Cell experiment:

U-2 OS cells seeded in 60-mm-diameter dishes are incubated with DMSO, 10 μM STS as an inducer of apoptosis, or increasing concentrations of Digoxin at 37℃ for 6 h. Cells are washed with fluorescence-activated cell sorter (FACS) buffer (PBS with 2% FBS) and stained with PI. Cell staining is quantified using a BD LSRII flow cytometer and FlowJo software. Alternatively, U-2 OS cells seeded in 96-well plates are incubated with DMSO, STS, or increasing concentrations of Digoxin at 37℃ for 6 or 24 h. PrestoBlue reagent is added to supernatants of compound-treated cells, and cells are incubated at 37℃ for 30 min. Fluorescence as a surrogate for cell viability is quantified using a Synergy H1 plate reader[1].

Digoxin is a potent inhibitor of Na+/K+-ATPase, clinically used to treat arrhythmia and heart failure.

Digoxin, a sodium-potassium ATPase inhibitor, has a negative Z score of –26.67, suggesting a function for the sodium-potassium ATPase in CHIKV infection. Relative to DMSO-treated cells, treatment of U-2 OS cells with Digoxin results in a dose-dependent decrease in CHIKV infection with a half-maximal effective concentration (EC50) of 48.8 nM. Digoxin treatment similarly decreases CHIKV infection of primary human synovial fibroblasts (HSFs) and Vero African green monkey kidney cells with EC50s of 43.9 nM and 67.3 nM, respectively. Digoxin treatment significantly diminishes CHIKV infection in these cell types, with EC50s of 16.2 μM in ST2 cells and 23.2 μM in C2C12 cells, values 330 and 475 times the EC50 of Digoxin in U-2 OS cells. Cell viability is only modestly impaired at 24 h posttreatment with 1 μM Digoxin, a dose 20 times the Digoxin EC50 for CHIKV antiviral activity in these cells[1].

References:
[1]. Ashbrook AW, et al. Antagonism of the Sodium-Potassium ATPase Impairs Chikungunya Virus Infection. MBio. 2016 May 24;7(3). pii: e00693-16.