Allaudeen H S, Kozarich J W, Bertino J R, De Clercq E
Proc Natl Acad Sci U S A. 1981 May;78(5):2698-702. doi: 10.1073/pnas.78.5.2698.
Bromovinyldeoxyuridine (BVdUrd) is a potent antiherpesvirus compound with low cytotoxicity. To gain an insight into its selectivity and mechanism of inhibition, we chemically synthesized the 5'-triphosphate of BVdUrd, BVdUTP, and tested its effect on the activities of DNA polymerases [DNA nucleotidyltransferase (DNA directed), EC 2.7.7.7] of two herpesviruses--i.e., herpes simplex virus type 1 (HSV-1) and Epstein-Barr virus (EBV)--as well as cellular DNA polymerases alpha, beta, and gamma. The effects on the DNA polymerases were determined under assay conditions optimal for the individual polymerases. We found that the BVdUTP was considerably more inhibityory to the utilization of dTTP by the HSV-1 DNA polymerase then by the cellular DNA polymerases. For instance, as little as 1 microM BVdUTP inhibited the utilization of dTTP by HSV-1 DNA polymerase 50%, whereas the same concentration inhibited the DNA polymerase alpha and the DNA polymerase beta activities only 9% and 3%, respectively. The BVdUTP inhibited DNA synthesis by competing with the natural substrate, dTTP. The Km for dTTP and the Ki for the BVdUTP of the HSV-1 DNA polymerase were 0.66 and 0.25 microM, respectively. Kinetic analyses with the DNA polymerases alpha and beta and the EBV DNA polymerase also reflected a similar difference in sensitivity between the HSV-1 enzyme and other enzymes. Increasing the concentration of either the DNA template or the enzyme in the reaction mixture did not bring about a significant change in the extent of inhibition. Preincubation of the inhibitor with the enzyme was not necessary for inhibition. Studies on time course of inhibition revealed that the compound is inhibitory even after the initiation of DNA synthesis. These studies indicate that the ability of BVdUTP to preferentially inhibit the HSV-1 DNA polymerase may contribute towards its selective inhibition of the viral DNA replication in infected cells.
溴乙烯基脱氧尿苷（BVdUrd）是一种具有低细胞毒性的强效抗疱疹病毒化合物。为深入了解其选择性和抑制机制，我们化学合成了BVdUrd的5'-三磷酸酯BVdUTP，并测试了其对两种疱疹病毒（即单纯疱疹病毒1型（HSV-1）和爱泼斯坦-巴尔病毒（EBV））以及细胞DNA聚合酶α、β和γ的DNA聚合酶[DNA核苷酸转移酶（DNA定向），EC 2.7.7.7]活性的影响。在适合各个聚合酶的测定条件下确定对DNA聚合酶的影响。我们发现，BVdUTP对HSV-1 DNA聚合酶利用dTTP的抑制作用比对细胞DNA聚合酶的抑制作用要强得多。例如，低至1 microM的BVdUTP可抑制HSV-1 DNA聚合酶对dTTP的利用达50%，而相同浓度仅分别抑制DNA聚合酶α和DNA聚合酶β活性9%和3%。BVdUTP通过与天然底物dTTP竞争来抑制DNA合成。HSV-1 DNA聚合酶对dTTP的Km和对BVdUTP的Ki分别为0.66和0.25 microM。对DNA聚合酶α和β以及EBV DNA聚合酶的动力学分析也反映出HSV-1酶与其他酶在敏感性上存在类似差异。增加反应混合物中DNA模板或酶的浓度不会使抑制程度发生显著变化。抑制剂与酶预孵育并非抑制所必需。对抑制时间进程的研究表明，即使在DNA合成开始后该化合物仍具有抑制作用。这些研究表明，BVdUTP优先抑制HSV-1 DNA聚合酶的能力可能有助于其对感染细胞中病毒DNA复制的选择性抑制。
