Creation and Validation of cell-based screening systems for SARS-CoV-2 drug targets

SARS-CoV-2 药物靶标细胞筛选系统的创建和验证

基本信息

批准号：
10618835
负责人：
THOMAS MELENDY
金额：
$ 19.94万
依托单位：
STATE UNIVERSITY OF NEW YORK AT BUFFALO
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-06 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10618835
关键词：
2019-nCoV Amino Acids Antiviral Agents Appearance Biological Assay Biosensor COVID-19 vaccine Cell Line Cells Chymase Code Complex Coronavirus Development Disease Outbreaks Dose Drug Screening Drug Targeting Economics Enzyme Inhibition Enzyme Inhibitor Drugs Enzymes Exhibits FDA approved Firefly Luciferases Fluorescence Future Genomic DNA Health Human Hydroxychloroquine Individual Infection Libraries Luciferases Lung Messenger RNA National Center for Advancing Translational Sciences Peptide Hydrolases Pharmaceutical Preparations Plasmids Point Mutation Polyproteins Proteins RNA Polymerase Inhibitor RNA-Directed RNA Polymerase Recovery Renilla Luciferases Research Personnel Ribonucleic Acid Regulatory Sequences SARS-CoV-2 antiviral SARS-CoV-2 infection SARS-CoV-2 inhibitor Series Site System Testing Therapeutic Transfection Translating Uncertainty United States National Institutes of Health Vaccines Validation Viral Viral Genome Viral Physiology Viral Proteins Virus Diseases antiviral drug development coronavirus antiviral coronavirus pandemic design enzyme activity expression vector improved in vitro activity inhibitor novel coronavirus red fluorescent protein resistant strain response screening screening program stable cell line vaccine acceptance vector zoonotic coronavirus

项目摘要

Project Summary/Abstract: Effective antivirals are sorely needed against SARS CoV-2 (CoV2), and coronaviruses (CoV) in general. Screening systems for drugs targeting CoV2 enzymes usually involve screening against purified enzymes. This can be technically challenging, and the vast majority of inhibitors identified ultimately lack utility as antivirals because: they may not be transported into and retained in cells, altered within cells such that they no longer inhibit the target effectively, or exhibit off-target effects that preclude their use as therapeutics. Using predicted drugs directly in viral infection assays can exhibit off-target effects against the host cell or other viral proteins and can be difficult to distinguish within the context of the viral infection (the inhibition of CoV2 infection by hydroxychloroquine is one such example). Ideally screens for viral enzymes isolate the enzyme from the viral infection, but evaluate function within human cells. We have constructed vectors for expression of four wild-type CoV2 proteins, and unique biosensors, to evaluate function of two CoV2 enzymatic functions, the CoV Main Protease and the CoV RNA-dependent RNA polymerase complex. We are completing assay validation and beginning to use the assays to evaluate the few known inhibitors of these enzymes. The protease assay will be converted to a stable cell line for large scale screening programs. The transfection assays will be evaluated using both established and experimental 3CL and RdRpC inhibitors, and 128 FDA- approved drugs that we have predicted target these enzymes, to validate the usefulness of these screening assays. Drugs that show inhibition of activity will be quantified using dose-response analysis in the cell-based assays, and subsequently analyzed for inhibition of CoV2 infection of cultured human lung cells across a similar dose-response range. Following validation, vectors and/or stable cells lines for the cell-based drug screening systems will be made readily available to other researchers, such as NIH’s NCATS, who are currently seeking CoV antiviral agents.

项目摘要/摘要：迫切需要有效的抗病毒药物来对抗 SARS CoV-2 (CoV2) 和冠状病毒 (CoV) 概述针对 CoV2 酶的药物筛选系统通常涉及筛选。这在技术上具有挑战性，并且已鉴定出绝大多数抑制剂。最终缺乏作为抗病毒药物的效用，因为：它们可能不会被转运到细胞中并保留在细胞中，细胞不再有效抑制靶标，或表现出脱靶效应直接在病毒感染测定中使用预测的药物可能会阻止它们用作治疗剂。针对宿主细胞或其他病毒蛋白的脱靶效应，并且很难在病毒感染的背景（羟氯喹抑制 CoV2 感染就是其中之一）理想的情况是筛选病毒酶，将酶从病毒感染中分离出来，但要进行评估。我们构建了用于表达四种野生型 CoV2 的载体。蛋白质和独特的生物传感器，以评估两种 CoV2 酶功能的功能，即 CoV Main 我们正在完成蛋白酶和 CoV RNA 依赖性 RNA 聚合酶复合物的检测验证。并开始使用测定法来评估这些酶的少数已知抑制剂。转染测定将转化为稳定的细胞系，用于大规模筛选计划。将使用已建立的和实验性的 3CL 和 RdRpC 抑制剂以及 128 种 FDA- 批准我们预测了这些酶的靶药物，以验证这些酶的有用性将使用剂量反应分析来量化显示活性抑制的药物。在基于细胞的检测中，随后分析了对培养人类的 CoV2 感染的抑制作用验证后，载体和/或稳定细胞系具有相似的剂量反应范围。基于细胞的药物筛选系统将易于其他研究人员使用，例如 NIH 的 NCATS 目前正在寻找 CoV 抗病毒药物。