HCMV EVALUATION, BIOCHEMISTRY, AND VIRAL GENETICS

研究项目3的总体目标有两个方面：(i)通过体外评估确定有可能成为有用抗病毒药物的新化合物；（ii）研究有希望的化合物在细胞、分子和生化水平上的作用，以发现和/或深入了解新的抗病毒靶点。我们将评估在Townsend博士实验室（研究项目I）制备的一系列新的核苷类似物（苯并咪唑和吲哚）和相关杂环化合物的抗病毒活性和细胞毒性，以及在Zemlicka博士实验室（研究项目2）制备的一系列非常不同的核苷类似物（亚甲基环丙烷类似物和相关前药）。所有化合物将进行抗人类巨细胞病毒（HCMV）和单纯疱疹病毒I型（HSV-I）活性测试。最有希望的化合物也将针对临床分离株和对更昔洛韦、苯并咪唑核苷或亚甲基环丙烷类似物耐药的HCMV菌株进行测试。将在科学核心实验室对其他疱疹病毒进行更广泛的评估。我们应该广泛研究具有活性和低细胞毒性的新化合物，以及我们实验室中似乎通过独特机制起作用的现有化合物，以确定它们的作用模式，并确定它们是否影响新的抗病毒靶点。具有最佳总体概况的化合物将被发送到我们的科学核心进行体内研究。上述发现的化合物的抗病毒作用模式，以及我们已经发现的现有抗病毒化合物的作用模式，将通过选择的生化表征、耐药突变病毒的分离、突变病毒的表型和基因型表征、耐药性作图、定点诱变以及野生型和耐药病毒的体外药物代谢来研究。我们已经发现，本文描述的不同系列的化合物以不同的机制起作用。它们可以根据它们在病毒复制周期中起作用的时间来分类。例如，已经发现的化合物(i)在HCMV复制周期的早期起作用（某些苯并咪唑），（ii）抑制病毒DNA合成（亚甲基环丙烷类似物）和（iii）阻止病毒DNA加工（其他苯并咪唑和吲哚）。我们已经鉴定出其中一些化合物靶向的病毒基因产物（UL56、UL89），但尚未鉴定出其他化合物靶向的病毒基因产物。这些研究将扩展我们对某些药物靶点的认识，并有助于发现新的药物靶点。

The overall objective of Research Project 3 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, molecular and biochemical level to discover and/or gain insight into new antiviral targets. We shall evaluate for antiviral activity and cytotoxicity new series of nucleoside analogs (benzimidazoles and indoles) and related heterocyclics prepared in the laboratory of Dr. Townsend (Research Project I) and a very different series of nucleoside analogs (methylenecyclopropane analogs and related prodrugs) prepared in the laboratory of Dr. Zemlicka (Research Project 2). All compounds will be tested for activity against human cytomegalovirus (HCMV) and herpes simplex virus type I (HSV-I). The most promising compounds also will be tested against clinical isolates and HCMV strains which are resistant to ganciclovir, benzimidazole nucleosides, or methylenecyclopropane analogs. A broader evaluation against other herpesviruses will be performed in the Scientific Core laboratory. New compounds which are active and low in cytotoxicity - plus existing compounds from our laboratories that appear to act by unique mechanisms - shall be studied extensively to determine their mode of action and to determine if they affect a new antiviral target. Compounds with the best overall profiles will be sent to our Scientific Core for in vivo studies. The modes of antiviral action of compounds discovered as described above, and the modes of action of existing antiviral compounds which we have discovered, will be investigated using selected biochemical characterization, Isolation of drug-resistant mutant virus, phenotypic and genotypic characterization of mutant virus, drug resistance mapping, site- directed mutagenesis, and in vitro drug metabolism with wild-type and resistant viruses. We already have found that the different series of compounds described herein act by different mechanisms. They can be categorized by the time in the viral replication cycle at which they act. For example, compounds have been found which (i) act early in the HCMV replication cycle (certain benzimidazoles), (ii) inhibit viral DNA synthesis (methylenecyclopropane analogs) and (iii) stop viral DNA processing (other benzimidazoles and indoles). We have identified the viral gene products (UL56, UL89) targeted by some of these compounds but have not yet identified those targeted by others. These studies will extend our knowledge about certain drug targets and help to identify new ones.