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

• 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
• 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

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) 一般的。针对 CoV2 酶的药物筛选系统通常涉及筛选 纯化的酶。这在技术上具有挑战性，并且绝大多数抑制剂已被识别 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， 细胞内发生改变，使其不再有效抑制靶标，或表现出脱靶效应 阻止它们用作治疗药物。直接在病毒感染测定中使用预测药物可以表现出 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） 例子）。理想的情况是筛选病毒酶，将酶从病毒感染中分离出来，但评估 在人体细胞内发挥作用。我们构建了表达四种野生型 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 蛋白酶和 CoV RNA 依赖性 RNA 聚合酶复合物。我们正在完成分析验证 并开始使用测定法来评估这些酶的少数已知抑制剂。蛋白酶 检测将转化为稳定的细胞系，用于大规模筛选计划。转染测定 将使用已建立的和实验性的 3CL 和 RdRpC 抑制剂以及 128 种 FDA- 我们预测以这些酶为目标的批准药物，以验证这些酶的有用性 筛选分析。将使用剂量反应分析对表现出活性抑制作用的药物进行定量 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 肺细胞具有相似的剂量反应范围。验证后，载体和/或稳定细胞系 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。