严重急性呼吸系统综合症冠状病毒-2(SARS-CoV-2,SARS2)是COVID-19大流行和感染的原因,这些感染继续影响着全球数百万人的生活,尤其是那些年龄较大和/或免疫功能低下的人。SARS2主要蛋白酶,Mpro(也称为3C样蛋白酶,3CLpro),是一种真正的药物靶标,尼马特雷韦和安替特雷韦的有效抑制作用证明了这一点,药物Paxlovid和Xocova的活性成分,分别。然而,耐尼曲韦和耐安曲韦分离株的存在强调了开发具有不同耐药谱和/或不同作用机制的下一代药物的必要性.这里,我们报告了使用细胞信号增益测定法对649,568种化合物进行高通量筛选的结果.在这个试验中,Mpro抑制荧光素酶报告基因的表达,和8,777个小分子被认为是命中,导致荧光素酶活性的增加比样品场活性高3倍SD(相对于100µMGC376为6.8%的信号增益)。单一浓度和剂量-响应信号增益实验证实了3,522/8,762种化合物作为候选抑制剂。并行,所有初始高通量筛选命中均在肽切割试验中用纯化的Mpro进行了测试,只有39/8,762显示出抑制.重要的是,19/39化合物(49%)在两个SARS2检测中重新测试为阳性,包括两种先前报道的Mpro抑制剂,证明整体筛查策略的有效性。这种方法导致重新发现已知的Mpro抑制剂,例如钙蛋白酶抑制剂II,以及发现新的化合物,为未来的药物开发工作提供化学信息。
Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2, SARS2) is responsible for the COVID-19 pandemic and infections that continue to affect the lives of millions of people worldwide, especially those who are older and/or immunocompromised. The SARS2 main protease enzyme, Mpro (also called 3C-like protease, 3CLpro), is a bona fide drug target as evidenced by potent inhibition with nirmatrelvir and ensitrelvir, the active components of the drugs Paxlovid and Xocova, respectively. However, the existence of nirmatrelvir and ensitrelvir-resistant isolates underscores the need to develop next-generation drugs with different resistance profiles and/or distinct mechanisms of action. Here, we report the results of a high-throughput screen of 649,568 compounds using a cellular gain-of-signal assay. In this assay, Mpro inhibits expression of a luciferase reporter, and 8,777 small molecules were considered hits by causing a gain in luciferase activity 3x SD above the sample field activity (6.8% gain-of-signal relative to 100 µM GC376). Single concentration and dose-response gain-of-signal experiments confirmed 3,522/8,762 compounds as candidate inhibitors. In parallel, all initial high-throughput screening hits were tested in a peptide cleavage assay with purified Mpro and only 39/8,762 showed inhibition. Importantly, 19/39 compounds (49%) re-tested positive in both SARS2 assays, including two previously reported Mpro inhibitors, demonstrating the efficacy of the overall screening strategy. This approach led to the rediscovery of known Mpro inhibitors such as calpain inhibitor II, as well as to the discovery of novel compounds that provide chemical information for future drug development efforts.