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

项目摘要/摘要： SARS CoV-2(CoV2)和冠状病毒(CoV)急需有效的抗病毒药物 将军。针对CoV2酶的药物筛选系统通常涉及对 纯化的酶。这在技术上可能是具有挑战性的，而且绝大多数抑制剂都已确定 最终缺乏作为抗病毒药物的效用，因为：它们可能不被输送到细胞中并保留在细胞中， 在细胞内发生改变，使得它们不再有效地抑制目标，或表现出偏离目标的效果 排除了它们被用作治疗的可能性。直接在病毒感染检测中使用预测药物可能会显示 对宿主细胞或其他病毒蛋白的非靶标效应，在 病毒感染的背景(羟氯喹对CoV2感染的抑制就是其中之一 例如)。理想的情况是对病毒酶进行筛选，将酶从病毒感染中分离出来，但要评估 在人体细胞内发挥作用。我们构建了四个野生型CoV2的表达载体 蛋白质和独特的生物传感器，以评估CoV2的两种酶功能，CoV Main 蛋白水解酶和冠状病毒RNA依赖的RNA聚合酶复合体。我们正在完成化验验证 并开始使用这些分析来评估这些酶的为数不多的已知抑制剂。水解酶 试验将转化为稳定的细胞系，用于大规模筛选计划。转染法 将使用已建立的和实验性的3CL和RdRpC抑制剂以及128 FDA- 我们预测的针对这些酶的批准药物，以验证这些酶的有效性 筛选化验。显示活性抑制的药物将使用剂量-反应分析进行量化。 在基于细胞的检测中，随后分析了对培养的人CoV2感染的抑制作用 在相似的剂量反应范围内的肺细胞。在验证后，载体和/或稳定的细胞系 对于基于细胞的药物筛选系统，其他研究人员将很容易获得，例如 他们目前正在寻找冠状病毒抗病毒药物。

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.