title: Defining ligand binding modes of the orphan G protein-coupled receptor GPR84
Creators: name: Mahmud, Zobaer  Al
Publisher: name: University of Glasgow
Year: 2019
Subjects: subject: GPR84, ligand binding modes, allosteric modulation, positive allosteric modulator.

description: Pro-inflammatory and pro-fibrotic G protein-coupled receptor 84 (GPR84), a member of the rhodopsin-like Class A GPCR family, recently has attracted interest as a potential drug target for chronic inflammation-associated diseases including ulcerative colitis, neuropathic pain, atherosclerosis and fibrosis-associated diseases including idiopathic pulmonary fibrosis. However, GPR84 still remains poorly characterized in terms of its signal transduction pathways and pathophysiological roles and is officially considered as ‘orphan’ receptor as the putative endogenous agonists MCFAs are weak activators of the receptor with a poorly defined mode of interaction with the receptor. The modes of ligand binding and mechanism(s) of action of the currently available pharmacological tool compounds are also very limited which have hindered the target validation process. Herein, the G protein coupling selectivity of GPR84, ligand-GPR84 interactions and modes of action of orthosteric and allosteric GPR84 ligands along with their orthologue selectivity were investigated in an attempt to characterize this enigmatic receptor. Employing a series of BRET-based GPR84 SPASM biosensors, GPR84 activation was found to couple to the Gαi/o G protein family with preferential recruitment of Gαi1/2 and Gαi3 over Gαo and Gαz G proteins. Based on homology modelling and site-directed mutagenesis studies, integral roles of arginine 172 of extracellular loop 2 in orthosteric ligand recognition and functions were identified wherein this residue acted as the putative charge partner for the carboxylate of MCFAs or the hydrophilic head groups of embelin or embelin-like ligands. Homology modelling, mutational analysis and subsequent docking studies also suggested that phenylalanine 170EL2, phenylalanine 335F6.51 and tryptophan 3607.43 might be associated with orthosteric ligand detection. 3,3´-diindolylmethane (DIM) and DIM analogue di(5,7-difluoro-1H-indole-3-yl)methane (PSB-16671) were found to bind to a site which is topographically distinct from the orthosteric site as both ligands retained their agonist functions upon mutation of all these residues to alanine where the activity of orthosteric agonists was either lost completely or reduced significantly. Functional studies with GPR84 antagonist compound-107 and radioligand binding studies with a chemically related radiolabelled antagonist, [3H]-G9543 suggested that this class of antagonists bind to a site which is different from the orthosteric and allosteric binding sites, indicating three spatiotemporally distinct ligand binding sites within GPR84. Antagonists but not agonists of GPR84 displays significant variation in pharmacology between human and mouse orthologues as evidenced from either loss of activity (compound-837) or markedly reduced potencies at mouse GPR84. GPR84 ligands display similar pharmacology in mouse monocyte-macrophage cell line RAW264.7 as those observed in transfected cells expressing mouse GPR84. DIM, analogue 3a (5,5´-dimethoxy-3,3´-diindolylmethane) and particularly PSB-16671 acted as highly effective positive allosteric modulators (PAM) of the function of orthosteric agonists through a mechanism that includes both affinity and efficacy modulation for embelin and C-10 or predominantly governed by affinity modulation with marginal effect on efficacy for full agonists including compound-1. These ligands display PAM activity in a ‘probe-dependent’ manner wherein the degree of affinity cooperativity tracks with the orthosteric agonist efficacy although little ‘probe-dependence’ was observed when net affinity/efficacy cooperativity was considered. DIM but not PSB-16671 showed significant variation in allosteric interactions with orthosteric agonists between human and mouse GPR84. Mathematical analysis of allosteric interactions showed that PSB-16671 binds human GPR84 with 360 and 5-fold higher affinity than decanoic acid and DIM, respectively while compound-1 displays 14-fold higher avidity than PSB-16671. The analysis also showed that the estimated binding affinities of these ligands for mouse GPR84 were equivalent to those observed for the human orthologue. In summary, the research studies presented herein provides new insights into ligand-GPR84 interactions and mode(s) of pharmacological actions of the GPR84 tool compounds which might be useful to accelerate structure-based drug design identifying further improved ligands or for translational studies assessing the therapeutic potential of this receptor.