While published data around the functional significance of G

While published data around the functional significance of GPR84 are quite sparse, details of signaling pathways initiated by the receptor are even more limited, including in recombinant systems. In recombinant cells, GPR84 activation reportedly leads to Gi pathway activation, resulting in pertussis toxin sensitive inhibition of cAMP [5]. Furthermore, treatment with putative GPR84 ligands results in internalization of the receptor from the cell surface [10]. More importantly, no significant studies of the signaling properties of GPR84 in physiologically relevant cells, particularly of immune origin, have been reported. Here, we have endeavored to perform a comprehensive study of GPR84 receptor signaling and function in recombinant and primary immune cells, particularly macrophages and neutrophils, to decipher how GPR84 activation modulates important physiological functions of these cells.
Materials and methods
Results
Discussion In this report we performed a comprehensive study of GPR84 signaling and function in recombinant and primary immune cells, including macrophages and neutrophils. These studies were enabled by the identification of embelin as a highly potent and selective agonist for GPR84 in a high-throughput screen of a small molecule library in a melanophore based screening platform. This finding was initially disclosed by our group in Patent Application WO2007027661 A2, 2007 [23] and the agonist activity of embelin at GPR84 has since been confirmed by others [[24], [25], [26]]. Embelin is a natural product derived from Embelia Ribes, a plant which has been used for thousands of years in traditional Chinese medicine for treatment of a diverse array of diseases, including gastrointestinal and inflammatory ailments [27,28]. Previous studies have shown that embelin has numerous biological activities, including inhibition of cell proliferation, induction of apoptosis and tumor cell migration [[28], [29], [30]]. Whether any of embelin’s previously described in vitro or in vivo activities are mediated through GPR84 remains unknown. The biological activity of embelin has also been linked to its only other known molecular target – Angiotensin 1/2 (1-5) australia of X-linked inhibitor of apoptosis protein (XIAP), of which embelin is an inhibitor [28]. Our own screening and profiling efforts showed that embelin is a highly potent and efficacious human and mouse GPR84 agonist in assays measuring inhibition of cAMP, with little activity at numerous additional GPCRs. Structure-activity relationship studies with respect to the length of the embelin alkyl side chain revealed a U-shaped relationship, with the optimal side chain length being 7 or 8 carbons and a complete loss of activity with very short (C3) or long (C15) side chains. The C7 and C8 embelin analogs exhibited similar potencies at human and mouse GPR84. Moreover, the C3 analog retaining the polar, hydroxyquinone head group that acts as a carboxylate mimetic completely lacked GPR84 activity and served as a useful control for off-target activities that might be mediated by the head group. These tools allowed us to observe correlations of rank order of potencies in primary cells to those in recombinant cells. The use of these tools, along with G-protein pathway inhibitors and cells derived from GPR84 deficient mice enabled us to strongly correlate signaling and functional activities observed for embelin in primary cells with its agonist action at the GPR84 receptor. Medium chain fatty acids, particularly capric, undecanoic and lauric acids with carbon chain lengths of 10, 11 and 12 carbons, respectively, have been previously characterized as GPR84 ligands [5]. The potencies of these fatty acids at GPR84 in various assay platforms are in the tens to hundreds of micromolar range [5]; concentrations significantly higher than those reported in physiological systems under normal conditions. This raises concerns regarding their role as true natural ligands for GPR84 in vivo. Interestingly, among all tested fatty acids, those with 10, 11, or 12 carbon alkyl chain lengths were the most potent for GPR84, while shorter acids with 8 carbons were inactive [5,10]. Among synthetic analogs, the most potent were ones with side chain lengths of 7 or 8. We hypothesize therefore that the dihydroxybenzoquinone head group present in embelin provides both a mimetic of the acid group of MCFAs as well as functioning as a spacer equivalent to around 2–3 carbons to bring the total length into the same range as the more active MCFAs. The combination of these two features though, provides analogues with significantly greater potency for GPR84 compared to MCFAs. A similar SAR has been described recently using an alternative heterocyclic head group, 4,6-dihydroxypyrimidine. As in our study, a U-shaped chain length-response was observed and in this case the optimal chain length was again 7 or 8 carbons [31]. Also of note is the report that 6-n-octylamino uracil (6-OAU), with yet another alternative head group and an 8-carbon chain, has also been reported to be a potent agonist of GPR84 [10]. Thus, there is some commonality across a range of acidic and acid mimetic compounds with appended linear carbon chains and their ability to activate GPR84. Based on this assessment it can be speculated that physiologically relevant ligand(s) for GPR84 are likely to be of lipid or fatty acid nature, however the precise identity of a true endogenous or physiologically relevant ligand for GPR84 remains to be elucidated.