Atazanavir inhibits SARS-CoV-2 replication and pro-inflammatory cytokine production

Natalia Fintelman-Rodrigues; Carolina Q. Sacramento; Carlyle Ribeiro Lima; Franklin Souza da Silva; André C. Ferreira; Mayara Mattos; Caroline S. de Freitas; Vinicius Cardoso Soares; Suelen da Silva Gomes Dias; Jairo R. Temerozo; Milene Miranda; Aline R. Matos; Fernando A. Bozza; Nicolas Carels; Carlos Roberto Alves; Marilda M. Siqueira; Patrícia T. Bozza; Thiago Moreno L. Souza

Protein extracts containing SARS-CoV-2 Mpro activity were obtained from Vero cell monolayers at 25 cm 2 flasks that were infected for 1h with an MOI of 0.1 at 37 °C and 5% CO 2 . After 1 or 2 days of infection, the supernatant was harvested and monolayers were washed 3 times with in sterile cold PBS (pH 7.2). Next, cells were suspended into 1 mL of lysis buffer (100 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10% glycerol and 0.6% Triton X-100) and kept at 4 °C. The soluble protein fraction was isolated as the supernatant after centrifugation (100,000 x g, 30 min, 4 °C) and stored at -20°C until further use. The protein concentrations of the samples were determined using the BCA protein assay kit.