyribonucleosides; D054306:Dideoxynucleotides; D012367:RNA, Viral; D003843:Deoxycytidine Monophosphate; C101329:2',3'-dideoxycytidine monophosphate; C024107:2'-deoxycytidine 5'-triphosphate; D019259:Lamivudine; C037406:2',3'-dideoxycytidine 5'-triphosphate; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H