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）为非节段阴性- 链病毒（NNSV），是全球婴儿急性呼吸道感染的主要原因。在 此外，hRSV是老年人群中疾病的重要原因，并且对于患有hRSV的患者通常是致命的。 免疫系统受损目前没有疫苗可用，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®）表现出较差的功效并存在安全性问题。的 开发更安全更有效的治疗剂是一个主要的未满足的医疗需求。该项目的目标是 通过发现和开发用于治疗的hRSV和hMPV RNA合成的抑制剂来解决这一需求。 通过靶向病毒核蛋白（N）和病毒P蛋白之间的相互作用， 病毒聚合酶（L）。这种相互作用对病毒RNA合成至关重要;在感染NNSV的细胞中，L-N 复合物是复制所必需的，P介导L和N-RNA模板之间的相互作用。的 策略是建立和应用基于hRSV和hMPV N/P相互作用的抑制剂的生物化学筛选， 关于荧光偏振。这种方法是基于一项成功的抗埃博拉病毒筛查工作， 该团队的研究旨在确定埃博拉病毒核蛋白（eNP）和埃博拉病毒P 蛋白质等价物，称为eVP 35。一次荧光偏振仪的研制及应用 检测（FPA），然后进行二次检测，包括基于不相关相互作用的计数筛选FPA 结果发现了6种特异性eVP 35/eNP相互作用抑制剂，IC 50值范围为1 µM至 35 µM。其中两种化合物在一项称为“细胞抑制剂”的细胞试验中抑制埃博拉病毒RNA的合成。 微基因组复制测定。在第一阶段，这些努力将扩大到针对这种保守的病毒 通过关注具有广泛临床重要性的hRSV和hMPV，研究了它们之间的相互作用。主FPA屏幕， hRSV和hMPV N-蛋白相互作用的抑制剂与荧光团标记的肽， 将开发相应的P蛋白。此外，生物化学（例如，生物层干涉测量法，BLI）和 蜂窝（例如，将构建具有正交读数的二次测定，以验证初始 命中并评估细胞渗透性和作用机制。主要和次要试验将 适用于超过40万种不同的化合物。通过确定以下因素来验证已确认的强效、选择性抑制剂： 它们抑制感染性病毒测定的能力，并确保它们没有细胞毒性。体外ADME试验 初步的SAR将优先考虑类似物，以便进一步优化。这项建议的优点包括： 高效、协作的研究团队;高灵敏度、均匀的FPA屏幕; FPA反屏幕， 快速识别和消除假阳性;有可能鉴定靶向hRSV和hMPV的广泛抑制剂; 和细胞测定以建立靶特异性功能。在第二阶段，优先验证的抑制剂将是 化学优化为先导化合物，用于动物模型中的功效和毒性测试。