Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease

Chunlong Ma; Brett Hurst; Yanmei Hu; Tommy Szeto; Bart Tarbet; Jun Wang

. CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.20.051581 doi: bioRxiv preprint k2/KI is commonly used to evaluate the efficacy for covalent inhibitor. We observed substrate depletion when proteolytic reactions progress longer than 90 min, therefore only first 90 min of the progress curves were used in the curve fitting ( Figure 6 middle column). In this study, we could not accurately determine the k2 for the protease inhibitors: Calpain inhibitor II, MG132, Boceprevir, and Calpain inhibitor XII, due to the very slow k2 in these case: significant substrate depletion before the establishment of the equilibrium between EI and EI*. In these cases, KI was Cytotoxicity measurement. A549, MDCK, HCT-8, Caco-2, Vero, and BEAS2B cells for cytotoxicity CPE assays were seeded and grown overnight at 37 °C in a 5% CO2 atmosphere to ∼90% confluence on the next day. Cells were washed with PBS buffer and 200 µl DMEM with 2% FBS and 1% penicillin−streptomycin, and various concentration of protease inhibitors was added to each well. 48 hrs after addition the protease inhibitors, cells were stained with 66 μg/ mL neutral red for 2 h, and neutral red uptake was measured at an absorbance at 540 nm using a . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.20.051581 doi: bioRxiv preprint Multiskan FC microplate photometer (Thermo Fisher Scientific). The CC50 values were calculated from best-fit dose−response curves using GraphPad Prism 5 software. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.20.051581 doi: bioRxiv preprint cells 6 days after infection. From these data, 90% virus inhibitory concentrations (EC90) were determined by regression analysis.