| Assay Method Information | |
| | Enzyme Assay |
| Description: | For select test compounds, the fluorogenic enzymatic assay measuring inhibition of SARS-CoV-2 Mpro was followed by a subsequent chromatographic purification by HPLC to remove any potential fluorescence interference from the test compound. Assays contained test compound, 20 nM Mpro, 10 uM substrate (DABCYL-KTSAVLQSGFRKME-EDANS, SEQ ID NO:1), 1 uL test compound in 100% DMSO, in a final volume of 100 uL of assay buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM DTT, 0.005% Tween20). After 1 hr, the reaction was stopped by the addition of 10 uL of 100 uM GC-376 (10 uM final concentration). Test compounds, substrate and product were then separated using an Agilent 1260 Infinity HPLC system with a Luna 5 um 50×4.6 mm C8 LC column (Phenomenex) using 10 mM sodium acetate, pH 4.0 as the aqueous phase and acetonitrile as the organic phase. Both absorbance at 420 nm and fluorescence at 355 nm excitation and 460 mm emission were followed. Product peak area was then normalized to % inhibition where 0% was equal to the peak area in the absence of test compound and 100% was equal to the peak area in the absence of enzyme, which was typically not detectable. Positive control, GC-376 (IC50=10.7 nM), was tested in parallel with the test compounds to ensure the accuracy of the assay. |
| Affinity data for this assay | |
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