| Assay Method Information | |
| | Mpro In Vitro Screening Assay |
| Description: | To determine potency and selectivity index of identified hits, compounds were tested in 8-point dose response with a 3-fold step dilution at concentration ranging from 30-0.01 μM and four replicates. N-hydroxy cytidine (NHC), an antiviral with known anti SARS-CoV-2 activity, was used as a reference inhibitor.All infections with virulent strains were performed in a BSL-3 laboratory in accordance with CDC and US Army safety regulations. To identify small molecule inhibitors of SARS-CoV-2, VeroE6 cells (ATCC CRL-1586) were seeded at a density of 4000 cells/well in a 384 well imaging plates (Aurora Microplates, ABE2-31101A). Next day cells were pre-treated with the compound for two hours and then infected with SARS-CoV-2 (USA-WA1/2020) at a multiplicity of infection (MOI) of 0.01. After 32 hours following infection, cells were fixed in 10% formalin. To detect the viral antigen, immunofluorescence staining was performed wherein formalin fixed cells were washed three times with Phosphate buffered saline (PBS) and then incubated at room temperature (RT) with 50 μl of a combination cell permeabilization and blocking buffer (3% BSA, 0.1% Triton X-100 in PBS). After 1 hour, blocking buffer was replaced with 50 μl primary antibody solution (SARS-CoV/SARS-CoV-2 Nucleocapsid Rabbit Mab, Sino Biological, Cat 40143-R001) diluted 1:1000 in PBS and allowed to bind for 1 hour at RT. After two washes with 50 μl PBS, cells were stained for 30 minutes with 1:500 dilution of Alexa 488 anti-rabbit IgG (Invitrogen A11031). After 30 minutes, cells were washed three times with PBS. In the final step, PBS was replaced with 50 μl per well of 1:10000 Hoechst nuclear dye (Invitrogen H3570) and 5 mg/ml HCS Cellmask Deep Red (Invitrogen H32721), a cytoplasmic stain, all diluted with PBS. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |