| Assay Method Information | |
| | TMPRSS2 pericellular activity screening assay |
| Description: | Vero E6 cells were transfected with mock (pcDNA3.1), TMPRSS2 (pcDNA3.1/TMPRSS2 Uniprot: O15393-1), or TMPRSS2-S441A (pcDNA3.1/TMPRSS2-S441A) using Lipofectamine 3000 (ThermoFisher) in 12-well plates. For the mouse TMPRSS2 assay, empty vector (pCMV6-Entry, Origene #PS100001) and TMPRSS2-Myc-DDK (Origene: #MR207852) were used. After 24 hr transfection, cells were washed with PBS and media replaced with HCell-100 media (Wisent) containing 200 uM Boc-QAR-AMC (Rd Systems) and either vehicle (0.01% DMSO) or compounds at the indicated concentration for 24 hr. To measure proteolytic activity, 90 uL of cell media was transferred to a black 96-well plate, and fluorescence was measured at room temperature (excitation: 360 nm, emission: 460 nm) using an FLx800 TBE microplate reader (Bio-Tek Instruments). Background subtracted (mock-transfected cells) proteolytic activities are presented as percentage of activity relative to vehicle-treated cells (screen at 10 nM). IC50 values were determined after generating a nonlinear regression analysis from a log([Compound]) vs. a proteolytic activity plot using GraphPad Prism software (version 9.0.1). GraphPad Prism was used to identify and eliminate outliers (Q = 1) and assess the goodness of the fits. IC50 values presented are the mean +- standard deviation (SD) of at least three independent experiments. |
| Affinity data for this assay | |
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