Drug repurposing screens to identify potential drugs for chronic kidney disease by targeting prostaglandin E2 receptor

Fig. 1. Several genes and pathways were affected by the ECE inhibitor (phosphoramidon) in HK-2 cells. (A) The procedure of NGS data obtained from human kidney cells after treatment with or without phosphoramidon. Figures were created with BioRender.com. (B) The significantly up- and downregulated genes at a threshold of 1.5-fold change were highlighted for the mRNA levels after treatment with phosphoramidon. (C) The prediction of the PTGER2 network was generated from a comparison between the NGS data and the IPA database with a 1.5-fold change cutoff from HK-2 cells. The activating or inhibiting are colored orange or blue after phosphoramidon treatment, respectively.

Fig. 2. Homology modeling of the EP2 protein structure according to the 3D conformation of the EP4 crystal structure via the I-TASSER web server. (A) The most reasonable model of the EP2 protein structure. (B) Ramachandran plot of each amino acid of the EP2 modeling protein. (C) Sequence comparison between the target protein (EP2) and template structure (EP4). The identity and similarity scores are 30.0% and 47.2%, respectively. (D) The binding site of the EP2 structure is defined by the site of the antagonist on the EP4 crystal structure.

Fig. 3. Docking poses of selected candidates on the binding site of the EP2 protein structure: (A) dobutamine, (B) dofetilide, (C) citalopram, and (D) ritodrine. Chemical interactions such as conventional H-bonds, carbon H-bonds, pi-donor H-bonds, pi-cation, pi-alkyl, pi-sulfur, and van der Waals interactions are colored green, cyan, purple, orange, pink, and yellow, respectively.

Fig. 4. Cell viability of HK-2 cells after treatment with four drug candidates for 1 d. The cell viability data were obtained using the SRB assay. Each cell viability data point was normalized to the control group at a concentration of 0 μM and represented as the percentage of cell viability (%). Data are shown as the means ± standard deviations of three individual experiments, n = 3, * p < 0.05 between the indicated groups.

Fig. 5. The autophagy-related protein levels of LC3-I, LC3-II, Beclin 1, and p62 were measured in drug-treated HK-2 cells at concentrations of 0, 10, 20, and 40 μM for 24 h: (A) dobutamine, (B) dofetilide, (C) citalopram, and (D) ritodrine. GAPDH was regarded as the internal control. Data are shown as the means ± standard deviations of three individual experiments, n = 3. *p < 0.05 compared with the control.

Fig. 6. Western blot analysis of NLRP3 inflammasome-related protein expression in drug-treated HK-2 cells at concentrations of 0, 10, 20, and 40 μM: (A) dobutamine, (B) dofetilide, (C) citalopram, and (D) ritodrine. GAPDH was used as the internal control. The LPS+ATP-induced NLRP3 inflammasome in HK-2 cells was incubated with the test compounds and LPS (2 μg/mL) for 24 h and subsequently treated with ATP (4 mM) for 2 h. # p < 0.05 compared with the LPS+ATP group. * p < 0.05 compared with the control group. Data are shown as the means ± standard deviations of three individual experiments, n = 3.

Fig. 7. Expression levels of fibrosis-related proteins in HK-2 cells via western blot analysis. Cells were treated with (A) dobutamine, (B) dofetilide, (C) citalopram, and (D) ritodrine at concentrations of 0, 10, 20, and 40 μM for 24 h. GAPDH was regarded as the internal control. The fibrosis in drug-treated HK-2 cells was treated with TGF-β1 (15 ng/mL) and subsequently incubated for 24 h. # p < 0.05 compared with the TGF-β1 group. * p < 0.05 compared with the control group. Data are shown as the means ± standard deviations of three individual experiments, n = 3.