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

The main protease (Mpro) 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 Mpro 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 Mpro from herbal extracts and decipher their synergistic anti-Mpro mechanisms. Preliminary screening identified Rhodiola crenulata root (RCR), a well-known Tibetan herb, showing the most potent time-dependent inhibition against SARS-CoV-2 Mpro. By integrating fluorescence resonance energy transfer (FRET)-based biochemical assay with phytochemical and chemoproteomic profiling, we efficiently identified thirteen Mpro covalent inhibitors from the crude extract of RCR. Among these, rhodiosin and gallic acid were validated as the key anti-Mpro constituents, due to their strong anti-Mpro effects and high abundance in RCR. Remarkably, their combination exhibited a pronounced synergy in Mpro 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 Mpro dimerization and significantly facilitating the covalent modification of Mpro by gallic acid. Collectively, the covalent SARS-CoV-2 Mpro inhibitors are efficiently identified from a Tibetan herb, while a phytochemical combination with synergistic anti-Mpro effects and their unique allosteric-induced cooperative modification mechanism are revealed.