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Assay Method Information

Assay Name:  RTCA Assay for Evaluating In Vitro Inhibition of SARS-CoV-2 Replication by Mpro Inhibitors
Description:  Inhibition of SARS-CoV-2 replication in VERO E6 and HEK hACE2 cells was measured using an xCELLigence RTCA HT Analyzer (Agilent Technologies), tracking the virus-induced cytopathic effect (CPE) on the cellular growth kinetics. Applied methods were similar to those recently described elsewhere. After a background reading of the 96-well E-plate using 50 μL only of media, 1 104 cells were seeded in each well, using 100 μL of culture media (VERO E6 cells: DMEM + 1 penicillin/streptomycin + 10% FBS or HEK-hACE2 cells: DMEM + 1 penicillin/streptomycin + 10% FBS + 10 mM Hepes). The E-plate was incubated for 30 min at RT to minimize edging effects. After the cells settled, the E-plate was transferred to an xCELLigence instrument for real-time analysis of cell proliferation for 24 h. Drug candidates dissolved in DMSO were twofold serially diluted in cell culture media in a 96-deep-well plate. Drug samples were prepared in duplicate and DMSO was included as buffer control. An equal volume of cell culture media containing SARS-CoV-2 (USA_WA1/2020 isolate) was added to the drugs. Final concentrations of small drug tested ranged from 0.2 μM to 50 μM, and wells containing virus only or cell culture media only were added as controls. The plate was incubated for 1 h at 37 C in 5% CO2. After the incubation, the E-plate was removed from the xCELLigence instrument and the cell culture media in the E-plate was replaced by 250 μL of the drugs candidates/virus mixture from the deep-well plate. The E-plate was then transferred back to the xCeLLigence instrument and real-time analysis of the CPE was continued for an additional 75 h. Data were analyzed using the RTCA software (Agilent Technologies). Duplicate wells were averaged, and the cell index was normalized to the last measured time point before the addition of the drugs/virus mixture Using Prism9 software (GraphPad) the normalized cell index data were plotted versus time, and the IC50 of each drug candidate was calculated.
Affinity data for this assay
 

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