Development of Cell-based Assays to Identify SARS-CoV-2 Protease Inhibitor

冠状病毒是正链RNA病毒，基因组特别大（27-32 kb）。所有冠状病毒都在基因1a和1b中编码其复制所需的酶。基因1a和1b被翻译成多蛋白；1b在1a开放阅读帧的末端用-1移帧表示。在SARS-CoV和SARS-CoV-2中，1a多蛋白含有两种蛋白酶，一种是木瓜蛋白酶样蛋白酶（PL），另一种是3-凝乳胰蛋白酶样蛋白酶（3CL）。这些蛋白酶在14个位点（PL蛋白酶3个位点，3CL蛋白酶11个位点）切割多蛋白，产生16个非结构蛋白（nsps）。这些蛋白质包括RNA复制所需的病毒酶，包括RNA依赖的RNA聚合酶。RNA复制机制不仅产生更多的病毒基因组拷贝，而且还产生亚基因组RNA，作为结构蛋白翻译的mrna。因此，PL和3CL蛋白酶对病毒复制至关重要，这使它们成为抗病毒药物的极佳靶点。我们将开发基于人类细胞的检测方法，可作为鉴定SARS-CoV-2 3CL蛋白酶抑制剂的管道。这些检测将比目前基于蛋白质的检测具有优势，后者经常受到体外检测条件的影响，并且不能考虑化合物的吸收和毒性问题。此外，这些检测被设计用于BSL-2遏制，使它们比现有的BSL-3复制能力病毒检测更容易获得，后者不适合高通量平台。

Coronaviruses are positive-strand RNA viruses with exceptionally large genomes (27-32 kb). All coronaviruses encode the enzymes required for their replication in genes 1a and 1b. Genes 1a and 1b are translated as polyproteins; 1b is expressed by -1 frameshifting at the end of the 1a open reading frame. In both SARS-CoV and SARS-CoV-2, the 1a polyprotein contains two proteases, a papain-like (PL) and a 3-chymotrypsin-like (3CL) protease. Together, these proteases cleave polyprotein at 14 sites (3 for PL protease, 11 for 3CL protease), generating 16 nonstructural proteins (nsps). These proteins include viral enzymes required for RNA replication including RNA-dependent RNA polymerase. The RNA replication machinery not only generates more copies of the viral genome but also generates subgenomic RNAs that act as mRNAs for the translation of structural proteins. Thus, the PL and 3CL proteases are critical for viral replication, which make them excellent targets for antivirals. ___We will develop human cell-based assays that can be used as pipelines to identify inhibitors of the SARS-CoV-2 3CL protease. These assays will have advantages over current protein-based assays that are often affected by in vitro assay conditions and do not account for compound absorption and toxicity issues. Furthermore, these assays are designed to be used in BSL-2 containment, making them far more accessible than the existing BSL-3 replication-competent virus assays, which are not amenable to high-throughput platforms.