Uncovering the covalent inhibitors of SARS-CoV-2 Mpro in Tibetan edible herb Rhodiola crenulata and their synergistic anti-Mpro mechanism

作者：Guanghao Zhu, Yani Zhang, Yuan Xiong, Xudong Hou, Qingguang Zhang, Zhao-Qin Zhang, Xiaoyu Zhuang, Weidong Zhang, Guangbo Ge · 发表于：Journal of Pharmaceutical Analysis · 年份：2025 · DOI：10.1016/j.jpha.2025.101224 · 被引用次数：4 · 研究领域：Medicinal Plants and Bioactive Compounds、Pharmacological Effects of Natural Compounds、Plant-based Medicinal Research

The main protease (M pro ) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been validated as a therapeutic target for antiviral drug development, given its critical role in the viral life cycle. SARS-CoV-2 M pro contains 12 cysteine residues, which are susceptible to covalent modification by nucleophilic entities. In this study, we showcase an efficient strategy to uncover the key covalent inhibitors of SARS-CoV-2 M pro from herbal extracts and decipher their synergistic anti-M pro mechanisms. Preliminary screening identified Rhodiola crenulata root (RCR), a well-known Tibetan herb, showing the most potent time-dependent inhibition against SARS-CoV-2 M pro . By integrating fluorescence resonance energy transfer (FRET)-based biochemical assay with phytochemical and chemoproteomic profiling, we efficiently identified thirteen M pro covalent inhibitors from the crude extract of RCR. Among these, rhodiosin and gallic acid were validated as the key anti-M pro constituents, due to their strong anti-M pro effects and high abundance in RCR. Remarkably, their combination exhibited a pronounced synergy in M pro inhibition. Further intact protein mass measurements and top-down mass spectrometry (MS) analysis, complemented by biophysical methods, elucidated how these two compounds work in concert. Our findings revealed that rhodiosin functions as an allosteric inhibitor, disrupting M pro dimerization and significantly facilitating the covalent modification of M pro by...