Boc qar amc

The cell-based enzyme activity assay of TMPRSS2 was established in the previous study (Jin et al., 2020 (link)). 293T and 293T-TMPRSS2-expressing cells were seeded in a black, 96-well plate (20,000 cells/well). The next day, the growth medium was replaced with 80 μl of compounds or PBS alone to the wells in the indicated concentrations and incubated at room temperature for 1 hr. The 100 μM fluorogenic substrate Boc-QAR-AMC (R&D Biosystems) was added to each well and put in a 37℃ incubator for another 2 hr. Fluorescence (excitation 360 nm, emission 410 nm) was measured using Synergy H1 hybrid multi-mode microplate reader (BioTek Instruments, Inc).

Twenty-four hours after transfection, the media was replaced with 80 µL of phosphate-buffered saline (PBS). Inhibitors or PBS alone were added to the wells in the indicated concentrations and incubated at 25°C for 15 minutes. The fluorogenic substrate BOC-QAR-AMC (R&D Biosystems) was then added to each well to a final concentration of 100µM. Fluorescence (excitation 365 nm, emission 410 nm) was immediately measured every 15 minutes at 37°C using a GloMax plate reader (Promega).

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 (Thermo Fisher Scientific) 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 h transfection, cells were washed with PBS and the medium replaced with HCell-100 medium (Wisent) containing 200 µM Boc-QAR-AMC (R&D Systems) and either vehicle (0.01% DMSO) or compounds at the indicated concentration for 24 h. To measure proteolytic activity, 90 µl of cell medium 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 the 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]) versus a proteolytic activity plot using GraphPad Prism software (v.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 ± s.d. of at least three independent experiments.