SARS-CoV-2 Antiviral Screening Assay

One day prior to infection, 5 × 104 VeroE6 cells were seeded in 100µL assay medium (containing 2.5% FCS) in 96 well plates. The next day, seven twofold serial dilutions of compounds (0.6–40 µM, in triplicate) were added to the cells (25 µL/well, in assay medium). Four virus control wells were supplemented with 25 µL of assay medium. After 15 min, 25 µL of a virus mix diluted in medium was added to the wells. The amount of virus working stock used was calibrated prior to the assay, based on a replication kinetics, so that the replication growth is still in the exponential growth phase for the readout as previously described^{10 (link),11 (link)}. Four cell control wells (i.e. with no virus) were supplemented with 50 µL of assay medium. On each plate a control compound (Remdesivir, BLDPHARM, Shanghai, China) was added in duplicate with seven twofold serial dilutions (0.16–20 µM, in duplicate). Plates were incubated for 2 days at 37 °C prior to quantification of the viral genome by real-time RT-PCR. To do so, 100 µL of viral supernatant was collected in S-Block (QIAGEN, Hilden, Germany) previously loaded with VXL lysis buffer containing proteinase K and RNA carrier. RNA extraction was performed using the Qiacube HT automat and the Cador Pathogen 96 HT kit following manufacturer instruction. Viral RNA was quantified by real-time RT-qPCR (EXPRESS One-Step Superscript qRT-PCR Kit, universal Invitrogen using 3.5 µL of RNA and 6.5 µL of RT qPCR mix and standard fast cycling parameters, i.e., 10 min at 50 °C, 2 min at 95 °C, and 40 amplification cycles (95 °C for 3 s followed by 30 s at 60 °C). Quantification was provided by four 2 log serial dilutions of an appropriate T7-generated synthetic RNA standard of known quantities (10² to 10⁸ copies). RT-qPCR reactions were performed on QuantStudio 12K Flex Real-Time PCR System (APPLIED BIOSYSTEMS, Waltham, USA) and analyzed using QuantStudio 12 K Flex Applied Biosystems software v1.2.3. Primers and probe sequences, which target SARS-CoV-2N gene, were: Fw: GGCCGCAAATTGCACAAT; Rev: CCAATGCGCGACATTCC; Probe: FAM-CCCCCAGCGCTTCAGCGTTCT-BHQ1. The 50% and 90% effective concentrations (EC50, EC90; compound concentration required to inhibit viral RNA replication by 50% and 90%) were determined using logarithmic interpolation as previously described^{11 (link)}. For the evaluation of the CC50 (the concentration that reduces the total cell number by 50%), the same culture conditions were set as for the determination of the EC50, without addition of the virus, and cell viability was measured using CellTiter Blue (PROMEGA, Fitchburg, USA) as previously described for the screening. CC50 was determined using logarithmic interpolation. All data obtained were analyzed using GraphPad Prism software version 7.0 (GRAPH PAD software Inc, California, USA). All graphical representations were also performed on GraphPad Prism software version 7.0 (https://graphpad-prism.software.informer.com/7.0/).