The important hydrogen bonds with Glu166 are formed by the 6-hydroxyl group attached to the chromen-4-one moiety (2.778 ?) and the 5-hydroxyl group attached to the glucuronate moiety (3.331 ?). This study suggests important scaffolds to design 3CLpro inhibitors to develop antiviral agents or health-foods and dietary supplements to cope with SARS-CoV-2. BL21 (DE3) for protein ROCK inhibitor-2 expression. BL21 (DE3) cells were grown on LuriaCBertani (LB) agar plates containing 150?(6500 rev min?1) for 10?min in a high-speed refrigerated centrifuge at 277?K. The cultured cell paste was resuspended in 25?ml of a buffer consisting of 50?mM TrisCHCl pH 8.0, 100?mM NaCl, 10?mM imidazole, 1?mM phenylmethylsulfonyl fluoride (PMSF), 10?(15,000 rev min?1) for 30?min in a high-speed refrigerated ultra-centrifuge at 277?K. The protein was purified by affinity chromatography using a 5?ml Hi-Trap Q His column (GE Healthcare, Piscataway, New Jersey, USA). The column was equilibrated with a buffer consisting of 20?mM Bis-Tris pH ROCK inhibitor-2 7.5. The column was eluted using a linear NaCl gradient to 1 1.0?M NaCl and the protein was eluted at 0.18?M NaCl. The purified protein was buffer exchanged into 20?mM Bis-Tris pH 7.5 using Vivaspin 20 MWCO 10?kDa (GE Healthcare), a centrifugal device. SDSCPAGE showed one band around 34?kDa (33796.64?Da), corresponding to the molecular weight of SARS-CoV-2 3CLpro. FRET protease assays with SARS-CoV-2 3CLpro The custom-synthesised fluorogenic substrate, DABCYL-KTSAVLQSGFRKME-EDANS (ANYGEN, Gwangju, Korea), was used as a substrate for the proteolytic assay using the SARS-CoV-2 3CLpro9. This substrate contains the nsp4/nsp5 cleavage sequence, GVLQSG10, and works as a generic peptide substrate for many coronaviruses including the SARS-CoV-2 3CLpro. The peptide was dissolved in distilled water and incubated with each protease. A SpectraMax i3x Multi-mode microplate reader (Molecular Devices) was used to measure spectral-based fluorescence. The proteolytic activity was determined at 310?K by following the increase in fluorescence PDGFRA (excitation = 340?nm, emission = 490?nm, bandwidths = 9, 15?nm, respectively) of EDANS upon peptide hydrolysis as a function of time. Assays were conducted in black, 96-well plates (Nunc) in 300?culture. The amount of purified protein synthesised was 29.85?mg. For storage and assay, the protein solution was concentrated to 99.5?mg ml?1. The concentrate was diluted to 2?percent fluorescence inhibition (Figure 2). The compound showed the severely reduced fluorescent ROCK inhibitor-2 intensity and thus represented their SARS-CoV-2 3CLpro inhibitory activity. The IC50 values were calculated from the dose-dependent inhibitory curves of baicalin, herbacetin and pectolinarin. The measured values were 34.71?docking study for baicalin, herbacetin and pectolinarin was performed to deduce their binding mode and binding affinity. The glide scores of three compounds were ?8.776, ?8.738 and ?10.969, respectively. The binding modes of herbacetin and pectolinarin were similar to those obtained from the docking study of the catalytic domain of SARS-CoV 3CLpro21. In the case of herbacetin, the phenyl moiety occupies the S1 site of SARS-CoV-2 with the aid of Glu166 and the chromen-4-one scaffold locates in the S2 site with hydrogen bonds with His41 and Gln189. The binding mode of pectolinarin showed that the l-mannopyranosyl -d-glucopyranoside moiety occupies the S1 and S2 sites and the chromen-4-one moiety locates in the S2 and S3 site similar to the binding mode observed in SARS-CoV 3CLpro. The binding mode of baicalin is described in ROCK inhibitor-2 Figure 4. Due to the presence of the glucuronate moiety, its binding mode is more similar to that of pectolinarin than herbacetin. The important hydrogen bonds with Glu166 are formed by the 6-hydroxyl group attached to the chromen-4-one moiety (2.778 ?) and the 5-hydroxyl group attached to the glucuronate moiety (3.331 ?). As a result, more than half of the compound was docked in the S1 site. The two hydrogen bonds of the 3-hydroxyl and carboxylic acid groups from the glucuronate moiety formed with Gly143 (2.962 ?) and Asn142 (2.883 ?), respectively, stabilise the overall interaction. The C stacking between His41 and.