Autophagy activation – a novel strategy for inhibition of human cytomegalovirus

项目概要/摘要 人巨细胞病毒（HCMV）是世界范围内最常见的先天性感染，也是主要的传染性疾病。 实体器官和骨髓移植后并发症。一些抗病毒药物用于HCMV 治疗，都是针对病毒DNA聚合酶。更昔洛韦及其口服制剂缬更昔洛韦是最 常用的代理商。一种末端酶抑制剂最近被批准用于骨髓移植后的HCMV预防。 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，在长期治疗过程中出现耐药病毒突变体，缺乏 抗病毒联合方案，以及对所有当前治疗方案无反应的难治性HCMV疾病病例， 抗病毒药这些严重的问题推动了HCMV的药物开发管道。新病毒的鉴定 靶点和病毒-宿主相互作用将导致更好地控制HCMV，并将降低发病率和死亡率 从这种病原体中分离出来 使用包含1280种活性化合物的LOPAC库，我们确定了几种新的命中物， 在低微摩尔浓度下体外抑制HCMV复制。化合物ARP 101抑制了几种 HCMV株以及人疱疹病毒（HSV）1和2。此外，ARP 101抑制了更昔洛韦的表达。 抗HCMV，表明其作用机制不同于GCV。初步数据显示， ARP 101诱导自噬，而HCMV主要旨在抑制自噬。尽管有自噬诱导， 底物p62也在用ARP 101处理的感染细胞中被诱导。因此，我们假设， 自噬底物p62是ARP 101抑制HCMV的主要枢纽，导致非典型的 自噬相关途径，克服HCMV复制。 我们的目标是：1）详细研究ARP 101的抗HCMV活性，并确定其用于治疗HCMV感染时的活性。 与批准的抗HCMV剂更昔洛韦和莱特莫韦组合; 2）描绘自噬蛋白 由ARP 101诱导的HCMV抑制，特别关注p62，和3）鉴定细胞 使用ARP 101的细胞可渗透/生物正交/光标记衍生物，下拉ARP 101的靶标 分析和质谱法。 该项目的成功将导致深入的目标验证研究，这反过来又将使合理设计成为可能 ARP 101的改进衍生物用于HCMV抑制。我们的研究将增强我们对 HCMV感染过程中自噬的作用及其对HCMV治疗剂的潜在靶向作用， 直接作用的抗病毒剂。

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.