Using systems medicine to identify a therapeutic agent with potential for repurposing in inflammatory bowel disease

Abstract

Inflammatory bowel diseases (IBDs) cause significant morbidity and mortality. Aberrant NF-κB signalling is strongly associated with these conditions, and several established drugs influence the NF-κB signalling network to exert their effect. This study aimed to identify drugs that alter NF-κB signalling and could be repositioned for use in IBD. The SysmedIBD Consortium established a novel drug-repurposing pipeline based on a combination of in silico drug discovery and biological assays targeted at demonstrating an impact on NF-κB signalling, and a murine model of IBD. The drug discovery algorithm identified several drugs already established in IBD, including corticosteroids. The highest-ranked drug was the macrolide antibiotic clarithromycin, which has previously been reported to have anti-inflammatory effects in aseptic conditions. The effects of clarithromycin effects were validated in several experiments: it influenced NF-κB-mediated transcription in murine peritoneal macrophages and intestinal enteroids; it suppressed NF-κB protein shuttling in murine reporter enteroids; it suppressed NF-κB (p65) DNA binding in the small intestine of mice exposed to lipopolysaccharide; and it reduced the severity of dextran sulphate sodium-induced colitis in C57BL/6 mice. Clarithromycin also suppressed NF-κB (p65) nuclear translocation in human intestinal enteroids. These findings demonstrate that in silico drug repositioning algorithms can viably be allied to laboratory validation assays in the context of IBD, and that further clinical assessment of clarithromycin in the management of IBD is required.This article has an associated First Person interview with the joint first authors of the paper.

Keywords: Crohn's disease; Drug repositioning; Inflammatory bowel diseases; Interdisciplinary research; Macrolide; NF-κB; Organoids; Ulcerative colitis.

Fig. 1.

Schematic representation of the bioinformatic approach used to identify drugs with the potential to modulate IBD and NF-κB signalling. The black and white panels indicate the two different analysis streams, merging in the grey-shaded areas. GWAS, genome-wide association studies; IBD, inflammatory bowel disease; MEALR, motif enrichment analysis by logistic regression.

Fig. 2.

CLA suppresses stimulus-induced NF-κB activation in primary murine cell cultures. (A-D) Luciferase activation curves for peritoneal macrophages extracted from HTNF.LucBAC mice stimulated with 10 ng/ml lipopolysaccharide (LPS) (A), 40 ng/ml TNF (B), 10 µg/ml muramyl dipeptide (MDP) (C) or 500 ng/ml flagellin (D). Solid lines indicate the responses for cells pre-treated with drug vehicle (DMSO); dashed and dotted lines indicate the responses generated from cells pre-treated with 10 µM or 100 µM clarithromycin (CLA), respectively. (E) Median fluorescence intensity for anti-phopsho-p65 antibody-stained peritoneal macrophages from C57BL/6 mice either unstimulated or stimulated with LPS and pre-treated with 10 µM CLA or DMSO vehicle. N=4 mice. Statistically significant differences tested by one-way ANOVA and Dunnett's post hoc test.

Fig. 3.

CLA suppresses TNF-induced luciferase activity in enteroids. (A,B) Representative luciferase activation curves for enteroids derived from HTNF.LucBAC mice either unstimulated (A) or stimulated with 100 ng/ml TNF (B), and without pre-treatment (solid line), or with 30 min pre-treatment with DMSO vehicle (dashed line), 1 µM CLA (dotted line) or 10 µM CLA (dotted and dashed line). (C) Area under the curve (AUC) calculations for the same experiment. N=3. *P<0.05, **P<0.01. Statistically significant differences tested by two-way ANOVA and Dunnett's post hoc test.

Fig. 4.

CLA suppresses TNF-induced p65 nuclear translocation in enteroids. (A) Representative images of brightfield (top row) and red channel (bottom row) images of dynamic, live-cell imaging studies of enteroids derived from p65-DsRedxp/ IκBα-eGFP mice, either untreated, treated with CLA alone, treated with 100 ng/ml TNF alone, or pre-treated with 10 µM CLA and subsequently stimulated with TNF. (B,C) Relative nuclear red fluorescence curves for individual cells (grey lines), mean (solid red lines) and 1 s.d. above and below the mean (dashed red lines) over time. Cells were pre-treated with DMSO vehicle (B) or CLA (C) at time −35 min and stimulated with TNF at time 0 min. (D) AUC calculations for individual cells between time 0 min and time 90 min for B and C. Statistically significant differences tested by Mann–Whitney U-test. (E) Time to peak fluorescence, post-TNF stimulation for individual cells; statistically significant differences tested by Mann–Whitney U-test. (F) Difference of individual cells’ peak fluorescence from the median value; statistically significant differences tested by Mann–Whitney U-test. N=88 vehicle pre-treated cells, N=62 CLA pre-treated cells for D-F; lines represent median and IQR for these panels. Scale bars: 100 µm.

Fig. 5.

CLA attenuates NF-κB activation and dextran sulphate sodium (DSS) colitis in C57BL/6 mice. (A) Relative p65 DNA binding activity in whole-cell lysates from the proximal small intestine of C57BL/6 mice pre-treated for 3 days with 50 mg/kg CLA or saline vehicle, and subsequently injected i.p. with 0.125 mg/kg LPS or vehicle. N=5-6. (B-F) Effect of CLA on outcomes of DSS colitis in C57BL/6 mice. (B) Weight loss plotted over time. (C) AUC analysis of weight loss. (D) Histology severity scores. (E) Number of surviving crypts per colonic circumference. (F) Representative photomicrographs of the colonic mucosa of DSS-treated mice co-treated with saline vehicle or 10 mg/kg CLA by oro-gastric gavage. N=9-10. Statistically significant differences tested by one-way ANOVA and Dunnett's post hoc test in all panels. Scale bars: 200 µm (top row in F) and 100 µm (bottom row in F).

Fig. 6.

CLA attenuates NF-κB activation in human enteric organoids. (A) Representative photomicrographs of human ileal enteroids either unstimulated or stimulated with 100 ng/ml TNF, and either co-administered DMSO vehicle or 10 µM CLA and immunostained for total p65. (B) Quantification of nuclear p65 staining of human enteroids. Statistically significant differences tested by Kruskal–Wallis one-way ANOVA and Dunn's post hoc. N=6. Scale bars: 50 µm.