Fluorogenic Assays for Protease Inhibitors

The 3CLpro (SARS-CoV-2) assay kit (BPS Bioscience, San Diego CA, cat. no. 78042-2) is designed to measure 3CLpro activity and identify inhibitors of this enzyme, while human cathepsin L inhibitor screening kit (abcam, cat. Cambridge, UK, No. ab197012) and human calpain activity assay kit (AnaSpec. Inc. Fremont CA, cat. No. AS-72149) are designed to measure human cysteine protease activity and identify inhibitors of these enzymes. These assays were performed in a 96-well plate using a fluorogenic substrate. Briefly, a solution of each enzyme was prepared according to the manufacturer’s protocol in assay buffer. Separately, solutions of test compounds necessary to generate a seven-point dose response curve were prepared in half-log serial dilution. Test compounds were added to the plate, and the mixture was preincubated for 30 min at room temperature. A blank well (no enzyme) was included to assess the background signal, while the known inhibitors GC376, FF-FMK and B27-WT were used as positive controls for SARS-CoV-2 M^pro/3CL^pro, cathepsin L, and calpain, respectively. The plates of SARS-CoV-2 M^pro, cathepsin L, and calpain were incubated with each fluorogenic substrate for 4, 0.3, and 1 h, respectively, at room temperature. Then fluorescence intensity was measured in a Cytation 5 cell imaging multi-mode reader (BioTek, Winooski, VT, USA)(excitation/emission: 360/460 nm, excitation/emission: 400/505 nm, and excitation/emission: 490/520 nm), respectively. End point fluorescence intensities were measured, and the blank value were subtracted from all values.