Fig. 1: A primary cell-based HCI assay identifies compounds active against SARS-CoV-2 infection.

a Simplified assay workflow. b Representative images from dimethyl sulfoxide (DMSO)-, remdesivir- or apilimod-treated wells. The entire imaged area per well (four fields of view taken with a ×10 objective and stitched together) is shown for each treatment, as well as an 8-fold magnified segment demarcated with a white box. DNA signal [4′,6-diamidino-2-phenylindole (DAPI)] is colored green, and the virus visualized with immunofluorescence is colored magenta. Infected (arrow) and uninfected (arrowhead) cells are indicated; 500 µm and 50 µm scale bars are shown in the composite and magnified images, respectively. Raw and normalized (Norm.) values calculated from the images are shown. c Box and whiskers plot of SARS-CoV-2 assay control EC₅₀s obtained from n = 9 independent biological experiments with all data points shown. Whiskers indicate minimums and maximums, the box extends from the 25th to 75th percentile, the center of the box is the median and geomeans are reported. d Heat map images of normalized data from 1.9 µM ReFRAME screening plates. Normalized activity values for percentage infected cells and total cell numbers are indicated according to the scale bar and density plot for compound and control wells is shown. DMSO-treated wells are in column 24 and positive control-treated wells (blocks of wells with 2.4 µM remdesivir, 2.4 µM apilimod, or 9.6 µM puromycin) in column 23. Density plots representing the frequency of values associated with each well type are shown on the right. e Distribution of 1.9 µM ReFRAME screen data for compound and control wells. f Screen hit selection thresholds. g Library dose-response reconfirmation results, with the SARS-CoV-2 EC₅₀ of each compound plotted against its host-cell toxicity CC₅₀ as assessed in uninfected HeLa-ACE2 cells. Gray area defines non-selective compounds, where the selectivity index (CC₅₀/EC₅₀) is < 10. Dotted lines represent maximal concentrations tested in dose-response studies for the assay compounds (40 µM) and controls apilimod and remdesivir (9.6 µM). Activities of controls (black diamonds) and assay compounds (pink diamonds) are shown. Activity of the ReFRAME library copy of puromycin that was screened as part of this hit reconfirmation is also indicated (red diamond). Source data are provided as a Source Data file.