COVID-19: Assay Development, HTS and Drug Repurposing to identify potential therapeutics against SARS-CoV-2

COVID-19：检测开发、HTS 和药物再利用，以确定针对 SARS-CoV-2 的潜在疗法

• 批准号: 10700705
• 负责人: Matthew Hall
• 金额: $ 408.8万
• 依托单位: NATIONAL CENTER FOR ADVANCING TRANSLATIONAL SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10700705
• 关键词: 2019-nCoV; ACE2; Address; Affinity; Antibodies; Area; Bacteriophages; Binding; Biochemical; Biological Assay; Biology; COVID-19; COVID-19 assay; COVID-19 pandemic; COVID-19 patient; COVID-19 pneumonia; COVID-19 therapeutics; Cells; Clinical; Collaborations; Communities; Data; Development; Disease; Effectiveness; Enzymes; Extramural Activities; FDA approved; Fluorescence; Goals; Human; Journals; Laboratories; Libraries; Measures; Mechanical ventilation; Modeling; National Center for Advancing Translational Sciences; Peptide Hydrolases; Pharmaceutical Preparations; Positioning Attribute; Proteins; Public Health; Publications; Pulmonary Fibrosis; Recombinants; Reporting; Research; Research Activity; Research Contracts; Research Personnel; Research Support; Resources; Running; SARS-CoV-2 infection; SARS-CoV-2 spike protein; Scientist; Screening Result; Site; Societies; Speed; TMPRSS2 gene; Testing; Therapeutic; Therapeutic Intervention; Thrombin; Translational Research; Translations; Vaccines; Variant; Viral; Virus; Virus Diseases; Work; antibody engineering; assay development; atypical pneumonia; base; betacoronavirus; clinical candidate; clinical efficacy; design; drug repurposing; effective therapy; follow-up; high throughput screening; inhibitor; innovation; insight; member; mortality; nanobodies; neutralizing antibody; novel; pandemic disease; public health emergency; receptor; response; screening; small molecular inhibitor; small molecule; supplemental oxygen; symposium; therapeutic development; therapeutically effective; viral entry inhibitor

During this past reporting period, NCATS continued development of a number of target-based SARS-CoV-2 assays to screen for potential hits against the virus. Optimized assays were screened against a panel of drug repurposing, clinical candidate and antiviral libraries that have been assembled at NCATS. These results have been made freely available to public with unprecedented speed through the NCATS OpenData Portal and through a number of preprint/journal publications. These assays include both virus (e.g. Spike protein) and host (e.g. ACE2, TMPRSS) targets, as well as BSL3 live SARS-CoV-2 virus infectivity assays accessed through research contracts. Many assays have been solely developed in-house at NCATS, while a number of others have been designed in collaboration with both intramural and extramural laboratories with subject area expertise. The COVID-19 pandemic is a pressing public health emergency garnering rapid response from scientists across the globe, and NCATS scientists have been conducting screening and drug repurposing against a number of SARS-CoV-2-related targets in response: 1) Host cell invasion is initiated through direct binding of the viral spike protein to the host receptor ACE2. Members of this team developed a proximity-based AlphaLISA assay to measure binding of SARS-CoV-2 spike RBD protein to ACE2 and used it to run a drug-repurposing screen, yielding 25 high-quality, small-molecule hits worthy of follow-up in cell-based models. This established AlphaLISA RBD-ACE2 platform allows scientists to evaluate disruption of this viral-host interaction, and this assay has been reported at multiple conferences. 2) The TMPRSS2 enzyme has been shown to be critically important for priming SARS-CoV-2 for viral entry into host cells, yet no SARS-CoV-2-related biochemical assay for this target had been previously reported. To enable inhibitor discovery and profiling of FDA-approved therapeutics, this team developed a biochemcial assay using recombinant TMPRSS2 suitable for high-throughput screening applications. The team validated this important assay, and demonstrated effectiveness to quantify inhibition down to subnanomolar concentrations using clinically approved proteases. This TMPRSS2 work will enable further screening against this important antiviral host target, and provide insight into selectivity and potency of repurposing candidates. 3) Antibodies that physically block viral entry of SARS-CoV-2 may provide leads for further therapeutic development. This project team used an innovative combination of humanized nanobody library and enrichment strategies (also known as phage panning) to discover several clones of antibodies capable of high-affinity binding to SARS-CoV-2 spike protein. Because these highly engineered antibody variants already incorporate beneficial features such as similarity to human antibodies, they are expected to be developed into viable therapeutic leads faster than other types of neutralizing antibodies. 4) Pulmonary fibrosis is a major problem associated with severe cases of SARS-CoV-2 infections and the leading cause of mortality associated with COVID-19. Currently, the only treatment is supplemental oxygen and mechanical ventilation to hospitalized COVID patients. There is no effective therapeutic measure to mitigate the disease. The goal of this project is to develop small molecular inhibitors as therapeutic measures to mitigate COVID-19 associated lung fibrosis.The project's goal is to develop small molecular inhibitors as therapeutic measures to mitigate COVID-19 associated lung fibrosis. During this period, we tried to optimize the absorbance-based assay developed in our collaborator's lab and develop fluorescence-based thrombin activity assay.

