ON THE MECHANISM OF SELECTIVE-INHIBITION OF HERPESVIRUS REPLICATION BY (E)-5-(2-BROMOVINYL)-2'-DEOXYURIDINE
ON THE MECHANISM OF SELECTIVE-INHIBITION OF HERPESVIRUS REPLICATION BY (E)-5-(2-BROMOVINYL)-2'-DEOXYURIDINE
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DOI：
10.1073/pnas.78.5.2698
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发表时间：
1981-01-01
期刊：
影响因子：
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通讯作者：
DECLERCQ, E
中科院分区：
文献类型：
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作者：
ALLAUDEEN, HS;KOZARICH, JW;DECLERCQ, E
溴乙烯脱氧尿苷（BVdUrd）是一种有效的抗疱疹病毒化合物，具有低细胞毒性。为了深入了解其选择性和抑制机制，化学合成了BVdUrd的5“-三磷酸，BVdUTP，并测试了其对2种疱疹病毒的DNA聚合酶[DNA核苷酸转移酶（DNA定向的），EC 2.7.7.7]活性的影响，单纯疱疹病毒1型（HSV-1）和EB病毒（EBV），以及细胞DNA聚合酶α，β。和γ。在对单个聚合酶最佳的测定条件下测定对DNA聚合酶的影响。与细胞DNA聚合酶相比，BVdUTP对HSV-1 DNA聚合酶利用dTTP的抑制作用更强。例如，低至1 μ M的BVdUTP抑制HSV-1 DNA聚合酶对dTTP的利用50%;相同浓度抑制DNA聚合酶α。和DNA聚合酶β。活动分别只有9和3%。BVdUTP通过与天然底物dTTP竞争抑制DNA合成。HSV-1 DNA聚合酶的dTTP的Km和BVdUTP的Ki分别为0.66和0.25 μ M。用DNA聚合酶α进行动力学分析。和βEBVDNA聚合酶也反映了HSV-1酶和其他酶之间敏感性的类似差异。增加反应混合物中DNA模板或酶的浓度不会引起抑制程度的显著变化。预孵育的抑制剂与酶是没有必要的抑制。对抑制时间进程的研究表明，该化合物即使在DNA合成开始后也具有抑制作用。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选择性抑制感染细胞中的病毒DNA复制。
Bromovinyldeoxyuridine (BVdUrd) is a potent antiherpesvirus compound with low cytotoxicity. To gain an insight into its selectivity and mechanism of inhibition, the 5''-triphosphate of BVdUrd, BVdUTP was chemically synthesized and its effect was tested on the activites of DNA polymerases [DNA nucleotidyltransferase (DNA directed), EC 2.7.7.7] of 2 herpesviruses, i.e., herpes simplex virus type 1 (HSV-1) and Epstein-Barr virus (EBV), and cellular DNA polymerases .alpha., .beta. and .gamma.. The effects on the DNA polymerases were determined under assay conditions optimal for the individual polymerases. The BVdUTP was considerably more inhibitory to the utilization of dTTP by the HSV-1 DNA polymerase than by the cellular DNA polymerases. For instance, as little as 1 .mu.M BVdUTP inhibited the utilization of dTTP by HSV-1 DNA polymerase 50%; 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 .mu.M, 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 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.