Identification of human cytomegalovirus life cycle stage-specific therapeutics

研究总结 人巨细胞病毒是一种疱疹病毒，它会增加人的发病率和死亡率。 免疫功能受损的人，如新生儿、移植受者、艾滋病患者和老年人。 巨细胞病毒病在一些免疫受损的宿主中的常见表现包括 移植中的婴儿和胃肠道疾病、肺炎、巨细胞病毒综合征和终末器官疾病 收件人。巨细胞病毒是导致出生缺陷的主要原因，影响1-2.5%的新生儿，约有40,000 美国每年新增巨细胞病毒感染病例，范围从固体器官移植的8%-50%不等 接受者(全球145,000人)患有巨细胞病毒相关疾病。FDA批准的抗CMV药物， 更昔洛韦、伐更昔洛韦、磷甲酸钠、西多福韦和最近的莱特莫韦显示出有限的疗效和 严重的缺点包括口服生物利用度差，与剂量相关的毒性，并促进药物的选择 分别为耐药病毒。CMV是一项重大的卫生挑战，需要多方面的发展 为产生有效和安全的治疗方法来限制CMV的增殖。该计划的目标是 目前的资助是开发和利用高通量CMV感染性分析来识别化学抑制剂， 针对CMV生命周期的不同步骤。我们的中心假设是，针对多重目标的化合物 病毒生命周期的各个步骤可以发展成有效的治疗方法，阻止感染、复制和 传播。CMV的生命周期(~96小时)是一个复杂的过程，需要病毒因子来操纵细胞 产生感染性粒子的途径，通过无细胞感染或细胞间传播传播。至 识别病毒生命周期不同阶段的抑制剂，我们将使用表达两个生命的CMV变体- 周期阶段特异性报告蛋白与黄色荧光蛋白(YFP)融合：1)即刻早期(IE)-2 基因产物(AD169IE2-YFP)和2)临床样菌株中的早期膜蛋白唯一短(US)28 (TB40/EUS28-YFP)。与MicroBiotix合作，我们计划目标1：进行高含量筛选分析，以 确定巨细胞病毒感染早期阶段的抑制物；目标2：开发和应用高通量筛查 用于鉴定感染和病毒传播的晚期阶段抑制物的分析；目标3：验证 并使用一组正交表根据效力、特异性和类似药物的属性对确认的命中结果进行优先排序 分析；和目标4：确定优先命中化合物的作用机制。中国的发展和 利用几种强大的高通量分析方法将发现针对多个步骤的 CMV的生命周期。已鉴定的Hit化合物有可能发展成为CMV的先导探针。 感染和传播。优先考虑的抑制剂将限制巨细胞病毒在患者体内的感染和传播 巨细胞病毒相关疾病的风险。

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.