| Assay Method Information | |
| | Biochemical Assay |
| Description: | Compounds (as 10 mM DMSO stock) were serial diluted 4-fold using 100% DMSO in a LabCyte 384-well LDV plate and acoustically transferred using a LabCyte ECHO 550 into Corning 384-well black NBS plates. Standard 10-point IC50 384-well plate layout is as follows: 100 μM of 2-(1H-benzo[d][1,2,3]triazol-1-yl)-N-(4-(pyridin-3-yl)phenyl)-N-(thiophen-3-ylmethyl)acetamide was stamped into columns 1 and 24 (low control), DMSO was stamped into columns 2 and 23 (high control), and serial diluted compounds were stamped from high (100 μM) to low (0.38 nM) concentrations in columns 3-12 (replicate 1) and 13-22 (replicate 2). Protocol for running the assay is as follows: assay wells stamped with 0.25 μL of compound or DMSO were filled via a ThermoFisher Multidrop Combi liquid dispenser with 14.5 μL of 150 nM or 200 nM (concentration for 25 μL final reaction volume) of SARS-CoV-1 or SARS-CoV-2 3CLProM, respectively, in assay buffer (50 mM HEPES, 0.1 mg/ml BSA, 0.01% v/v TRITON X100, 2 mM DTT, pH 7.5). Assay plates were then centrifuged at 1,000 RPM (Eppendorf 5810R, S-4-104 rotor) for 1 minute, covered, and incubated at room temperature for 15 minutes. Reactions were initiated using the Multidrop Combi liquid dispenser to titrate 10 μL of 2 μM (concentration for 25 μL final reaction volume) of fluorophore-quencher peptide substrate (from AnaSpec, Inc. Catalog No. AS-65599) solubilized in assay buffer into each well. Assay plates were again centrifuged at 1,000 RPM for 1 minute, covered, and incubated at room temperature for 30 minutes. |
| Affinity data for this assay | |
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