Mechanistic studies comparing the incorporation of (+) and (-) isomers of 3TCTP by HIV-1 reverse transcriptase

Biochemistry. 1999 Jan 5;38(1):55-63. doi: 10.1021/bi982340r.


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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Anti-HIV Agents / chemistry*
  • Binding Sites
  • DNA Primers / chemistry
  • DNA, Viral / chemistry
  • Deoxycytidine Monophosphate / chemistry
  • Deoxycytosine Nucleotides / chemistry*
  • Deoxyribonucleosides / chemistry
  • Dideoxynucleotides
  • HIV Reverse Transcriptase / chemistry*
  • Hydrolysis
  • Kinetics
  • Lamivudine / chemistry*
  • Models, Molecular
  • RNA, Viral / chemistry
  • Ribonuclease H / chemistry
  • Stereoisomerism
  • Templates, Genetic


  • Anti-HIV Agents
  • DNA Primers
  • DNA, Viral
  • Deoxycytosine Nucleotides
  • Deoxyribonucleosides
  • Dideoxynucleotides
  • RNA, Viral
  • Deoxycytidine Monophosphate
  • 2',3'-dideoxycytidine monophosphate
  • 2'-deoxycytidine 5'-triphosphate
  • Lamivudine
  • 2',3'-dideoxycytidine 5'-triphosphate
  • HIV Reverse Transcriptase
  • Ribonuclease H