Inhibition of SARS-CoV 3CL protease by flavonoids

Jo, Seri; Kim, Suwon; Shin, Dong Hae; Kim, Mi-Sun

The custom-synthesised fluorogenic substrate, DABCYL-KTSAVLQSGFRKME-EDANS (ANYGEN, Gwangju, Korea), was used as a substrate for the proteolytic assay using the SARS-CoV 3CLpro 18 . This substrate contains the nsp4/nsp5 cleavage sequence, GVLQ#SG 19 , and works as a generic peptide substrate for many coronavirus including the SARS-CoV 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 (k excitation ¼ 340 nm, k emission ¼ 490 nm, bandwidths ¼ 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 ll assay buffers containing protease and substrate as follow; For the SARS-CoV 3CLpro assay, 4.05 ll of 0.074 mM protease containing 50 mM Tris pH 6.5 was incubated with 7.5 ll of 0.1 mM substrate at 310 K for 2 h before measuring Relative Fluorescence Unit (RFU). Before the assay, the emission spectra of 64 flavonoids were surveyed after illuminating at 340 nm to avoid the overlapping with the emission spectrum of EDANS. Every compound was suitable to be tested. The final concentration of the protease, peptide and chemical used at the assay was 1, 2.5 and 20 lM each. At first, the SARS-CoV 3CLpro and chemical were mixed and pre-incubated at room temperature for 1 h. The reaction was initiated by the addition of the substrate and each well was incubated at 310 K for 16 h. After that, we measured the fluorescence of the mixture on the black 96-well plate using the endpoint mode of SpectraMax i3x where the excitation wavelength was fixed to 340 nm and the emission wavelength was set to 490 nm using 9, 15 nm bandwidth, respectively. All reactions were carried out in triplicate. Among the first 64 flavonoids (Supplementary Table 1 ), three of them were picked up to further assay at a concentration range of 2-320 lM. The IC 50 value which is the value causing 50% inhibition of the catalytic activity of the SARS-CoV 3CLpro was calculated by nonlinear regression analysis using GraphPad Prism 7.03 (GraphPad Software, San Diego, CA, USA).