| Assay Method Information | |
| | In Vitro Assay (SARS-CoV-2 Mpro) Enzymatic Assay |
| Description: | The C-His6-tagged SARS-CoV-2 MPRO (NC_045512) was cloned, expressed in E. coli, and purified by WuXi. The substrate of Dabcyl-KTSAVLQIISGFRKME-(Edans) was synthesized by Genscript. The assay buffer contained 20 mM of Tris-HCl (pH=7.3), 100 mM of NaCl, 1 mM of EDTA, 5 mM of TCEP and 0.1% BSA. The final concentrations of the Mpro protein and substrate were 25 nM and M, respectively, in the MPRO enzymatic assay. Reference compound GC376 was provided by WuXi AppTec and was included in each plate to ensure assay robustness. Test compounds were tested at single dose or 10 doses titration, in duplicate. Compounds were added to an assay plate (384w format) using ECHO, in duplicate wells. The final concentration is 10 μM for the single dose experiment. As for the full dose response experiment, samples were 3-fold serially diluted starting from 25 μM for 10 doses and added to an assay plate, in duplicate wells. The final concentrations (M) of each compound was 25, 8.33, 2.778, 0.926, 0.309, 0.103, 0.034, 0.011, 0.0038, and 0.0013. MPRO protein (25 μL, 30 nM) was added to an assay plate containing test compounds using a Multidrop. The test compound and MPRO protein were pre-incubated at RT for 30 min. Then, substrate (5 μL, 150 μM) was added to an assay plate. For 100% inhibition controls (HPE, high percent effect), 1 μM of GC376 was added. For no inhibition controls (ZPE, zero percent effect), the same volume of DMSO was added. The final DMSO concentration was 1%. Each activity testing point had a relevant background control without the enzyme to remove the fluorescence interference of the compound. After 60 min incubation at 30° C., the fluorescence signal (RFU) was detected using a microplate reader M2e (SpectraMax) at Ex/Em=340 nm/490 nm. |
| Affinity data for this assay | |
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