Huang L, Ishii K K, Zuccola H, Gehring A M, Hwang C B, Hogle J, Coen D M
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Proc Natl Acad Sci U S A. 1999 Jan 19;96(2):447-52. doi: 10.1073/pnas.96.2.447.
Acyclovir (ACV), like many antiviral drugs, is a nucleoside analog. In vitro, ACV triphosphate inhibits herpesvirus DNA polymerase by means of binding, incorporation into primer/template, and dead-end complex formation in the presence of the next deoxynucleoside triphosphate. However, it is not known whether this mechanism operates in vivo. To address this and other questions, we analyzed eight mutant polymerases encoded by drug-resistant viruses, each altered in a region conserved among alpha-like DNA polymerases. We measured Km and kcat values for dGTP and ACV triphosphate incorporation and Ki values of ACV triphosphate for dGTP incorporation for each mutant. Certain mutants showed increased Km values for ACV triphosphate incorporation, suggesting a defect in inhibitor binding. Other mutants showed reduced kcat values for ACV triphosphate incorporation, suggesting a defect in incorporation of inhibitor into DNA, while the rest of the mutants exhibited both altered km and kcat values. In most cases, the fold increase in Ki of ACV triphosphate for dGTP incorporation relative to wild-type polymerase was similar to fold resistance conferred by the mutation in vivo; however, one mutation conferred a much greater increase in resistance than in Ki. The effects of mutations on enzyme kinetics could be explained by using a model of an alpha-like DNA polymerase active site bound to primer/template and inhibitor. The results have implications for mechanisms of action and resistance of antiviral nucleoside analogs in vivo, in particular for the importance of incorporation into DNA and for the functional roles of conserved regions of polymerases.
阿昔洛韦（ACV）与许多抗病毒药物一样，是一种核苷类似物。在体外，阿昔洛韦三磷酸酯通过结合、掺入引物/模板以及在存在下一个脱氧核苷三磷酸时形成终止复合物来抑制疱疹病毒DNA聚合酶。然而，尚不清楚这种机制在体内是否起作用。为了解决这个问题及其他问题，我们分析了由耐药病毒编码的8种突变聚合酶，每种在α样DNA聚合酶保守区域发生了改变。我们测量了每种突变体掺入dGTP和阿昔洛韦三磷酸酯的Km和kcat值以及阿昔洛韦三磷酸酯对掺入dGTP的Ki值。某些突变体显示阿昔洛韦三磷酸酯掺入的Km值增加，表明抑制剂结合存在缺陷。其他突变体显示阿昔洛韦三磷酸酯掺入的kcat值降低，表明抑制剂掺入DNA存在缺陷，而其余突变体的Km和kcat值均发生改变。在大多数情况下，相对于野生型聚合酶，阿昔洛韦三磷酸酯对掺入dGTP的Ki增加倍数与体内突变赋予的耐药倍数相似；然而，一种突变赋予的耐药增加幅度远大于Ki增加幅度。突变对酶动力学的影响可以通过使用与引物/模板和抑制剂结合的α样DNA聚合酶活性位点模型来解释。这些结果对抗病毒核苷类似物在体内的作用机制和耐药性具有启示意义，特别是对于掺入DNA的重要性以及聚合酶保守区域的功能作用。
