| Assay Method Information | |
| | SARS-CoV-1 and -2 3CLpro Biochemical Assay |
| Description: | The protease activity and subsequent 10-point IC50 curves were spectroscopically determined using a scaled down, end point assay adapted from a previously described peptide-based Forster resonance energy transfer (FRET) assay. Compounds (as 10 mM DMSO stocks) were serially diluted 4-fold using 100% DMSO in a LabCyte 384-well LDV plate and acoustically transferred using a LabCyte ECHO 550 instrument into Corning 384-well black NBS plates. The standard 10-point IC50 384-well plate layout is as follows. First, 100 μM 8 was stamped into columns 1 and 24 (low control), DMSO was stamped into columns 2 and 23 (high control), and serially 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). To run the assay, 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 or 200 nM (concentration for a 25 μL final reaction volume) SARS-CoV-1 or SARS-CoV-2 3CLpro, respectively, in assay buffer [50 mM HEPES, 0.1 mg/mL BSA, 0.01% (v/v) TRITON X100, and 2 mM DTT (pH 7.5)]. Assay plates were then centrifuged at 1000 rpm (Eppendorf 5810R, S-4-104 rotor) for 1 min, covered, and incubated at room temperature for 15 min. Reactions were initiated using the Multidrop Combi liquid dispenser to titrate 10 μL of 2 μM (concentration for a 25 μL final reaction volume) fluorophore-quencher peptide substrate [HiyteFluor-488ESATLQSGLRKAK-(QXL)-NH2 from AnaSpec, Inc. catalog no. AS-65599] solubilized in assay buffer into each well. Assay plates were again centrifuged at 1000 rpm for 1 min, covered, and incubated at room temperature for 30 min. Biochemical assays were quenched through the addition of 5 μL of 500 mM acetic acid via a Multidrop Combi liquid dispenser. Assay plates were then centrifuged at 1000 rpm for 1 min, and the resulting fluorescence intensity was measured on a BioTek Cytation 5 multimode plate reader (λex = 485 nm; λem = 528 nm). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |