Identification of Drug Repurposing Candidates for Coxsackievirus B3 Infection in iPSC-Derived Brain-like Endothelial Cells

Figures

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  Figure 1. A previously published CVB-infected iBEC RNA sequencing dataset was queried and downloaded from Gene Expression Omnibus (GEO) (GSE269413). Whole transcriptome analysis was then run using a suite of bioinformatics tools in the “3PodR” package, including differential gene expression, pathway analysis, and perturbagen analysis [19]. Visualizations were subsequently generated from this analysis. The “3PodR” tool package (pre-release, https://doi.org/10.5281/zenodo.8190833, accessed on 8 August 2024) enabled the interpretation of transcriptomic changes, biomolecular pathways, and possible perturbagens were identified, providing a unique transcriptional analysis of this dataset. Created in Biorender. Jacob Wood. (2025) https://BioRender.com (accessed on 10 January 2025).

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  Figure 2. (A,B) Volcano plots representing differential gene expression at 2 (A) and 5 dpi (B), with genes color-coded and annotated. A threshold-adjusted p-value of padj < 0.05 was used. The y-axis represents a −log10 p-value scale. The x-axis represents log2 fold change, the y-axis represents adjusted p-value. Upregulated genes are red dots; downregulated genes are blue dots. Highlighted genes are represented as yellow dots. (C) Heatmap showing the top differentially expressed genes in iBECs following coxsackievirus B3 (CVB) at 2 and 5 dpi. The left panel shows the log2 fold change in gene expression in cells infected at 2 dpi compared to mock-infected controls; the right panel shows gene expression changes at 5 dpi relative to their respective mock controls. Color intensity reflects log2 fold change values, with positive values indicating upregulation and negative values reflecting downregulation. Gene names are listed accordingly.

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  Figure 3. Gene Set Enrichment Analysis (GSEA) of differentially expressed genes for 2 and 5 dpi. (A) Ridge plots displaying the distribution of log2FC for DEGs in the top 39 significantly (padj < 0.05) shared GSEA pathways between 2 and 5 dpi. These 37 pathways were selected by identifying an overlap among the top 75 GSEA pathways for 2 and 5 dpi. The x-axis displays log2 fold change values, while the y-axis lists GSEA pathway names corresponding to those in panels A and B. The ridges within the plot are colored, where blue is 2 dpi, and yellow is 5 dpi. (B) Heatmap showing the normalized enrichment scores (NES) for the top 39 significantly (padj < 0.05) shared GSEA pathways. Pathways are clustered hierarchically by normalized enrichment scores (NES), with a color scale ranging from light blue (lower enrichment) to dark blue (higher enrichment).

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  Figure 4. (A) Hierarchically clustered Tanimoto similarity heatmap comparing chemical structures of the top 22 candidate compounds identified at 2 dpi by iLINCs. Each cell represents the paired Tanimoto coefficient between paired compounds, with higher values indicating greater structural similarity, with a color scale representation. (B) UMAP plot visualizing the compounds from 2 and 5 dpi based on 5-group Kmeans clustering of Tanimoto coefficients, highlighting structurally related molecules, with 4 drugs labeled as followed: erlotinib, pelitinib, neratinib, and tyrphostin_AG-1478. (C) Representative chemical scaffold (“Backbone”) shared among EGFR inhibitors (erlotinib, pelitinib, and neratinib), as well as Tyrphostin_AG-1478’s chemical structure, indicated as structurally similar top hits at 2 dpi.

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  Figure 5. (A) Substructure of closely related EGFR inhibitors and related analogs. (B) Common structures of an additional cluster of RTK inhibitors. (C) Common substructure of MEK inhibitors is shown in red. Trametinib is also shown, which clusters with the MEK1/2 inhibitors based on Tanimoto coefficient. (D) Four-way Venn diagram showing neratinib, pelitinib, tyrphostin AG-1478, erlotinib and their numerical value for unique and shared SwissTargetPrediction docking gene targets. (E) Tables from iLINCs discordant drug signatures at 2 dpi showing EGFR and MEK1/2 inhibitors, with FDA-approved drugs highlighted in red text, and corresponding discordant score color scale in green. Abbreviations: Tyr: tyrphostin AG-1478. (F) Table of 18 SwissTargetPrediction docking gene targets shared across neratinib, pelitinib, tyrphostin AG-1478, and erlotinib, with docking probability scores with representative green and red color scale.