Assay development to discover therapeutics against human cytomegalovirus

描述（由申请方提供）：人巨细胞病毒（HCMV）在60-90%的人群中引起潜伏性和持续性感染。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、AIDS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，以限制HCMV的增殖。目前针对HCMV的治疗是靶向病毒聚合酶的抗病毒药物，如更昔洛韦、缬更昔洛韦、膦甲酸和西多福韦。这些药物的一个主要缺点是它们的毒性、药物间的相互作用以及在长期治疗后病毒耐药性菌株的发展。本申请提出开发和利用高通量筛选测定来鉴定响应于PA-10-213“用于探针和治疗前发现的高通量筛选测定的开发”而限制HCMV增殖和传播的先导化合物。“我们的中心假设是，靶向HCMV增殖的多个阶段将通过有效抑制HCMV生命周期显着减少HCMV疾病。因此，我们建议通过开发高通量筛选测定来发现阻断HCMV增殖的不同步骤的化合物。这些试验将利用表达与黄色荧光蛋白或荧光素酶融合的嵌合病毒蛋白的HCMV变体。此外，用于确定命中化合物的患者来源的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测定是实验室中的常规方案。开发用于鉴定抗HCMV药物的稳健的高通量筛选是产生针对HCMV和相关疾病的有效疗法的重要第一步。潜在的先导化合物可以靶向HCMV生命周期的各个步骤，例如病毒进入、病毒蛋白表达、病毒外出和病毒颗粒从感染的cII释放。这些化合物与当前疗法组合作为多药物鸡尾酒可能在治疗具有高HCMV病毒载量的患者方面非常有效，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.