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

The levels of TNF-α, IL-6 and LDH were quantified in the monocyte supernatants from infected and uninfected cells. ELISA for TNF-α and IL-6 required 100 µL of supernatants to be exposed to capture antibody in 96-well plates. After a 2h incubation period at room temperature (RT), the detection antibody was added. Plates were incubated for another 2h at RT. Streptavidin-HRP and its substrate were added, incubated for 20 minutes and the optical density was determined using a microplate reader set to 450 nm. Extracellular lactate dehydrogenase (LDH) was quantified using Doles ® kit according to manufacturer's` instructions. Supernatant was centrifuged at 5,000 rpm for 1 minute, to remove cellular debris. A total of 25 µL of supernatant was placed into 96well plates and incubated with 5 µL of ferric alum and 100 µL of LDH substrate for 3 minutes at 37 °C. Nicotinamide adenine dinucleotide (NAD, oxidized form) was added followed by the addition of a stabilizing solution. After a 10 min incubation, plates were measured in a spectrophotometer at 492 nm. The docking of ligands was performed in a box of 10 Å edges with its mass center matching that of the complexed peptide. Each scan produced 20 conformations for each ligand with the best score being used for molecular dynamics simulations.