Autophagy activation – a novel strategy for inhibition of human cytomegalovirus

自噬激活——抑制人类巨细胞病毒的新策略

• 批准号: 10442936
• 负责人: Ravit Boger
• 金额: $ 19万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10442936
• 关键词: Affect; Affinity; Antioxidants; Antiviral Agents; Autophagocytosis; Binding Proteins; Biological Assay; Biology; Bone Marrow; Bone Marrow Transplantation; Cells; Chemicals; Child; Child Health; Cidofovir; Combined Modality Therapy; Complex; Complication; Cytomegalovirus; DNA Polymerase Inhibitor; DNA-Directed DNA Polymerase; Data; Deterioration; Development; Disease; Drug Combinations; Excision; FRAP1 gene; Formulation; Foscarnet; Future; GCLC gene; Ganciclovir; Goals; Health; Hearing; Herpesvirus 1; Hour; Human; In Vitro; Infection; Intravenous; Laboratories; Lead; Libraries; Mass Spectrum Analysis; Measures; Molecular Target; Morbidity - disease rate; Oral; Organ; Outcome; Pathway interactions; Permeability; Pharmaceutical Preparations; Pharmacology; Phosphorylation; Phosphotransferases; Pregnancy; Prophylactic treatment; Proteins; Proteomics; Refractory; Regimen; Resistance; Resolution; Response Elements; Role; Solid; Therapeutic; Toxic effect; Transplant Recipients; Transplantation; Valganciclovir; Viral; Virus; base; cancer cell; cellular targeting; congenital infection; design; drug development; experimental study; high throughput screening; improved; improved outcome; inhibitor; mortality; mutant; nephrotoxicity; novel; novel strategies; novel therapeutic intervention; nucleoside analog; pathogen; prevent; side effect; stem; success; terminase; validation studies; viral DNA; virus host interaction

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.

项目概要/摘要 人类巨细胞病毒（HCMV）是世界范围内最常见的先天性感染，也是一种主要传染病 实体器官和骨髓移植后的并发症。一些抗病毒药物可用于治疗 HCMV 治疗方法均针对病毒 DNA 聚合酶。更昔洛韦及其口服制剂缬更昔洛韦是最 常用药剂。一种终止酶抑制剂最近被批准用于骨髓后 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 抗病毒联合治疗方案，以及对目前所有药物均无反应的难治性 HCMV 疾病病例 抗病毒药物。这些严重的问题推动了 HCMV 的药物开发。新型病毒的鉴定 目标和病毒-宿主相互作用将更好地控制 HCMV，并降低发病率和死亡率 来自这种病原体。 利用包含 1280 种药理活性化合物的 LOPAC 库，我们确定了几种新的热门化合物 在体外以低微摩尔浓度抑制 HCMV 复制。化合物 ARP101 抑制多种 HCMV 株以及人疱疹病毒 (HSV) 1 和 2。此外，ARP101 还抑制更昔洛韦 HCMV具有耐药性，说明其作用机制与GCV不同。我们的初步数据表明 ARP101诱导自噬，而HCMV主要旨在抑制自噬。尽管有自噬诱导作用，但 底物 p62 也在用 ARP101 处理的感染细胞中被诱导。因此我们假设 自噬底物 p62 是 ARP101 抑制 HCMV 的主要枢纽，导致非典型的 克服 HCMV 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，并定义其用于以下用途时的活性： 与已批准的抗 HCMV 药物更昔洛韦和莱特莫韦联合使用； 2) 描述自噬蛋白 由 ARP101 诱导以抑制 HCMV，特别关注 p62，以及 3) 识别细胞 使用 ARP101 的细胞渗透性/生物正交/光标记衍生物来确定 ARP101 的目标，下拉 分析和质谱分析。 该项目的成功将导致深入的目标验证研究，从而实现基本原理设计 用于抑制 HCMV 的 ARP101 改良衍生物。我们的研究将增强我们对这个角色的理解 HCMV 感染期间的自噬及其与 HCMV 联合治疗的潜在靶向 直接作用的抗病毒药物。