Feng J Y, Anderson K S
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520-8066, USA.
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.
在临床上用作抗HIV药物、靶向HIV-1逆转录酶（RT）的核苷抑制剂中，（-）-2'，3'-二脱氧-3'-硫代胞苷[（-）SddC或3TC]是唯一具有非天然L（-）核苷构型的类似物。已证明3TC比具有天然核苷构型的D（+）异构体（+）SddC更有效且毒性更低。尽管许多因素可能明显起作用，包括摄取、代谢活化、降解和转运的差异，但异构SddC化合物的立体化学选择性和差异毒性的机制基础尚未完全理解。我们使用预稳态动力学分析来确定（-）和（+）异构SddCTP化合物以及相应的双脱氧和天然核苷三磷酸使用HIV-1逆转录酶掺入引物-模板复合物中的最大掺入速率kpol、核苷酸结合亲和力Kd和掺入效率kpol/Kd。与DNA/DNA相比，DNA/RNA引物-模板的dNTP亲和力（Kd）更紧密，并且酶掺入引物-模板复合物的效率（kpol/Kd）更高。最大掺入速率kpol遵循dCTP>ddCTP>（+）SddCTP>（-）SddCTP的趋势，而针对DNA/RNA引物-模板确定的Kd值遵循（-）SddCTP≡（+）SddCTP≡ddCTP>dCTP的顺序。相应的掺入效率遵循dCTP>ddCTP>（+）SddCTP>（-）SddCTP的趋势。这些数据表明，胞苷类似物核糖环上的扰动（C→S）会降低掺入速率和效率，但会增强结合亲和力。在RT、DNA/RNA引物-模板和SddCTP类似物的三元复合物的计算机模拟结构的背景下讨论了这些结果，以及对构效关系和进一步药物设计的影响。该信息为理解3TC对HIV-1逆转录酶的抑制提供了机制基础。
