SARS-CoV-2 main protease (Mpro) autocatalytically releases itself out of the viral polyprotein to form a fully active mature dimer in a manner that is not fully understood. Here, we introduce several tools to help elucidate differences between cis (intramolecular) and trans (intermolecular) proteolytic processing and to evaluate inhibition of precursor Mpro. We found that many mutations at the P1 position of the N-terminal autoprocessing site do not block cis autoprocessing but do inhibit trans processing. Notably, substituting the wild-type glutamine at the P1 position with isoleucine retains Mpro in an unprocessed precursor form that can be purified and further studied. We also developed a cell-based reporter assay suitable for compound library screening and evaluation in HEK293T cells. This assay can detect both overall Mpro inhibition and the fraction of uncleaved preMpro through separable fluorescent signals. We observed that inhibitory compounds preferentially block mature Mpro. Bofutrelvir and a novel compound designed in-house showed the lowest selectivity between precursor and mature Mpro, indicating that inhibition of both forms may be possible. Additionally, we observed positive modulation of precursor activity at low concentrations of inhibitors. Our findings help expand understanding of the SARS-CoV-2 viral life cycle and may facilitate development of strategies to target preMpro for inhibition or premature activation of Mpro.

中文翻译：

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SARS-CoV-2 主要蛋白酶的产酶形式：药物发现的离散靶标。


SARS-CoV-2 主要蛋白酶 （Mpro） 自催化地从病毒多蛋白中释放出来，以一种尚不完全清楚的方式形成完全活性的成熟二聚体。在这里，我们介绍了几种工具来帮助阐明顺式（分子内）和反式（分子间）蛋白水解加工之间的差异，并评估前体 Mpro 的抑制作用。我们发现 N 端自动加工位点 P1 位点的许多突变不会阻止顺式自动加工，但会抑制反式加工。值得注意的是，用异亮氨酸取代 P1 位的野生型谷氨酰胺可将 Mpro 保留为未加工的前体形式，可以进行纯化和进一步研究。我们还开发了一种基于细胞的报告基因检测，适用于HEK293T细胞中的化合物库筛选和评估。该测定可以通过可分离的荧光信号检测总体 Mpro 抑制和未裂解的 preMpro 的分数。我们观察到抑制性化合物优先阻断成熟的 Mpro。Bofutrelvir 和内部设计的新型化合物在前体和成熟 Mpro 之间的选择性最低，表明两种形式的抑制都是可能的。此外，我们观察到在低浓度抑制剂下前体活性的正调节。我们的研究结果有助于扩大对 SARS-CoV-2 病毒生命周期的理解，并可能有助于开发靶向 preMpro 以抑制或过早激活 Mpro 的策略。