IN VITRO EVALUATION AND BIOCHEMISTRY

该项目的总体目标有两个方面：（一）通过 体外评价有潜力成为有用的新化合物 抗病毒药物和（ii）研究有前途的化合物如何在 细胞和生化水平。 我们将评估抗病毒活性 新系列核苷类似物和相关杂环化合物 （苯并咪唑氨基甲酸酯和咪唑）在Dr. L.B.汤森，如研究项目I中所述。 评价将 在体外进行抗人巨细胞病毒（HCMV）的活性， 其他疱疹病毒和未感染细胞中的细胞毒性。 新 这些化合物对任一种病毒都有活性， 应评价细胞毒性和已发现的有前景的化合物 更广泛地包括研究，以阐明他们的生化模式， 行动上 将对研究项目I中制备的所有新化合物进行评价 在一系列体外试验中， 在空斑减少或ELISA测定中检测HCMV的复制。 许多化合物 也将针对其他人类疱疹病毒进行测试，包括 更昔洛韦（DHPG）耐药HCMV。 所有化合物也将在 利用视觉评分进行细胞毒性体外试验，对细胞生长的影响， 以及对标记前体摄取的影响。 这些化合物显示出 抗病毒活性和低毒性将进一步测试，包括病毒 产量降低测定、未感染细胞生长速率和细胞铺板 效率 这些化合物具有有效的和选择性的活性， HCMV将被送到我们在Burroughs Wellcome的合作者进行评估 在体外对人窄祖细胞的生长和我们的核心 体内抗病毒评价实验室。 新发现的和现有的有希望的抗病毒作用方式 还将研究抗病毒化合物。 因为我们已经知道 两个系列的活性核苷类似物通过不同的机制起作用 因为我们将研究新的非核苷杂环， 将根据化合物的性质使用这些方法。 方法将包括但不限于以下方面： 测定活性化合物的体外代谢。 （二） 确定活性化合物是否为病毒DNA的选择性抑制剂 合成. (iii)病毒DNA在细胞中的作用机制的表征 合成被抑制。 研究将包括三磷酸盐的影响 HCMV DNA聚合酶活性核苷类似物和哺乳动物 DNA聚合酶。 (iv)研究化合物的影响，如 苯并咪唑类化合物通过DNA合成以外的机制发挥作用， 抑制作用 这些研究将包括分离耐药突变体， 病毒颗粒的电子显微镜研究， 抗性病毒、选定的生化表征和标记拯救 问题研究 我们还将研究细胞毒性的机制， 抗病毒化合物，包括DNA流式细胞术，标记的前体摄取， 和激酶底物特异性研究。

The overall objective of this Project is two-fold: (i) to identify by in vitro evaluation new compounds which have the potential to become useful antiviral drugs and (ii) to investigate how promising compounds act at the cellular and biochemical level. We shall evaluate for antiviral activity and cytotoxicity new series of nucleoside analogs and related heterocyclics (benzimidazole carbamates and imidazoles) prepared in the laboratory of Dr. L.B. Townsend as described in Research Project I. Evaluations will be performed in vitro for activity against human cytomegalovirus (HCMV) plus other herpesviruses and for cytotoxicity in uninfected cells. New compounds which are active against either of the viruses and low in cytotoxicity plus promising compounds already discovered shall be evaluated more extensively including studies to elucidate their biochemical mode of action. All new compounds prepared in Research Project I will be evaluated in a series of primary in vitro tests for their capacity to inhibit the replication of HCMV in plaque reduction or ELISA assays. Many compounds also will be tested against other human herpesviruses--including ganciclovir (DHPG)-resistant HCMV. All compounds also will be tested in vitro for cytotoxicity utilizing visual scoring, effects on cell growth, and effects on labeled precursor uptake. Those compounds which show potent antiviral activity and low toxicity will be tested further including virus yield reduction assays, uninfected cell growth rates, and cell plating efficiency. Those compounds with potent and selective activity against HCMV will be sent to our collaborators at Burroughs Wellcome for evaluation in vitro on the growth of human narrow progenitor cells and to our Core Laboratory for in vivo antiviral evaluation. The mode of antiviral action of newly discovered and existing promising antiviral compounds also will be investigated. Because we already know that two series of active nucleoside analogs act by different mechanisms and because we shall be studying new non-nucleoside heterocycles, different approaches will be used depending on the nature of the compound. Approaches will include but not be limited to the following: (i) determination of the in vitro metabolism of active compounds. (ii) Determination if active compounds are selective inhibitors of viral DNA synthesis. (iii) Characterization of the mechanism by which viral DNA synthesis is inhibited. Studies would include the effects of triphosphates of the active nucleoside analogs on HCMV DNA polymerase and the mammalian DNA polymerases. (iv) Investigation of the effects of compounds such as the benzimidazoles which act by mechanisms other than DNA synthesis inhibition. Such studies will include isolation of drug-resistant mutants, electron microscopic studies of viral particles, drug metabolism in resistant viruses, selected biochemical characterization, and marker rescue studies. We also shall investigate mechanisms of cytotoxicity in selected antiviral compounds including DNA flow cytometry, labeled precursor uptake, and kinase substrate specificity studies.