Assay development to discover therapeutics against human cytomegalovirus

描述（由申请人提供）：人类巨细胞病毒（HCMV）在60-90%的人群中引起潜伏和持续感染。病毒增殖显著增加免疫功能低下个体（如新生儿、器官移植受者、艾滋病患者以及老年人）的发病率和/或死亡率。HCMV是一个重大的健康挑战，需要开发一种多方面的方法来产生有效和安全的治疗方法来限制HCMV的增殖。目前针对HCMV的治疗方法是抗病毒药物，如针对病毒聚合酶的更昔洛韦、缬更昔洛韦、膦酸钠和西多福韦。这些药物的一个主要缺点是它们的毒性、药物-药物相互作用以及长期治疗后病毒耐药菌株的发展。目前的申请建议开发和利用高通量筛选分析方法来识别限制HCMV增殖和传播的先导化合物，以响应PA-10-213，“开发用于探针和治疗前发现的高通量筛选分析方法”。我们的中心假设是，通过有效抑制HCMV生命周期，靶向HCMV增殖的多个阶段将显著减少HCMV疾病。因此，我们建议通过开发高通量筛选试验来发现阻断HCMV增殖不同步骤的化合物。这些检测将利用表达嵌合病毒蛋白与黄色荧光蛋白或荧光素酶融合的HCMV变体。此外，用于确定hit化合物的患者衍生临床菌株的病毒和细胞特异性以及化合物的一般作用模式的二次分析是实验室的常规方案。开发强大的高通量筛选来鉴定抗HCMV药物是产生针对HCMV和相关疾病的有效疗法的重要的第一步。潜在的先导化合物可能针对HCMV生命周期的各个步骤，如病毒进入、病毒蛋白表达、病毒输出和病毒颗粒从感染细胞释放。这些化合物与目前的多药鸡尾酒疗法相结合，可能非常有效地治疗HCMV病毒载量高的患者，并限制HCMV相关疾病。

DESCRIPTION (provided by applicant): Human cytomegalovirus (HCMV) causes latent and persistent infections in 60-90% of the population. Virus proliferation significantly increases the morbidity and/or mortality in immunocompromised individuals such as newborns, organ transplant recipients, AIDS patients, as well as the elderly. HCMV is a significant health challenge requiring the development of a multi-facet approach for the generation of effective and safe treatments to limit HCMV proliferation. The current treatments against HCMV are anti-viral drugs such as ganciclovir, valganciclovir, foscarnet, and cidofovir that target the viral polymerase. A major draw-back to these drugs is their toxicity, drug-drug interactions, and the development of drug resistance strains of the virus upon prolonged treatment. The current application proposes to develop and utilize high-throughput screening assays to identify lead compounds that limit HCMV proliferation and dissemination in response to PA-10-213, 'Development of Assays for High-Throughput screening for use in Probe and Pre-therapeutic Discovery.' Our central hypothesis is that targeting multiple stages of HCMV proliferation would significantly reduce HCMV disease through the effective inhibition of the HCMV life cycle. Thus, we propose to discover compounds that block different steps of HCMV proliferation through the development of high-throughput screening assays. These assays will utilize HCMV variants that express chimeric viral proteins fused to yellow fluorescent proteins or luciferase. In addition, secondary assays to determine the viral and cellular specificity for patient-derived clinical strais of hit compounds and the compound's general mode of action are routine protocols in the lab. The development of robust high-throughput screens for the identification of anti-HCMV drugs is an essential first step to generating effective therapies against HCMV and the associated diseases. Potential lead compounds may target various steps of the HCMV life cycle such as viral entry, viral protein expression, viral egress, and virus particle release from the infected cll. These compounds in combination with current therapy as a multi-drug cocktail would likely be very effective at treating patients with a high HCMV viral load and limit HCMV associated diseases.