3.4 Anti-SARS-CoV-2 Mpro effects of the newly identified covalent binders
Next, the anti-Mpro effects of these newly identified covalent binders from LJ were validated by using the standards or the purified compounds. Herein, a total of 20 naturally occurring Mpro binders isolated from LJ were collected and their anti-Mpro effects were tested one by one. As demonstrated in Fig. 4 , among all tested natural covalent binders, nine compounds exhibited relatively strong inhibition to the proteolytic activity of Mpro in both time- and dose- dependent manners. Notably, gallic acid and quercetin exhibited the highest anti-Mpro potency with an IC50value of 6.18 μM and 9.44 μM after preincubation for 63 min. The IC50 values for the other compounds and the positive inhibitor (myricetin) are listed in Table 2 . The structures of all the above constituents are shown in Fig. S4 .
3.5 Inactivation kinetics of gallic acid and quercetin against SARS-CoV-2 Mpro
Next, the inactivation kinetics of gallic acid and quercetin were studied via performing a suit of SARS-CoV-2 Mpro inhibition assays. The residual activity of gallic acid and quercetin were measured by varying inhibitor concentrations at various time intervals. The obtained data was analyzed to determine the rate and mechanism of inactivation. As illustrated inFig. 5 , gallic acid and quercetin could inactivate the activity of SARS-CoV-2 Mpro in dose- and time- dependent manners. The KI value of gallic acid was determined to be 5.63 μM, and the Kinact value was calculated to be 0.05 min-1. In contrast, the inactivation of Mpro by quercetin was relatively weak with theKI value of 75.98 μM and theKinact value of 0.05 min-1.