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: 10615151
• 负责人: Ravit Boger
• 金额: $ 22.8万
• 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 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10615151
• 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; Cytomegalovirus Infections; 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; Laboratories; Libraries; Mass Spectrum Analysis; Measures; Molecular Target; Morbidity - disease rate; Oral; Organ; Outcome; Pathway interactions; Permeability; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Pregnancy; Prophylactic treatment; Proteins; Proteomics; Refractory; Regimen; Resistance; Resolution; Response Elements; Role; Solid; Therapeutic; Toxic effect; Transplant Recipients; Transplantation; Valganciclovir; Viral; Viral Physiology; Virus; cancer cell; cellular targeting; congenital infection; design; drug development; experimental study; high throughput screening; improved; improved outcome; inhibition of autophagy; inhibitor; intravenous administration; mortality; mutant; nephrotoxicity; novel; novel strategies; novel therapeutic intervention; nucleoside analog; pathogen; pharmacologic; 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.

项目摘要/摘要 人类巨细胞病毒（HCMV）是全球最常见的先天性感染，是主要感染性的 固体器官和骨髓移植后并发症。 HCMV使用了一些抗病毒剂 治疗，全部靶向病毒DNA聚合酶。 Ganciclovir及其口服配方Valganciclovir是最多的 常用代理。骨髓骨髓后，最近批准了末端酶抑制剂的HCMV预防 移植。正在进行的治疗挑战源于与DNA相关的副作用 聚合酶抑制剂，长期治疗过程中抗性病毒突变体的出现，缺乏 抗病毒组合方案和难治性HCMV疾病的病例，对所有电流无反应 抗病毒药。这些严重的问题推动了HCMV的药物开发管道。新型病毒的识别 目标和病毒宿主相互作用将导致更好地控制HCMV，并降低发病率和死亡率 从这个病原体。 使用1280个药理学活性化合物的LOPAC库，我们确定了几个新命中 在低微摩尔浓度下，体外抑制HCMV复制。复合ARP101抑制了几个 HCMV的菌株以及人类疱疹病毒（HSV）1和2。此外，ARP101抑制了Ganciclovir 抗性HCMV，表明其作用机理与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。尽管诱导自噬，但 在用ARP101处理的受感染细胞中也诱导了底物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 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，并在使用中定义其活性 与批准的抗HCMV代理Ganciclovir和Letermovir结合； 2）描绘自噬蛋白 由ARP101诱导的HCMV抑制作用，特定于p62，3）确定细胞 ARP101的靶标使用ARP101的细胞渗透/生物异构/光标记衍生物，下拉 测定和质谱法。 该项目的成功将导致深入的目标验证研究，这反过来又可以使理由设计 用于HCMV抑制作用的ARP101的衍生物的改进。我们的研究将增强我们对角色的理解 HCMV感染期间的自噬及其对HCMV治疗剂的潜在靶向 直接作用抗病毒剂。