PDF(Pubmed)
Abstract：
While research has identified several inhibitors of the main protease (Mpro) of SARS-CoV-2, a significant portion of these compounds exhibit reduced activity in the presence of reducing agents, raising concerns about their effectiveness in vivo. Furthermore, the conventional biosafety level 3 (BSL-3) for cellular assays using viral particles poses a limitation for the widespread evaluation of Mpro inhibitor efficacy in a cell-based assay. Here, we established a BSL-1 compatible cellular assay to evaluate the in vivo potential of Mpro inhibitors. This assay utilizes mammalian cells expressing a tagged Mpro construct containing N-terminal glutathione S-transferase (GST) and C-terminal hemagglutinin (HA) tags and monitors Mpro autodigestion. Using this method, GC376 and boceprevir effectively inhibited Mpro autodigestion, suggesting their potential in vivo activity. Conversely, carmofur and ebselen did not exhibit significant inhibitory effects in this assay. We further investigated the inhibitory potential of selenoneine on Mpro using this approach. Computational analyses of binding energies suggest that noncovalent interactions play a critical role in facilitating the covalent modification of the C145 residue, leading to Mpro inhibition. Our method is straightforward, cost-effective, and readily applicable in standard laboratories, making it accessible to researchers with varying levels of expertise in infectious diseases.
摘要：
虽然研究已经确定了SARS-CoV-2的主要蛋白酶（Mpro）的几种抑制剂，但这些化合物的很大一部分在还原剂存在下表现出降低的活性。引起人们对其体内有效性的担忧。此外，使用病毒颗粒的细胞试验的常规生物安全3级(BSL-3)对基于细胞的试验中Mpro抑制剂功效的广泛评估造成了限制.这里,我们建立了一个与BSL-1相容的细胞试验来评估Mpro抑制剂的体内潜力.该测定利用表达含有N-末端谷胱甘肽S-转移酶(GST)和C-末端血凝素(HA)标签的标记的Mpro构建体的哺乳动物细胞并监测Mpro自身消化。使用此方法，GC376和boceprevir有效抑制Mpro自身消化，表明它们在体内的潜在活性。相反，卡莫富和依布硒在该测定中没有表现出明显的抑制作用。我们使用这种方法进一步研究了硒酮对Mpro的抑制潜力。结合能的计算分析表明，非共价相互作用在促进C145残基的共价修饰中起关键作用。导致Mpro抑制。我们的方法很简单，成本效益高,并容易适用于标准实验室，使具有不同传染病专业知识水平的研究人员可以使用它。
