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，以针对SARS COV-2（COV2）和冠状病毒（COV）一般的。针对COV2酶的药物的筛查系统通常涉及筛查纯化的酶。这在技术上可能具有挑战性，绝大多数抑制剂确定最终缺乏作为抗病毒药的效用，因为：它们可能不会被转运到细胞中，并保留在细胞中，在细胞内改变，使它们不再有效地抑制目标，或暴露于脱靶效应排除他们用作治疗的使用。直接在病毒感染测定中使用预测的药物可以显示针对宿主细胞或其他病毒蛋白的脱靶作用，可能很难区分病毒感染的背景（羟氯喹抑制COV2感染就是这样一种例子）。理想情况下，病毒酶的筛选将酶从病毒感染中分离出来，但评估在人类细胞中的功能。我们已经构建了表达四种野生型COV2的向量蛋白质和独特的生物传感器，以评估两个COV2酶函数的功能，COV主蛋白酶和COV RNA依赖性RNA聚合酶复合物。我们正在完成测定验证并开始使用阿萨斯评估这些酶的少数已知抑制剂。蛋白酶测定将转换为大规模筛选程序的稳定单元线。翻译测定法将使用已建立和实验性3Cl和RDRPC抑制剂以及128 FDA-进行评估我们已经预测的批准针对这些酶的药物，以验证这些酶的实用性筛选分析。表现出抑制活性的药物将通过剂量反应分析进行定量在基于细胞的测定中，随后分析了对培养的人的COV2感染的抑制肺细胞跨越相似的剂量反应范围。验证后，向量和/或稳定的单元线线对于基于细胞的药物筛查系统，将容易为其他研究人员提供 NIH的NCAT，目前正在寻找COV抗病毒药。