| Assay Method Information | |
| | Determination of inhibition of SARS-CoV-2 Mproby 11r, 13a,and 13b |
| Description: | A fluorescent substrate harboring the cleavage site (indicated by the arrow, ↓) of SARS-CoV-2 Mpro(Dabcyl-KTSAVLQ↓SGFRKM-E(Edans)-NH2; GL Biochem) and buffer composed of 20 mM Tris, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.3 was used for the inhibition assay. In the fluorescence resonance energy transfer (FRET)-based cleavage assay (14), the fluorescence signal of the Edans generated due to the cleavage of the substrate by the Mprowas monitored at an emission wavelength of 460 nm with excitation at 360 nm, using a Flx800 fluorescence spectrophotometer (BioTek).Initially, 2.5 uL of the SARS-CoV-2 or SARS-CoV Mproat the final concentration of 2.0 uM was pipetted into a 96-well plate containing pre-pipetted 22.5 uLreaction buffer. Afterwards, the reaction was initiated by addition of 25 uL of thesubstrate dissolved in the reaction buffer to 50 uL final volume, at different final concentrations varied from 5 to 640 uM (5, 10, 20, 40, 80, 160, 320, 640 uM). A calibration curve was generated by measurement of varied concentrations (from 0.15 to 20 uM) of free Edans in a final volume of 50 uL reaction buffer. Initial velocities were determined from the linear section of the curve, and the corresponding relative fluorescence units per unit of time (RFU/s) was converted to the amount of the cleaved substrate per unit of time (uM/s) by fitting to the calibration curve of free Edans.Inner-filter effect corrections were applied for the kinetic measurement saccording to (30). The fluorescence of the substrate (in RFU) dissolved in 50 uL final volume of reaction buffer at the corresponding concentrations used for the kinetic assay was measured and defined as f(substrate). Afterwards, 1 uL free Edans was added (final concentration: 5 uM) to each well, and the fluorescence reading was taken as f(substrate + Edans). Simultaneously, are ference value (in RFU)was measured with the same concentration of free Edans in 50 uL of reaction buffer, giving f(reference). Stock solutions of the compounds were prepared with 100% DMSO. For the determination of the IC50, 0.5 uM of SARS-CoV-2Mprowas incubated with 11r, 13a, or 13bat various concentrations from 0 to 100 uM in reaction buffer at 37 C for 10 min. Afterwards, the FRET substrate at a final concentration of 20 uM was added to each well at a final total volume of 50 uL to initiate the reaction. The GraphPad Prism 6.0 software (GraphPad) was used for the calculation of the IC50 values. Measurements of inhibitory activities of the compounds were performed in triplicate and are presented as the mean +/- SD. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |