High Throughput Screening for Identification of Human Cytomegalovirus Inhibitors

描述：人类巨细胞病毒(CMV)感染仍然是孕妇、实体器官移植接受者和艾滋病毒/艾滋病患者的主要威胁。先天性巨细胞病毒是导致智力低下和耳聋的主要感染性原因。虽然CMV的治疗方法可以抑制病毒复制，但长期使用会导致严重的副作用和出现耐药病毒。现有的CMV抑制剂具有相同的作用机制--抑制病毒DNA聚合酶。寻找新的巨细胞病毒抑制剂和开发巨细胞病毒的治疗策略是绝对必要的。巨细胞病毒新疗法的开发可以降低先天性感染儿童和实体器官移植人群的发病率和死亡率。由于形成为高通量筛选(HTS)系统的大型化学库的可用性，鉴定用于巨细胞病毒治疗的新化合物现在是可能的。本申请(PAR-12-058)的总体目标是使用定量HTS和荧光素酶-重组CMV试验鉴定抑制CMV复制的化合物，使用二次和三次试验验证化合物对CMV的特异性，并通过HTS发现抗CMV药物的组合以了解其抑制CMV的机制。在过去的几年里，我们建立和验证了灵敏和重复性好的CMV复制抑制定量方法，包括荧光素酶重组CMV、荧光素酶抗性CMV、病毒产量和基于实时荧光PCR的DNA复制。这些技术已经被广泛用于我们对CMV抑制剂的研究，如青蒿素和心脏糖苷，这两个不同的CMV抑制剂家族 在他们的作用模式和盐霉素。我们的检测方法可用于与美国国立卫生研究院合作筛选最大的化学探针库，并识别病毒复制阶段的化学探针，在该阶段，这些化合物进行二次和三次分析。拟议的调查可能会成功，因为我们在抗病毒检测方面已经积累了经验，而且NIH在HTS方面的专业知识。从这一应用中获得的信息将对CMV治疗产生关键影响。通过使用抑制不同于病毒DNA聚合酶的靶点的化合物和实施联合抗CMV治疗，它可能导致CMV疗法中新概念的发展。

DESCRIPTION: Infection with human Cytomegalovirus (CMV) continues to be a major threat for pregnant women, solid organ transplant recipients and patients with HIV-AIDS. Congenital CMV is the leading infectious cause of mental retardation and deafness. Although therapies for CMV can suppress virus replication, their prolonged use results in serious side effects and the emergence of resistant viruses. The available CMV inhibitors share the same mechanism of action - inhibition of viral DNA polymerase. There is an absolute need to identify new CMV inhibitors and to develop treatment strategies for CMV. The development of novel therapies for CMV could reduce morbidity and mortality in congenitally-infected children and the solid organ transplant population. Identification of novel compounds for CMV therapy is now possible because of the availability of large chemical libraries formatted as high-throughput screening (HTS) systems. The overall goals of this application (PAR-12-058) are to identify compounds that inhibit CMV replication using a quantitative HTS and a luciferase-recombinant CMV assay, to validate the specificity of the compounds for CMV using secondary and tertiary assays and to discover combination of anti-CMV agents by HTS to understand their mechanism of CMV inhibition. During the last several years we developed and validated sensitive and reproducible assays for quantification of CMV replication inhibition including a luciferase-recombinant CMV, luciferase-recombinant ganciclovir-resistant CMV, virus yield and DNA replication based on real-time PCR. These techniques have already been used extensively in our studies of CMV inhibitors such as artemisinins, and cardiac glycosides, two families of CMV inhibitors that differ in their mode of action and salinomycin. Our assays can be applied to screen the largest library of chemical probes in collaboration with the NIH and to identify chemical probes for the stage of virus replication in which these compounds act secondary and tertiary assays. The proposed investigations are likely to succeed because of our established experience in anti-viral assays and the expertise of NIH in HTS. The information derived from this application will have critical impact on CMV therapy. It may lead to the development of new concepts in CMV therapeutics by using compounds that inhibit a target different from the viral DNA polymerase and by implementing combination anti-CMV therapy.