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

项目概要/摘要： 在中国，迫切需要有效的抗病毒药物来对抗SARS CoV-2（CoV 2）和冠状病毒（CoV）。 将军靶向CoV 2酶的药物筛选系统通常涉及针对以下各项的筛选： 纯化酶这在技术上是具有挑战性的，而且绝大多数已鉴定的抑制剂 最终缺乏作为抗病毒药物的实用性，因为：它们不能被转运并保留在细胞中， 在细胞内改变，使得它们不再有效地抑制靶标，或表现出脱靶效应， 排除了它们作为治疗剂的用途。在病毒感染测定中直接使用预测的药物可以表现出 针对宿主细胞或其他病毒蛋白的脱靶效应，并且可能难以在细胞内区分。 在病毒感染的背景下（通过羟氯喹抑制CoV 2感染是这样一种 示例）。理想的病毒酶筛选分离酶从病毒感染，但评估 在人体细胞中发挥作用。我们构建了表达四种野生型CoV 2的载体， 蛋白质和独特的生物传感器，以评估两种CoV 2酶功能的功能，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 并开始使用这些分析来评估这些酶的少数已知抑制剂。蛋白酶 试验将转化为稳定的细胞系用于大规模筛选程序。转染试验 将使用已建立的和实验性的3CL和RdRpC抑制剂以及128种FDA- 我们预测的靶向这些酶的批准药物，以验证这些酶的有效性， 筛选测定。将使用剂量反应分析定量显示活性抑制的药物 在基于细胞的测定中，并随后分析培养的人的CoV 2感染的抑制 肺细胞在相似的剂量反应范围内。验证后，载体和/或稳定细胞系 对于基于细胞的药物筛选系统将很容易提供给其他研究人员，如 NIH的NCATS，他们目前正在寻找CoV抗病毒剂。

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.