| Assay Method Information | |
| | Mpro mutant enzymatic assays |
| Description: | Compounds are 3 fold serially diluted for 10 doses and added to an assay plate (384w format) using ECHO, in duplicate wells. The test concentration of test compounds is defined by sponsor. For 100% inhibition control (HPE, hundred percent effect), 2 pM of GC376 is added. For no inhibition control (ZPE, zero percent effect), DMSO is added. 25 pL of Mpro proteins are added to the assay plates containing compounds, respectively, using Multidrop. The compounds and Mpro proteins are pre-incubated at room temperature for 30 min. Then 5 pL of Mpro substrates are added to the related assay plates, respectively. Each activity testing point has a relevant background control to normalize the fluorescence interference of compound. After 60 min incubation at 30°C, the fluorescence signal (RFU) is detected using a microplate reader M2e (SpectraMax) at Ex/Em=340nm/490nm. The inhibition activity is calculated using the formula below, IC50 values will be calculated using the Inhibition% data. |
| Affinity data for this assay | |
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