DOP035 Anti-inflammatory potential of novel tethered agonists of the adhesion G protein-coupled receptor F5
Authors: Jacenik D , Wnorowski A , Pietrzak D , Mudgal A , Scrofani L , Jóźwiak K , Fichna J 
Published: January 2025

Abstract

Background
An accumulating data suggest that the adhesion G protein-coupled receptor F5 (ADGRF5) is fundamental for human health and diseases1-3. It was noted that ADGRF5 mediates immune response in cancers but its impact on colitis development is still unknown. Moreover, recent findings identified mature tethered agonist of ADGRF5 which corresponds to Stachel sequence of this receptor4. The purpose of this work was to design peptides corresponding to Stachel sequence able to activate ADGRF5 with higher activity than the mature ADGRF5 tethered agonist and evaluate them in colitis model.

Methods
Single amino acid substitutions in the mature ADGRF5 tethered agonist sequence combined with calcium accumulation and inositol phosphate conversion assays as well as ADGRF5 overexpressing cells were used to identify novel ADGRF5 tethered agonists. The downstream signalling pathway induced by ADGRF5 tethered agonists was estimated using Western blot technique. In vivo studies employing murine model of colitis induced by dextran sulphate sodium salt (DSS) administration were performed. Anti-inflammatory potential of novel ADGRF5 tethered agonists was assessed using macroscopic and microscopic damage score as well as total length of inflamed colon.

Results
The comparison of mature and modified ADGRF5 tethered agonist activity documented that substitution of the amino acids in position 11 and 13 improves their activity in ADGRF5 overexpressing cells when compared to wild-type cells. In vitro approach using calcium accumulation and inositol phosphate conversion assays revealed that novel ADGRF5 tethered agonists are characterized by 6-fold lower EC50 value than parent ADGRF5 tethered agonist. Moreover, our novel ADGRF5 tethered agonists are able to induce phosphorylation of protein kinase A and B as well as extracellular signal-regulated kinase signalling pathways in time and dose-dependent manner. Finally, administration of novel ADGRF5 tethered agonists in the murine model of colitis led to a significant improvement of macroscopic and microscopic damage score compared to animals treated with mature ADGRF5 tethered agonist. Additionally, the colons of mice treated with novel ADGRF5 tethered agonists but not with mature ADGRF5 tethered agonist were characterized by reduction of total length of inflammation and improvement of colon architecture.

Conclusion
We found critical positions in the Stachel sequence of ADGRF5 agonists which are required for their activity. New ADGRF5 tethered agonists displayed anti-inflammatory action highlighting that modulation of ADGRF5 activity may serve as an attractive target in colitis treatment.

References
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2. Einspahr JM, Tilley DG. Pathophysiological impact of the adhesion G protein-coupled receptor family. Am J Physiol Cell Physiol. 2022;323(2):C640-C647. doi:10.1152/ajpcell.00445.2021
3. Hamann J, Aust G, Araç D, et al. International Union of Basic and Clinical Pharmacology. XCIV. Adhesion G protein-coupled receptors. Pharmacol Rev. 2015;67(2):338-367. doi:10.1124/pr.114.009647
4. Bridges JP, Safina C, Pirard B, et al. Regulation of pulmonary surfactant by the adhesion GPCR GPR116/ADGRF5 requires a tethered agonist-mediated activation mechanism. Elife. 2022;11:e69061. Published 2022 Sep 8. doi:10.7554/eLife.69061