| Assay Method Information | |
| | Antiviral cell-based assays |
| Description: | Some compounds in the 6a to 6k and 7a to 7k series were also investigated for their antiviral activity against the replication of MERS-CoV, FIPV, or MHV-1 in Huh-7, CRFK, or L929 cells, respectively (19). Briefly, medium containing DMSO (<0.1%) or each compound (up to 100 μM) was added to confluent cells, which were immediately infected with viruses at a multiplicity of infection (MOI) of 0.01. After incubation of the cells at 37°C for 24 hours, viral titers were determined with the median tissure culture infectious dose (TCID50) method (FIPV or MHV) with the CRFK or L929 cells or plaque assay with Vero81 cells (MERS-CoV). For SARS-CoV-2, confluent Vero E6 cells were inoculated with ~50 to 100 PFU per well, and medium containing various concentrations of each compound and agar was applied to the cells. After 48 to 72 hours, plaques in each well were counted. EC50 values were determined by GraphPad Prism software using a variable slope (GraphPad, La Jolla, CA) (19). To confirm that these inhibitors also inhibit SARS-CoV-2 in primary human cells, differentiated human airway epithelial cells from three donors were used as previously described (36, 37). Two compounds (6j and 6e) were tested for their antiviral effects against SARS-CoV-2. Briefly, airway epithelial cells were washed with phosphate-buffered saline (PBS), and SARS-CoV-2 was inoculated at a MOI of 0.1 for 1 hour. After the inoculum was removed, media containing 6j (2 μM) or 6e (0.5 μM) was added. After 48 hours, cells were subjected to a freeze/thaw cycle, and virus titers were determined by plaque assay on Vero E6 cells. |
| Affinity data for this assay | |
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