title: Searching novel pharmacological tools for the pharmacology of orphan G protein-coupled receptor GPR84
Creators: name: Li, Yueming
Publisher: name: University of Glasgow
Year: 2025

description: G protein-coupled receptors (GPCRs), a large group of cell-surface receptors, are involved in numerous physiological and pathological processes. Among GPCRs, the pro-inflammatory orphan receptor GPR84, a member of the Class A receptor family, is an attractive drug target for inflammatory diseases and metabolic disorders. However, there remains a significant lack of GPR84 compounds, particularly GPR84 antagonists that can be used in mouse model studies as available GPR84 antagonists display marked species selectivity for human GPR84. The most direct method to overcome this challenge is through the screening and characterization of novel compounds for both human and mouse GPR84 to discover antagonists with similar affinities for both species. Compound 271 was characterized as a competitive orthosteric antagonist for both human and mouse GPR84, and the key residue Arg172ECL2 in the orthosteric binding pocket is not necessary for the binding of compound 271. However, previous studies have shown that the mutation of Arg172ECL2 to alanine or lysine abolished the function of medium-chain fatty acids while having no effect on the potency of the allosteric agonist 3,3’-diindolylmethane in GPR84. Therefore, studying the binding pocket of an antagonist to GPR84 would be essential for understanding the structural 
determinants of ligand recognition and receptor modulation. In addition to this time consuming method of screening a large number of compounds, generating a novel transgenic mouse strain expressing ‘chimeric’ human orthologue-like GPR84 could be another solution. Similar potencies of agonists at human and mouse orthologues, along with the high similarities between these two orthologues, provide a foundation for this idea. Herein, stable cells expressing each of HA-human GPR84, HA-mouse GPR84 or HA-humanised GPR84 were generated to characterize and compare the pharmacology of these forms using each of cAMP assays, radioligand binding assays and immunoblotting. Encouragingly, the ability of human GPR84 species selective antagonists compound 020, compound 140, compound 837 and GLPG1205 to block the activation of HA-humanised GPR84 was similar to that of HA-human GPR84. The phosphosite-specific antiserum pT263-pT264 recognized the 2-HTP induced phosphorylation of these three forms of receptors, which suggests that this antiserum can be used to detect the phosphorylation of GPR84 in ex vivo studies in the future. However, HA human GPR84 was constitutively phosphorylated at residues Ser221 and Ser224 while HA- humanised GPR84 was phosphorylated in a 2-HTP-dependent manner at these two residues. This result suggests that the differences between HA-human GPR84 and HA-humanised GPR84 still need to be considered carefully in future studies. 

In addition to lacking useful compound tools for pre-clinical models, basic research around GPR84 signalling also need to be improved. GPCR phosphorylation plays an important role in GPCR signalling and regulates the downstream signal transduction including desensitization and internalization of receptors. Thus, studying which GRK(s) might be involved in GPR84 phosphorylation is important for further understanding the signalling of the receptor. It was found that both GRK2 and GRK3 are involved in GPR84 phosphorylation, and Gai probably
influences the GRK subtypes that phosphorylate GPR84. Moreover, the recruitment and binding of arrestin 3 to GPR84 may not depend on GPR84 phosphorylation. The internalization of GPR84 was also tracked using BRET-based ‘Bystander’ assays and immunocytochemical staining experiments.

In summary, the studies presented herein characterize novel GPR84 antagonists and suggest the development of a ‘chimeric’ human orthologue-like GPR84 mouse model to explore the therapeutic potential of blocking this receptor. Moreover, the understandings on GPR84 phosphorylation and internalization mechanisms are also improved. The results in this thesis may help to better understand the therapeutic potential of GPR84.