Extended Data Fig. 2: Cell fusion inhibition assay.

a, Scheme of cell fusion inhibition assay. The assay is based on the co-culture, for 12 h, of Vero cells with U2OS cells transiently transfected to express spike protein. To permit quantitative assessment of cell fusion by high-content microscopy, the spike ‘donor’ U2OS cells are loaded with quantum dots (Q-dots) emitting at 525 nm (green) whereas the ‘acceptor’ Vero cells with Q-dots emitting at 800 nm (red). Screenings were performed in 384-well plates by co-plating Q-dot loaded ‘donor’ and ‘acceptor’ cells, followed by drug treatment at 10 μM standard concentration. After 24 h, cells were fixed and processed by high-content microscopy. Multiple control wells with 1% DMSO and no drugs were included in each plate. b, Fluorescent images of co-cultured U2OS (green Q-dots) and Vero (red Q-dots) cells. Cells are stained with HCS CellMask Blue; fused cells (heterokaryons) are identified as cells having more than two nuclei and containing both green and red Q-dots in their cytoplasm. Thus, the CFIA assay scores for the effect of drugs on the fusion of heterologous cells and detects inhibitors that block fusion of as few as two cells. The assay has a more than fivefold dynamic range. Scale bar, 100 μm c, CFIA image high content analysis pipeline. Images were analysed using the Harmony software (PerkinElmer). The cytoplasmic area, stained by the HCS Cell Mask Blue, was defined using the ‘Find Cytoplasm’ analysis module (Harmony) and the number of either green or red spots was counted using the ‘Find Spots’ analysis module (Harmony). All the cells that scored a nuclear area greater than five times the average area of a single nucleus and were simultaneously positive for at least two red and two green dots were considered as fused (highlighted in green). Data were expressed as a percentage of fused cells. d, Cumulative results of CFIA screening of FDA/EMA-approved drug libraries. We screened two FDA/EMA-approved drug libraries, The Spectrum Collection, MS Discovery System, and the Prestwick Chemical Library, Prestwick Chemical; 2,545 and 1,280 individual small molecules, respectively. The graph shows the cumulative results of the two screenings. The percentage of syncytia normalized on total cells is plotted as a z score. Compounds with a z-score ≤ −2.58 (red dotted line, 0.005% tail of the distribution) are shown in red, those between ≤ −1.96 (0.025% tail, blue dotted line) and −2.58 are in blue. In the Prestwick collection, 34 drugs inhibited S-mediated fusion with a z score <−1.96, of which 8 with a z score of <−2.58. In the MS Discovery Spectrum collection, 54 drugs performed at a z score <−1.96 of which 14 with a z score of <−2.58. All untreated controls for both libraries had a z score in the ±0.40 range. e. Analytic evaluation of CFIA results. A correlation chart between the percentage of syncytia per well and the total number of cells per well is shown for the two screened drug libraries. Drugs showing a total number of cells at ≥2 s.d. lower than the average of all drugs (red dotted line) considered toxic and not included in the subsequent analysis. Distribution of control wells (1% DMSO) is shown by a green box. f, Distribution frequency of the percentage of syncytia plotted as the number of drugs versus the z score of the percentage of syncytia per well; toxic cells were excluded as in e. Blue dotted lines are at z scores ± 1.96 and red dotted lines at z scores ± 2.56. The numbers of drugs are shown. g, Correlation chart for the common drugs between the two libraries. Data are plotted as the z score of the percentage of syncytia per well. Blue and red lines are as above.