Antiviral activity of curcumin and its analogs selected by an artificial intelligence-supported activity prediction system in SARS-CoV-2-infected VeroE6 cells

Cells African green monkey kidney cell line VeroE6 and VeroE6 expressing the transmembrane serine protease TMPRSS2 (VeroE6/TMPRSS2, Matsuyama et al. 2020) were purchased from the National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, Japan. VeroE6 cells were cultured in minimal essential medium (MEM; Sigma-Aldrich, MO, USA) supplemented with 10% fetal bovine serum (FBS) and antibiotics. VeroE6/TMPRSS2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, MA, USA) supplemented with 5% FBS and 1 mg/mL G418 (Nacalai, Kyoto, Japan). The cells were grown at 37°C in a CO2 incubator. Virus SARS-CoV-2 viral strain JPN/TY/WK/521 was provided by the National Institute of Infectious Diseases, Tokyo, Japan. Viruses were propagated in monolayers of VeroE6/TMPRSS2 cells in DMEM supplemented with 2% FBS and 1 mg/mL G418 at a multiplicity of infection of 0.01. Virus titration Infectivity was titrated by a focus-forming assay as described previously (Yasugi et al. 2013 ) with slight modifications. Viruses were serially diluted 10-fold in DMEM supplemented with 2% FBS and infected with confluent VeroE6 cells for 8 h. The cells were fixed with 4% paraformaldehyde for 15 min and washed with phosphate-buffered saline (PBS) three times. After the cells were permeabilized with 0.1% Triton ® X-100 for 15 min and washed with PBS three times, they were incubated with rabbit anti-nucleocapsid monoclonal antibody (Thermo Fisher Scientific) at a..