| Assay Method Information | |
| | Biochemical Assay |
| Description: | The potency of compounds against the SARS-CoV-2 Papain like protease (PLpro) was measured using a synthetic profluorogenic substrate Z-RLRGG-AMC. Compounds were serially diluted over 11 points using 100% DMSO as a diluent, from either a top concentration of 3 mM with a half-log dilution factor or a top dose of 0.1 mM with a 2-fold dilution factor. The top dose of compound in the assay was either 30 μM or 1 μM. The assay buffer contained 50 mM HEPES, pH 7.5, 1 mM TCEP and 0.1% BSA; final assay conditions included 1% DMSO, 6.25 nM PLPro enzyme, 25 μM peptide substrate and a 60 minute enzyme reaction time. Briefly, 250 nL of serially diluted compound was spotted into a white 384-well plate, followed by addition of SARS CoV2 PLpro enzyme (7.8 nM, 10 μL) in assay buffer. The compounds and PLpro enzyme were incubated for 30 minutes at room temperature. The reaction was then initiated by the addition of profluorogenic peptide substrate in assay buffer (5 μL, 125 μM Z-RLRGG-AMC). The reaction was allowed to progress for 60 minutes at 25° C. after which the plate was read on a Molecular Devices Spectramax M2e reader at an Ex/Em of 360 nm/460 nm. The no compound, zero percent inhibition (ZPE) control wells contained 1% DMSO vehicle with substrate and PLpro enzyme. The hundred percent effect (HPE) wells contained an internal Pfizer control compound at a dose sufficient to accomplish complete inhibition, 1% DMSO vehicle, substrate and PLpro enzyme. Data were analyzed with ActivityBase software (ID Business Solutions, Ltd). The raw data were transformed to percent activity values using the average from the ZPE and HPE control wells. |
| Affinity data for this assay | |
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