Abstract Background GPR68 (OGR1) is an acid-sensing G-protein-coupled receptor (GPCR) and, along with the related pH-sensing GPCRs GPR4 and GPR65 (TDAG8), has been implicated in Inflammatory Bowel Disease (IBD) through modulation of the inflammatory response and fibrotic phenotype. GPR68 expression is upregulated in inflamed versus non-inflamed tissue in Ulcerative Colitis and Crohn’s Disease patients1. Additionally, TNF-α, which is a characteristic cytokine in IBD, upregulates GPR68 in monocytes and GPR68 deficiency protects from spontaneous inflammation in the IL-10 KO model2. Increased GPR68 expression and activity in primary human and murine fibroblasts (non-IBD) subjected to extracellular acidic pH has also been described1 and intestinal activation of GPR68 contributes to fibrogenesis3. GPR68 negative allosteric modulators (NAMs) are thus predicted to be of therapeutic relevance in IBD through modulation of the inflammatory and fibrotic signalling pathways. Methods 1321N1 cells expressing recombinant human GPR68 or mouse GPR68 were stimulated with the historical EC80 pH6.8 shown to activate the cAMP response, or EC80 pH 7.4 shown to activate the inositol phosphate response in the cells, in the presence of test compound. The primary assay was also run in a NAM activity mode to establish alpha cooperativity factor and the compound KB (nM). Using the NAM assay mode, cells were stimulated with a dose response of pH6.2 – pH8.0 in the presence of three concentrations of compound from 31 nM to 3.1 μM for activity against both mouse and human GPR68 variants. CCD18Co cells, pre-treated with TNF-α to upregulate GPR68 expression, were treated with Compound 1 from 3nM to 3uM in neutral (pH7.4) or acidic (pH6.8) conditions and release of IL-6 and IL-11 was assessed by ELISA. Results In this work we have identified a novel, potent and highly selective NAM of GPR68. Compound 1 is shown to inhibit signalling via both Gs and Gq/11. We also demonstrate that Compound 1 produces similar data in colorectal fibroblasts to data that has been reported for GPR68 knockdown experiments in pancreatic cancer-associated fibroblasts (CAFs); where inhibition of GPR68 signalling reduces pro-inflammatory cytokine release. Moreover, Compound 1 is orally bioavailable in mice and significantly reduces the total colitis score, colon weight and spleen length in the acute Dextran Sulphate Sodium (DSS)-induced mouse model of Ulcerative Colitis. Conclusion In summary, we have demonstrated that TNF-α upregulates GPR68 expression in human colorectal fibroblasts and modulates the release of pro-inflammatory cytokines IL-6 and IL-11 under acidic pH. Moreover, modulation of GPR68 signalling using Compound 1 is efficacious in a (DSS)-induced mouse model of Ulcerative Colitis. References: 1) De Vallière C, Cosin-Roger J, Baebler K, et al. pH-Sensing G Protein-Coupled Receptor OGR1 (GPR68) Expression and Activation Increases in Intestinal Inflammation and Fibrosis. Int J Mol Sci. 2022;23(3):1419. doi:10.3390/ijms23031419 2) De Vallière C, Bäbler K, Busenhart P, et al. A Novel OGR1 (GPR68) Inhibitor Attenuates Inflammation in Murine Models of Colitis. Inflamm Intest Dis. 2021;6(3):140-153. doi:10.1159/000517474 3) Hutter S, Van Haaften WT, Hünerwadel A, et al. Intestinal Activation of pH-Sensing Receptor OGR1 GPR68 Contributes to Fibrogenesis. J Crohns Colitis. Published online August 25, 2018. doi:10.1093/ecco-jcc/jjy118 Conflict of interest: Dr. Bingham, Matilda: Matilda Bingham is an employee of Cumulus Oncology Cheng, Robert: No conflict of interest Sager, Christoph P.: No conflict of interest Remen, Luboš: No conflict of interest March, H Nikki: No conflict of interest Gallagher, Laura: Laura Gallagher is an employee of Cumulus Oncology Seuwen, Klaus: Klaus Seuwen is a consultant to Cumulus Oncology Hennig, Michael: Michael Hennig is founder and CEO of leadXpro Doris, Clare: Clare Doris is a shareholder in Cumulus Oncology
Bingham et al. (Thu,) studied this question.