High Throughput Screening for Identification of Human Cytomegalovirus Inhibitors

用于鉴定人类巨细胞病毒抑制剂的高通量筛选

• 批准号: 8870334
• 负责人: Ravit Boger
• 金额: $ 34.08万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-13 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8870334
• 关键词: Acquired Immunodeficiency Syndrome; Adult; Adverse effects; Antibodies; Area; Artemisinins; Binding; Biological; Biological Assay; Cardiac Glycosides; Cells; Chemicals; Child; Chronic; Cidofovir; Classification; Clinical; Collaborations; Cytomegalovirus; Cytomegalovirus Infections; Cytomegalovirus Vaccines; DNA Polymerase Inhibitor; DNA biosynthesis; DNA-Directed DNA Polymerase; Deterioration; Development; Dose; Drug Combinations; Drug Formulations; Drug resistance; Exclusion; Family; Foscarnet; Future; Ganciclovir; Goals; HIV; Health; Hepatitis C; Hour; Immunocompetent; Individual; Infection; Investigation; Laboratories; Lead; Libraries; Luciferases; Measures; Mental Retardation; Morbidity - disease rate; Oral; Organ Transplantation; Pathway interactions; Patients; Pharmaceutical Preparations; Population; Pregnant Women; Proteins; Recombinants; Reporter; Reporting; Resistance; Solid; Specificity; Staging; System; Techniques; Therapeutic Uses; Time; Toxic effect; Transplant Recipients; United States; United States National Institutes of Health; Valganciclovir; Viral; Virus; Virus Diseases; Virus Replication; Western Blotting; artemisinine; base; burden of illness; cohort; congenital cytomegalovirus; congenital infection; cost; cytotoxicity; deafness; experience; follow-up; hearing impairment; high throughput screening; indexing; inhibitor/antagonist; luminescence; maribavir; miniaturize; mortality; mutant; neoplastic; novel; novel therapeutics; pathogen; prevent; promoter; prophylactic; recombinant virus; response; salinomycin; screening; small molecule libraries; terminase; treatment strategy; viral DNA

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.

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原因。尽管 CMV 疗法可以抑制病毒复制，但长期使用会导致严重的副作用和耐药病毒的出现。现有的 CMV 抑制剂具有相同的作用机制——抑制病毒 DNA 聚合酶。绝对需要鉴定新的 CMV 抑制剂并制定 CMV 治疗策略。 CMV 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。由于采用高通量筛选 (HTS) 系统的大型化学库的可用性，现在可以鉴定用于 CMV 治疗的新型化合物。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 测定来鉴定抑制 CMV 复制的化合物，使用二级和三级测定验证化合物对 CMV 的特异性，并通过 HTS 发现抗 CMV 药物的组合，以了解其 CMV 抑制机制。在过去的几年中，我们开发并验证了灵敏且可重复的 CMV 复制抑制定量检测方法，包括荧光素酶重组 CMV、荧光素酶重组抗更昔洛韦 CMV、基于实时 PCR 的病毒产量和 DNA 复制。这些技术已广泛应用于我们对 CMV 抑制剂（如青蒿素和强心苷）的研究中，这两个 CMV 抑制剂家族的作用不同 其作用方式与盐霉素相同。我们的检测可用于与 NIH 合作筛选最大的化学探针库，并鉴定用于病毒复制阶段的化学探针，这些化合物在该阶段发挥二级和三级检测作用。由于我们在抗病毒检测方面的丰富经验以及 NIH 在 HTS 方面的专业知识，拟议的研究很可能会成功。从该应用程序中获得的信息将对 CMV 治疗产生至关重要的影响。通过使用抑制不同于病毒 DNA 聚合酶的靶标的化合物以及实施联合抗 CMV 治疗，它可能会导致 CMV 治疗新概念的发展。