IN VITRO EVALUATION AND BIOCHEMISTRY

该项目的总体目标有两个：(I)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)研究有希望的化合物如何在 细胞和生化水平。我们将评估抗病毒活性 和细胞毒性的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类)。 L.B.汤森在研究项目I中所述的评估将是 体外抗人巨细胞病毒(HCMV)PLUS活性 其他疱疹病毒和对未感染细胞的细胞毒性。新的 对这两种病毒都有活性且含量低的化合物 细胞毒性加上已发现的有前景的化合物应进行评估 更广泛地包括阐明它们的生化模式的研究 行动。将对研究项目I中准备的所有新化合物进行评估 在一系列初步的体外测试中，他们抑制了 空斑减少法或酶联免疫吸附试验检测人巨细胞病毒复制。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)耐药巨细胞病毒。所有化合物也将在 使用视觉评分的体外细胞毒性，对细胞生长的影响， 以及对标记前体摄取的影响。那些显示出强效的化合物 包括病毒在内的抗病毒活性和低毒作用将进一步测试 减产试验、未感染细胞生长率和细胞培养 效率。那些具有强烈和选择性的抗肿瘤活性的化合物 HCMV将被送往Burroughs Wellcome的合作者进行评估 人细小祖细胞的体外生长及对我们核心的影响 体内抗病毒评估实验室。 新发现和存在的有希望的抗病毒作用模式 抗病毒化合物也将被研究。因为我们已经知道 两个系列活性核苷类似物通过不同的机制发挥作用 因为我们将研究新的非核苷杂环，不同的 将根据化合物的性质使用不同的方法。 方法将包括但不限于以下内容：(I) 活性化合物体外代谢的测定。(Ii) 确定活性化合物是否是病毒DNA的选择性抑制剂 综合。(3)病毒DNA致病机制的特征 合成受到抑制。研究将包括三磷酸盐的影响。 活性核苷类似物对人巨细胞病毒DNA聚合酶和哺乳动物的影响 DNA聚合酶。(4)调查化合物的影响，例如 通过DNA合成以外的机制起作用的苯并咪唑 抑制力。这类研究将包括分离耐药突变株， 病毒颗粒、药物代谢的电子显微镜研究 耐药病毒、选定的生化特征和标记拯救 学习。我们还将研究选定的细胞毒性的机制 抗病毒化合物包括DNA流式细胞术，标记前体摄取， 和酶底物的特异性研究。

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.