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

The main protease (M^pro) of severe acute respiratory syndrome coronavirus 2 (SARSCoV-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 gallic acid. Collectively, the covalent SARS-CoV-2 M^pro inhibitors are efficiently identified from a Tibetan herb, while a phytochemical combination with synergistic anti-M^pro effects and their unique allosteric-induced cooperative modification mechanism are revealed.