在过去的报告期间，NCAT继续开发许多基于目标的SARS-COV-2分析，以筛选针对该病毒的潜在命中。对在NCATS组装的一组药物重新利用，临床候选和抗病毒库进行了优化测定。通过NCATS Opendata门户以及许多预印本/期刊出版物，这些结果以空前的速度自由地向公共场所提供。 这些测定法包括病毒（例如峰值蛋白）和宿主（例如ACE2，TMPRSS）靶标，以及通过研究合同访问的BSL3 Live SARS-COV-2病毒感染性检测分析。许多测定法在NCAT的内部开发，而其他许多测定方法是与具有主题领域专业知识的壁内和壁外实验室合作设计的。 COVID-19大流行是一个紧迫的公共卫生紧急紧急情况，该紧急情况得到了全球科学家的快速反应，而NCATS科学家一直在对许多与SARS-COV-2相关的目标进行筛查和药物重新使用： 1）宿主细胞侵袭是通过病毒尖峰蛋白与宿主受体ACE2的直接结合而引发的。该团队的成员开发了一种基于接近度的α分析，以测量SARS-COV-2 SPIKE RBD蛋白与ACE2的结合，并使用它来运行药物固定筛选，产生了25种高质量的，小分子在基于细胞模型中值得随访的小分子。这个已建立的Alphalisa RBD-ACE2平台允许科学家评估这种病毒宿主相互作用的破坏，并且在多个会议上报道了该测定法。 2）TMPRSS2酶已被证明对于启动SARS-COV-2对宿主细胞的启动至关重要，但先前没有报道过该靶标的SARS-COV-2与SARS-COV-2相关的生化测定法。为了实现FDA批准的治疗剂的抑制剂发现和分析，该团队使用适用于高通量筛查应用的重组TMPRSS2开发了生化测定。该团队验证了这项重要的测定法，并证明了使用临床认可的蛋白酶将抑制作用量化为亚洋摩尔浓度的有效性。这项TMPRSS2工作将使对这一重要的抗病毒宿主目标进行进一步的筛查，并洞悉重新利用候选人的选择性和效力。 3）物理阻断SARS-COV-2病毒进入的抗体可能为进一步的治疗发育提供潜在客户。该项目团队使用了人性化纳米库文库和富集策略（也称为噬菌体平鸟）的创新组合，发现了几种能够与SARS-COV-2尖峰蛋白具有高亲和力结合的抗体克隆。由于这些高度工程的抗体变体已经结合了有益的特征，例如与人类抗体的相似性，因此预计它们将比其他类型的中和抗体更快地发展为可行的治疗铅。 4）肺纤维化是与严重的SARS-COV-2感染病例以及与Covid-19相关的死亡率的主要原因有关的主要问题。目前，唯一的治疗方法是补充氧气和对住院的共同患者的机械通气。没有有效的治疗方法来减轻疾病。该项目的目的是开发小分子抑制剂，作为减轻199相关肺纤维化的治疗措施。该项目的目标是开发小分子抑制剂作为治疗措施，以减轻COVID-COVID-19S相关的肺纤维化。在此期间，我们试图优化合作者实验室中开发的基于吸光度的测定，并开发基于荧光的凝血酶活性测定。