Anti-Inflammatory Potential of Novel Tethered Agonists of the Adhesion G Protein-Coupled Receptor F5

The adhesion G protein-coupled receptor F5 (ADGRF5) has been implicated in modulating immune responses in cancer; however, its role in inflammatory bowel diseases (IBDs), particularly colitis, remains largely unexplored. In this study, we aimed to design and characterize novel peptide agonists derived from the ADGRF5 <i>Stachel</i> sequence, as well as to evaluate their therapeutic potential in preclinical colitis models. <i>In silico</i> analysis and single amino acid substitutions within the ADGRF5 tethered agonist sequence, combined with functional assays in ADGRF5-overexpressing cells, including calcium mobilization and inositol phosphate production, were employed to assess the activity of novel ADGRF5 agonists. Western blot technique and murine model of colitis were used to evaluate downstream signaling pathways and immunomodulatory effects of ADGRF5 ligands. We identified a series of peptides exhibiting significantly enhanced ADGRF5 agonist activity, achieving up to a 6-fold increase in potency over the wild-type version. We identified critical substitutions within the <i>Stachel</i> sequence, namely S11N and D13S, as essential for improving agonistic activity. Finally, using these novel ADGRF5 agonists, we demonstrated their potent anti-inflammatory effects <i>in vivo</i>, showing that ADGRF5 activation ameliorates experimental colitis, as evidenced by reduced macroscopic damage scores and improved colon architecture. These findings establish ADGRF5 as a potential therapeutic target for colitis and highlight the promise of <i>Stachel</i>-derived peptide agonists for the development of novel anti-inflammatory therapies.