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）和人元病毒（HMPV）是非细分的阴性 - Strand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是古老人口中疾病的重要原因，通常对患者致命 免疫系统受损。目前尚无疫苗，现有疗法（例如，利巴韦林， 免疫球蛋白或抗HRSV单克隆，下叶酸®）的效率较差，并且当前的安全问题。 开发更安全的疗法是一项主要的未满足医疗需求。这个项目的目标是 通过发现和开发HRSV和HMPV RNA合成的抑制剂来满足这种需求 通过靶向病毒核蛋白（N）和病毒P蛋白之间的相互作用，该相互作用是辅助因子 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 复制需要复合物，P介导L和N-RNA模板之间的相互作用。 策略是为基于HRSV和HMPV N/P相互作用的抑制剂构建和应用生化筛选 在荧光极化上。这种方法基于成功的抗欧博拉病毒筛查工作 该团队以识别埃博拉核蛋白（ENP）和埃博拉病毒P之间相互作用的抑制剂 蛋白质等效，称为EVP35。一级荧光极化的开发和应用 测定（FPA），然后进行次要测定，包括基于无关相互作用的反屏幕FPA 导致发现了六个特定的EVP35/ENP相互作用抑制剂，IC50值范围为1 µm至 35 µm。这些化合物中的两种抑制了基于细胞的测定中的埃博拉菌RNA合成 微型素组复制测定法。在第一阶段，这些努力将被延长以针对这种保守的病毒 通过关注HRSV和HMPV的相互作用，这是广泛的临床重要性。主要FPA屏幕 HRSV和HMPV N-蛋白与荧光团标记的辣椒的抑制剂的抑制剂 将开发相应的P蛋白。另外，生化（例如，生物层干涉法，BLI）和 将构建具有正交读取物的细胞（例如，分裂荧光素酶）辅助测定法 命中并评估细胞渗透性和作用机理。主要和次要测定将是 应用于> 400,000名潜水员化合物。确认的潜力，选择性抑制剂将通过确定来验证 它们抑制传染性病毒测定并确保它们不是细胞毒性的能力。体外ADME分析 和初步SAR将优先使用类似物以进一步优化。该提议的优势包括 生产，协作研究团队；高度敏感，均匀的FPA屏幕； FPA柜台屏幕 迅速识别并消除误报；鉴定针对HRSV和HMPV的广泛抑制剂的潜力； 和细胞刺激以建立目标特异性函数。在第二阶段，通过优先级验证的抑制剂将是 化学优化为铅化合物，以进行动物模型的效率和毒性测试。