Identification of human cytomegalovirus life cycle stage-specific therapeutics

研究综述 人巨细胞病毒（CMV）是一种可增加巨细胞病毒感染者发病率和死亡率的疱疹病毒。 免疫功能低下的个体，如新生儿、移植受者、AIDS患者和老年人。 在一些免疫受损宿主中CMV疾病的常见表现包括神经元缺陷， 婴儿和胃肠道疾病、肺炎、CMV综合征和移植中的终末器官疾病 受惠人士CMV是出生缺陷的主要原因，影响1-2.5%的新生儿，约有40，000 在美国，CMV感染的新病例每年在实体器官移植的8-50%之间 接受者（全球> 145，000）患有CMV相关疾病。FDA批准的抗巨细胞病毒药物 更昔洛韦、缬更昔洛韦、膦甲酸、西多福韦和最近的莱特莫韦显示出有限的功效， 严重的缺点包括口服生物利用度差、剂量相关性毒性、促进药物的选择等 抗病毒，分别。巨细胞病毒是一个重大的健康挑战，需要发展多方面的 该方法用于产生有效和安全的治疗以限制CMV增殖。的目的 目前的拨款是开发和利用高通量CMV感染性测定来鉴定化学抑制剂， 针对CMV生命周期的不同步骤。我们的中心假设是， 病毒生命周期的各个步骤可以被开发成有效的治疗剂， 传播。CMV生命周期（约96小时）是一个复杂的过程，需要病毒因子来操纵细胞 通过无细胞感染或细胞间传播产生用于传播的感染性颗粒的途径。到 为了确定病毒生命周期不同阶段的抑制剂，我们将使用表达两种生命周期的CMV变体， 与黄色荧光蛋白（YFP）融合的周期阶段特异性报告蛋白：1）立即早期（IE）-2 基因产物（AD 169 IE 2-YFP），和2）临床样菌株中的早期膜蛋白独特短（US）28 (TB40/EUS 28-YFP）。与Microbiotix合作，我们计划目标1：进行高含量筛选试验， 鉴定CMV感染早期阶段的抑制剂;目标2：开发和应用高通量筛选 鉴定感染和病毒传播晚期抑制剂的测定.目的3：抗病毒 并使用一组正交试验，根据效力、特异性和药物样特性对确认的命中进行优先排序。 目标4：定义优先命中化合物的作用机制。发展和 利用几种稳健的高通量测定将发现靶向多个步骤的新化合物， CMV生命周期这些化合物有可能成为CMV的先导探针 感染和传播。优先的抑制剂将限制CMV感染和传播的患者， CMV相关疾病的风险。

Research Summary Human cytomegalovirus (CMV) is a -herpes virus that increases morbidity and mortality of immunocompromised individuals such as newborns, transplant recipients, AIDS patients, and the elderly. Common manifestations of CMV disease in some immunocompromised hosts include neuronal defects in infants and gastrointestinal disorders, pneumonia, CMV syndrome, and end-organ disease in transplant recipients. CMV is the leading cause of birth defects affecting 1-2.5% of newborns with approximately 40,000 new cases of CMV infection annually in the United States and range from 8-50% of solid organ transplant recipients (>145,000 globally) present with CMV-associated diseases. The anti-CMV FDA-approved drugs, ganciclovir, valganciclovir, foscarnet, cidofovir, and recently letermovir demonstrate limited efficacy and have severe drawbacks including poor oral bioavailability, dose-related toxicity, and promote the selection of drug resistant viral, respectively. CMV is a major health challenge that requires the development of a multi-faceted approach for the generation of effective and safe treatments to limit CMV proliferation. The objective of the current grant is to develop and utilize high throughput CMV infectivity assays to identify chemical inhibitors that target the diverse steps of CMV life cycle. Our central hypothesis is that compounds targeting the multiple steps of the virus life cycle can be developed into effective therapeutics that block infection, replication, and dissemination. The CMV life cycle (~96hrs) is a complex process requiring viral factors to manipulate cellular pathways to generate an infectious particle for dissemination by cell-free infections or cell-to-cell spread. To identify inhibitors of the different steps of the virus life cycle, we will use CMV variants that express two life- cycle stage-specific reporter proteins fused to yellow fluorescent protein (YFP): 1) the immediate early (IE)-2 gene product (AD169IE2-YFP), and 2) the early membrane protein unique short (US) 28 in a clinical-like strain (TB40/EUS28-YFP). In collaboration with Microbiotix, we plan to Aim 1: Perform a high content screening assay to identify inhibitors of the early steps of a CMV infection; Aim 2: Develop and apply high-throughput screening assays for the identification of inhibitors of the late stages phases of infection and viral spread; Aim 3: Validate and prioritize confirmed hits based on potency, specificity, and drug-like properties using a panel of orthogonal assays; and Aim 4: Define the mechanism of action for prioritized hit compounds. The development and utilization of several robust high-throughput assays will discover novel compounds that target multiple steps of the CMV life-cycle. The identified hit compounds have the potential to be developed into lead probes for CMV infection and dissemination. The prioritized inhibitors would limit CMV infection and dissemination in patients at risk for CMV-associated diseases.