Fig 2: TEMPOL inhibits SARS-CoV-2 replication in human lung epithelial Calu-3 cells(A) Calu3 (ATCC, #HTB-55) cells were pretreated with TEMPOL for 2 h prior to continuous infection with SARS-CoV-2 (isolate USA WA1/2020) at MOI = 0.5. Forty-eight hours post-infection, cells were fixed, immunostained, and imaged by automated microscopy for infection (dsRNA + cells/total cell number) and cell number. TEMPOL-treated well data were normalized to aggregated DMSO control wells and plotted versus drug concentration to determine the IC50 (infection: blue) and CC50 (toxicity: green) (data are expressed as mean ± SD; n = 3 biological replicates). (Inset) Half-maximal inhibitory concentration (IC50), 50% cytotoxic concentration (CC50), selectivity index (SI) and 90% inhibitory concentration (IC90) of TEMPOL (units are 10−6 M).(B) Viability and percentage of infection in Calu-3 cells treated with the indicated concentrations of TEMPOL (0.01-20 μM) for 2 h before continuous infection with SARS-CoV-2 (isolate USA WA1/2020) at an of MOI = 0.5. Values represent average Percentage of Control (POC) = (TEMPOL well measurements/aggregated DMSO average) ∗ 100 (n = 3 biological replicates).(C) Calu-3 cell viability on treatment with up to 10 mM TEMPOL was assessed with the real-time ATP rate assay kit (Agilent). ATP production rates from glycolysis (glycoATP) and oxidative phosphorylation (mitoATP) were simultaneously determined with a Seahorse XFe96 Analyzer. As a control, the viability of cells treated with DMSO alone used to dissolve TEMPOL, were also included (data are expressed as mean ± SD; n = 3 biological replicates).

Fig 3: Carvedilol (A) and remdesivir (B) were tested in Calu-3 cells. Calu-3 (ATCC, HTB-55) cells were pretreated with test compounds for 2 h prior to continuous infection with SARS-CoV-2 (isolate USA WA1/2020) at MOI = 0.5. Cells were imaged 48 h after infection. EC50 (infection: blue) and CC50 (toxicity: green).