Autophagy activation – a novel strategy for inhibition of human cytomegalovirus

项目摘要/摘要 人巨细胞病毒是世界范围内最常见的先天性感染，也是一种主要的传染性疾病。 实体器官和骨髓移植后的并发症。几种抗病毒药物被用于治疗巨细胞病毒 治疗，都是针对病毒DNA聚合酶。更昔洛韦及其口服制剂万乃昔洛韦最多 常用的药物。一种终止酶抑制剂最近被批准用于预防骨髓后的人巨细胞病毒 移植。持续的治疗挑战源于与DNA相关的副作用 聚合酶抑制剂，在长时间的治疗过程中出现抗药性病毒突变，缺乏 抗病毒联合疗法，以及对所有电流均无反应的难治性HCMV病病例 抗病毒药物。这些严重的问题推动了HCMV的药物开发流水线。一株新型病毒的鉴定 靶向和病毒-宿主相互作用将导致更好地控制巨细胞病毒，并将降低发病率和死亡率。 来自这种病原体。 使用包含1280种药理活性化合物的Lopac文库，我们确定了几个新的热门药物 在体外低微摩尔浓度下抑制人巨细胞病毒复制。化合物ARP101抑制了几种 以及人类疱疹病毒(HSV)1和2。此外，ARP101还抑制更昔洛韦 抗HCMV，说明其作用机制与GCV不同。我们的初步数据显示， ARP101诱导自噬，而HCMV的主要目的是抑制自噬。尽管自噬诱导，但它的 在用ARP101处理的感染细胞中也能诱导底物p62。因此，我们假设 自噬底物p62是ARP101抑制HCMV的主要枢纽，导致非典型性 克服巨细胞病毒复制的自噬相关途径。 我们的目标是：1)详细验证ARP101的抗HCMV活性，并确定其在 与已批准的抗巨细胞病毒药物更昔洛韦和利特莫韦联合；2)描述自噬蛋白 由ARP101诱导的对HCMV的抑制，特别关注p62，以及3)鉴定细胞 使用ARP101的细胞渗透性/生物正交性/光标记衍生物的ARP101的靶点(S)，下拉 化验和质谱分析。 该项目的成功将导致深入的目标验证研究，这反过来将使基本设计成为可能 用于抑制巨细胞病毒的ARP101的改进衍生物。我们的学习将加深我们对这一角色的理解 人巨细胞病毒感染过程中自噬的研究及其与联合治疗的潜在靶向性 直接作用的抗病毒药物。

PROJECT SUMMARY/ABSTRACT Human cytomegalovirus (HCMV) is the most common congenital infection worldwide and a major infectious complication after solid organ and bone marrow transplantation. A few antiviral agents are used for HCMV therapy, all target the viral DNA polymerase. Ganciclovir, and its oral formulation valganciclovir are the most commonly used agents. A terminase inhibitor was recently approved for HCMV prophylaxis after bone marrow transplantation. Ongoing therapeutic challenges stem from the side effects associated with the DNA polymerase inhibitors, emergence of resistant virus mutants during prolonged treatment courses, lack of antiviral combination regimens, and cases of refractory HCMV disease that are non-responsive to all current antivirals. These serious problems drive the drug development pipeline for HCMV. Identification of novel viral targets and viral-host interactions will lead to better control of HCMV and will reduce morbidity and mortality from this pathogen. Using the LOPAC library of 1280 pharmacologically active compounds, we identified several new hits that inhibit HCMV replication in vitro at low micro-molar concentrations. Compound ARP101 inhibited several strains of HCMV, as well as human herpesvirus (HSV) 1 and 2. In addition, ARP101 inhibited a ganciclovir resistant HCMV, indicating its mechanism of action is different from GCV. Our preliminary data show that ARP101 induces autophagy, while HCMV mostly aims to inhibit autophagy. Despite autophagy induction, its substrate p62 is also induced in infected cells treated with ARP101. We therefore hypothesize that the autophagy substrate p62 is a major hub for HCMV inhibition by ARP101, leading to a non-canonical autophagy-related pathway that overcomes HCMV replication. Our goals are: 1) Validate in detail the anti-HCMV activity of ARP101 and define its activity when used in combination with approved anti-HCMV agents ganciclovir and letermovir; 2) Delineate the autophagic proteins that are induced by ARP101 for HCMV inhibition, with a specific focus on p62, and 3) Identify the cellular target(s) of ARP101 using a cell-permeable/bioorthogonal/photolabeling derivative of ARP101, pull down assays and mass spectrometry. Success of this project will lead to in-depth target validation studies, which will in turn enable rationale design of improved derivatives of ARP101 for HCMV inhibition. Our studies will enhance our understanding of the role of autophagy during HCMV infection and its potential targeting for HCMV therapeutics in combination with direct acting antiviral agents.