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)是非节段性阴性病毒。 链状病毒(NNSV)是全球婴儿急性呼吸道感染的主要原因。在……里面 此外，HRSV是老年人群疾病的重要原因，对患有以下疾病的患者往往是致命的 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单抗Synagis®)的疗效较差，且存在安全性问题。这个 开发更安全、更有效的治疗方法是一项尚未得到满足的主要医疗需求。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来使用，P蛋白是 病毒聚合酶(L)。这种相互作用对病毒核糖核酸的合成至关重要；在感染新城疫病毒的细胞中，L-N 复制需要复合体，P介导L与N-RNA模板之间的相互作用。这个 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化筛选 关于荧光偏振。这一方法是基于一项成功的抗埃博拉病毒筛查工作 该团队为确定埃博拉核蛋白(ENP)和埃博拉P病毒之间相互作用的抑制剂而开展的工作 相当于蛋白质，称为eVP35。一次荧光偏振仪的研制及应用 检测(FPA)之后进行二次检测，包括基于无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值从1微米到 35微米，其中两个化合物在一种基于细胞的检测中抑制埃博拉RNA的合成 微基因组复制试验。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HRSV和hMPV的相互作用，这两种病毒具有广泛的临床重要性。主要的FPA屏幕 HRSV和hMPV N蛋白与荧光团标记的多肽相互作用的抑制剂 相应的P-蛋白将被开发出来。此外，生化(例如，生物层干涉法，BLI)和 将构建具有正交读出的细胞(例如，分裂的荧光素酶)二次分析以验证初始 并评估细胞通透性和作用机制。一次和二次化验将是 适用于40万种不同的化合物。确认的有效的、选择性的抑制剂将通过确定 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测定法 初步的SAR将对类比进行优先排序，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；高度敏感、同质的FPA屏幕；FPA反屏幕，以 快速识别和消除假阳性；有可能识别针对HRSV和hMPV的广泛抑制物； 以及细胞检测，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 经过化学优化的先导化合物，用于动物模型的有效性和毒性测试。