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

基本信息

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
项目类别：
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

项目摘要

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。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目前没有可用的疫苗和现有的治疗方法（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。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)。这种相互作用对于感染 NNSV（L-N）的细胞中的病毒 RNA 合成至关重要。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。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效物，称为 eVP35 初级荧光偏振的开发和应用。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 35 µM。在第一阶段，这些努力将扩展到针对这种保守的病毒。重点关注 hRSV 和 hMPV 之间的相互作用，这两种病毒对于 FPA 筛查具有广泛的临床重要性。 hRSV 和 hMPV N 蛋白与荧光团标记的肽相互作用的抑制剂此外，还将开发相应的 P 蛋白，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将是主要和次要测定。已确认的有效、选择性抑制剂适用于超过 400,000 种化合物。它们抑制传染性病毒检测的能力并确保它们不具有体外 ADME 检测的细胞毒性。初步 SAR 将优先考虑类似物以进行进一步优化。高效、协作的研究团队；高度灵敏、均匀的 FPA 屏幕；快速识别并消除假阳性；有可能识别针对 hRSV 和 hMPV 的广泛抑制剂；在第二阶段，将优先进行经过验证的抑制剂。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。