Additional file 1 of A phenomics approach for antiviral drug discovery
Creators: name: Rietdijk, Jonne
name: Tampere, Marianna
name: Pettke, Aleksandra
name: Georgiev, Polina
name: Lapins, Maris
name: Warpman-Berglund, Ulrika
name: Spjuth, Ola
name: Puumalainen, Marjo-Riitta
name: Carreras-Puigvert, Jordi
Publisher: name: figshare
Year: 2021
Subjects: subject: Biochemistry
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Genetics
subject: Pharmacology
subject: Biotechnology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: 69999 Biological Sciences not elsewhere classified
subject: Cancer
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: 60506 Virology
subject: Computational Biology

description: Additional file 1: Fig. S1. Representative images of phenotypic reference compounds, calculated AUROC and phenomics approach overview. a Representative images of the phenotypic reference compounds Fenbendazole (Fen), Etoposide (Eto) and Metoclopramide (Met) to which MRC-5 cells were exposed for 48h at 5 μM, and stained with the indicated Cell Painting dyes or fluorophore conjugates. b Calculated area under the receiver operating characteristics curve (AUROC) = 0.98, which indicates that the PLS-DA loadings are representative for distinguishing infected from non-infected cells. c Detailed overview of the phenomics approach here described. Fig. S2. Randomized layouts and reproducibility assessment. a Two different randomized layouts were used for each biological replicate. Compounds are indicated by the colour scheme, concentrations are indicated by L (Low), M (Mid) or H (High), absence or presence of the virus is indicated by -/+ CoV-229E. b Correlation calculation between the two replicate/layouts assessed by Pearson correlation, with a 95% confidence interval, as well as simple linear regression (R2). c PCA of the morphological profiles induced by the phenotypic reference compounds for both biological replicate 1 (circle) and 2 (triangle). Fig. S3. Intraplate variation. a and b. Mean proportion of infected cells per condition and replicate, with calculated standard deviation SD indicated next to each corresponding dose when applicable. The proportion of infected cells in DMSO conditions was 91.4% +/- 6.9 SD for two biological replicates. Fig. S4. Survival of cells exposed to antiviral compounds and structures for the in-house synthesized compounds. a Nuclei count was used to assess the survival of MRC-5 cells exposed to the indicated compounds for 48h. Two-way ANOVA was performed to assess the statistical significance of each condition (*p&lt;0.02, **p&lt;0.002, ***p&lt;0.0002). b Chemical structures for the in-house synthesized compounds TH3289, TH5487 and TH6744. c Average nuclei count per image for DMSO or media conditions, in the presence or absence of CoV-229E. The use of DMSO as vehicle for the compounds did not result in alteration of the cytotoxicity effect of the virus, which resulted in 20% cytotoxicity. Data points are mean values ± SD from two biological duplicates and multiple technical replicates. Fig. S5. Morphological profiles and Pearson’s correlation coefficients. Individual morphological profiles presented as heatmaps of every tested compound at non-toxic concentrations, accompanied to the Pearson’s correlation coefficients for each condition in respect to DMSO in the presence or absence of CoV-229E. Fig. S6. Pairwise Pearson’s correlations and Euclidean distances calculated for each morphological profile. a Pairwise Pearson’s correlations coefficients for the mean averaged morphological profiles of each tested compound at non-toxic concentrations. The profiles of infected cells treated with Remdesivir and E-64d positively correlate with DMSO in the absence of the virus. b Euclidean distances calculated for each morphological profile in respect to DMSO conditions in the presence or absence of CoV-229E. The higher Euclidean distance of Remdesivir, E-64d, TH3289, TH6744 and TH5487, at the indicated concentrations, reflect antiviral activity. Fig. S7. Pairwise Pearson’s correlations coefficients for the morphological profiles of each tested compound at non-toxic concentrations. Fig. S8. PCA of the morphological features of each indicated compound. a - c PCA of morphological features upon treatment with Favipiravir, Cathepsin L inhibitor and Bafilomycin A1 in infected (+CoV-229E) conditions compared to DMSO control (-CoV-229E). Each dot in the PCA represents one image by taking the mean of all objects in the image. Percentage of variance explained is indicated by %. d UMAP analysis results in two clusters each containing either active or inactive antiviral compounds. Fig. S9. Representative images of the modified Cell Painting assay, including the virus-NP antibody, for the indicated treatments. Fig. S10. Quality control measures for all plates in the experiment. Images deviating more than five standard deviations from the median of FocusScore, MaxIntensity, MeanIntensity, PercentMaximal, PowerLogLogSlope and StdIntensity were flagged as outliers and removed from the analysis.