Inhibitors of the viral nucleoprotein-polymerase co-factor interaction for human RSV and MPV therapy

用于人类 RSV 和 MPV 治疗的病毒核蛋白-聚合酶辅因子相互作用的抑制剂

• 批准号: 9200084
• 负责人: Donald T Moir
• 金额: $ 30万
• 依托单位: MICROBIOTIX, INC
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9200084
• 关键词: 5 year old; Acute; Address; Animal Model; Biochemical; Biological; Biological Assay; Cause of Death; Cells; Cellular Assay; Child; Clinical; Complex; Development; Disease; Ebola virus; Elderly; Ensure; Exhibits; Family; Fluorescein-5-isothiocyanate; Fluorescence Polarization; Glycine decarboxylase; Goals; Human; Human Metapneumovirus; Immune system; Immunoglobulins; In Vitro; Infant; Interferometry; Label; Lead; Luciferases; Measures; Mediating; Medical; Monoclonal Antibodies; Mononegavirales; Morbidity - disease rate; N-terminal; Nucleoproteins; Palivizumab; Paramyxoviridae; Paramyxovirus; Patients; Peptides; Permeability; Phase; Pneumovirus; Polymerase; Population; Property; Proteins; RNA; RNA Viruses; RNA chemical synthesis; Reading; Research; Respiratory Tract Infections; Respiratory syncytial virus; Ribavirin; Safety; Series; Structure; Therapeutic; Therapeutic Uses; Toxicity Tests; Vaccines; Validation; Viral; Virus; Virus Inhibitors; Virus Replication; analog; base; cofactor; cytotoxic; efficacy testing; experience; fluorophore; high throughput screening; inhibitor/antagonist; mortality; novel; peptide P; physical property; screening; viral RNA

Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) are non-segmented negative- strand viruses (NNSV) and are the leading causes of acute respiratory tract infections in infants worldwide. In addition, hRSV is a significant cause of disease in elderly populations and can often be fatal for patients with compromised immune systems. Currently no vaccines are available, and existing therapeutics (e.g., ribavirin, immunoglobulin, or anti-hRSV monoclonal, Synagis®) exhibit poor efficacy and present safety concerns. The development of safer more effective therapeutics is a major unmet medical need. The goal of this project is to address this need by discovering and developing inhibitors of hRSV and hMPV RNA synthesis for therapeutic use by targeting the interaction between the viral nucleoprotein (N) and the viral P protein, a cofactor for the viral polymerase (L). This interaction is critical for viral RNA synthesis; in cells infected with NNSVs, an L-N complex is required for replication, and P mediates interactions between L and the N-RNA template. The strategy is to build and apply biochemical screens for inhibitors of the hRSV and hMPV N/P interaction based on fluorescence polarization. This approach is based on a successful anti-Ebola virus screening effort carried out by this team to identify inhibitors of the interaction between the Ebola nucleoprotein (eNP) and the Ebola P protein equivalent, known as eVP35. Development and application of a primary fluorescence polarization assay (FPA) followed by secondary assays including a counter-screen FPA based on an unrelated interaction resulted in the discovery of six specific eVP35/eNP interaction inhibitors with IC50 values ranging from 1 µM to 35 µM. Two of these compounds inhibited Ebola RNA synthesis in a cell based assay known as a minigenome replication assay. In Phase I, these efforts will be extended to target this conserved viral interaction by focusing on hRSV and hMPV, which are of broad clinical importance. Primary FPA screens for inhibitors of the hRSV and hMPV N-protein interactions with fluorophore-labeled peptides from the corresponding P-proteins will be developed. In addition, biochemical (e.g., biolayer interferometry, BLI) and cellular (e.g., split luciferase) secondary assays with orthogonal read-outs will be constructed to validate initial hits and to assess cellular permeability and mechanism of action. The primary and secondary assays will be applied to >400,000 diverse compounds. Confirmed potent, selective inhibitors will be validated by determining their ability to inhibit infectious viral assays and by ensuring that they are not cytotoxic. In vitro ADME assays and preliminary SAR will prioritize analogs for further optimization. Strengths of this proposal include the productive, collaborative research team; highly sensitive, homogeneous FPA screens; FPA counter-screens to rapidly recognize and eliminate false positives; potential to identify broad inhibitors targeting hRSV and hMPV; and cellular assays to establish the target-specific function. In Phase II, priority validated inhibitors will be chemically optimized into lead compounds for efficacy and toxicity testing in animal models.

人呼吸道合胞病毒（hRSV）和人偏肺病毒（hMPV）是不分节段负性病毒 链病毒（NNSV）是全世界婴儿急性呼吸道感染的主要原因。在 此外，hRSV 是老年人群疾病的一个重要原因，对于患有以下疾病的患者来说通常可能是致命的 免疫系统受损。目前还没有可用的疫苗，并且现有的治疗方法（例如利巴韦林、 免疫球蛋白或抗 hRSV 单克隆抗体 Synagis®）的疗效较差，并存在安全问题。这 开发更安全、更有效的治疗方法是一个未满足的主要医疗需求。该项目的目标是 通过发现和开发用于治疗的 hRSV 和 hMPV RNA 合成抑制剂来满足这一需求 通过靶向病毒核蛋白 (N) 和病毒 P 蛋白（病毒 P 蛋白的辅助因子）之间的相互作用来使用 病毒聚合酶（L）。这种相互作用对于病毒 RNA 合成至关重要。在感染 NNSV 的细胞中，L-N 复合体是复制所必需的，P 介导 L 和 N-RNA 模板之间的相互作用。这 策略是建立并应用基于 hRSV 和 hMPV N/P 相互作用的抑制剂的生化筛选 关于荧光偏振。该方法基于成功的抗埃博拉病毒筛查工作 该团队发现了埃博拉核蛋白 (eNP) 和埃博拉 P 之间相互作用的抑制剂 蛋白质等价物，称为 eVP35。初级荧光偏振的研制及应用 测定（FPA），然后进行二次测定，包括基于不相关相互作用的反筛选 FPA 结果发现了六种特定的 eVP35/eNP 相互作用抑制剂，其 IC50 值范围为 1 µM 至 35 µM。其中两种化合物在基于细胞的检测中抑制埃博拉 RNA 合成，称为 小基因组复制测定。在第一阶段，这些努力将扩大到针对这种保守的病毒 重点关注具有广泛临床重要性的 hRSV 和 hMPV 之间的相互作用。主要 FPA 屏幕 hRSV 和 hMPV N 蛋白与荧光团标记的肽相互作用的抑制剂 相应的P-蛋白将被开发。此外，生物化学（例如生物层干涉测量法，BLI）和 将构建具有正交读数的细胞（例如，分裂荧光素酶）二次测定以验证初始 命中并评估细胞渗透性和作用机制。主要和次要测定将是 适用于超过 400,000 种不同的化合物。已确认的有效、选择性抑制剂将通过确定来验证 它们抑制传染性病毒检测的能力并确保它们不具有细胞毒性。体外 ADME 测定 初步 SAR 将优先考虑类似物以进行进一步优化。该提案的优点包括 富有成效、协作的研究团队；高灵敏度、均匀的 FPA 屏幕； FPA 反筛选 快速识别并消除误报；识别针对 hRSV 和 hMPV 的广泛抑制剂的潜力； 和细胞测定以确定靶点特异性功能。在第二阶段，优先验证的抑制剂将是 经过化学优化成为先导化合物，用于动物模型中的功效和毒性测试。