The emerging pharmacology and function of GPR35 in the nervous system

G protein-coupled receptor 35 (GPR35) is an orphan G protein-coupled receptor (GPCR) that can be activated by kynurenic acid at high micromolar concentrations. A previously unappreciated mechanism of action of GPR35 has emerged as a Gαi/o-coupled inhibitor of synaptic transmission, a finding that has significant implications for the accepted role of kynurenic acid as a broad-spectrum antagonist of the NMDA, AMPA/kainite and α7 nicotinic receptors. In conjunction with previous findings that link agonism of GPR35 with significant reduction in nociceptive pain, GPR35 has emerged as a potential effector of regulation of mechanical sensitivity and analgesia of the Ret tyrosine kinase, and as a receptor involved in the transmission of anti-inflammatory effects of aspirin- potentially through affecting leucocyte rolling, adhesion and extravasation. Single nucleotide polymorphisms of GPR35 have linked this receptor to coronary artery calcification, inflammatory bowel disease and primary sclerosing cholangitis, while chromosomal aberrations of the 2q37.3 locus and altered copy number of GPR35 have been linked with autism, Albight's hereditary osteodystrophy-like syndrome, and congenital malformations, respectively. Herein, we present an update on both the pharmacology and potential function of GPR35, particularly pertaining to the nervous system. This review forms part of a special edition focussing on the role of lipid-sensing GPCRs in the nervous system

Novel and potent GPR35 agonists aid identification of residues important in ligand interaction

The orphan G protein coupled receptor GPR35 has emerged as a therapeutic target in a number of disease states including metabolic disorder, cardiovascular disease, inflammation and nociception. Efforts to characterize this receptor, however, have been complicated by marked differences in the pharmacology of GPR35 species orthologs. We have identified novel agonist compounds with which to dissect the pharmacology and function of GPR35. Selected compounds were chosen to illustrate the differences in ligand structure activity relationship between human, mouse and rat orthologs of GPR35. Sequence alignment and receptor homology modeling techniques guided site directed mutagenesis, which highlighted a number of residues involved in GPR35 ligand interaction and revealed differences between rodent and human orthologs. These findings will benefit future drug development programs and efforts to generate therapeutically relevant GPR35-specific pharmacological compounds

High-throughput identification and characterization of novel, species-selective GPR35 agonists

Drugs targeting the orphan receptor GPR35 have potential therapeutic application in a number of disease areas, including inflammation, metabolic disorders, nociception, and cardiovascular disease. Currently available surrogate GPR35 agonists identified from pharmacologically relevant compound libraries have limited utility due to the likelihood of off-target effects in vitro and in vivo and the variable potency that such ligands exhibit across species. We sought to identify and characterize novel GPR35 agonists to facilitate studies aimed at defining the physiologic role of GPR35. PathHunter β-arrestin recruitment technology was validated as a human GPR35 screening assay, and a high-throughput screen of 100,000 diverse low molecular weight compounds was conducted. Confirmed GPR35 agonists from five distinct chemotypes were selected for detailed characterization using both β-arrestin recruitment and G protein-dependent assays and each of the human, mouse, and rat GPR35 orthologs. These studies identified 4-{(Z)-[(2Z)-2-(2-fluorobenzylidene)-4-oxo-1,3-thiazolidin-5-ylidene]methyl}benzoic acid (compound 1) as the highest potency full agonist of human GPR35 yet described. As with certain other GPR35 agonists, compound 1 was markedly selective for human GPR35, but displayed elements of signal bias between β-arrestin-2 and G protein-dependent assays. Compound 1 also displayed competitive behavior when assessed against the human GPR35 antagonist, ML-145 (2-hydroxy-4-[4-(5Z)-5-[(E)-2-methyl-3-phenylprop-2-enylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]butanoylamino]benzoic acid). Of the other chemotypes studied, compounds 2 and 3 were selective for the human receptor, but compounds 4 and 5 demonstrated similar activity at human, rat, and mouse GPR35 orthologs. Further characterization of these compounds and related analogs is likely to facilitate a better understanding of GPR35 in health and disease

Antagonists of GPR35 display high species ortholog selectivity and varying modes of action

Variation in pharmacology and function of ligands at species orthologs can be a confounding feature in understanding the biology and role of poorly characterized receptors. Substantial selectivity in potency of a number of GPR35 agonists has previously been demonstrated between human and rat orthologs of this G protein-coupled receptor. Via a bioluminescence resonance energy transfer-based assay of induced interactions between GPR35 and β-arrestin-2, addition of the mouse ortholog to such studies indicated that, as for the rat ortholog, murine GPR35 displayed very low potency for pamoate, whereas potency for the reference GPR35 agonist zaprinast was intermediate between the rat and human orthologs. This pattern was replicated in receptor internalization and G protein activation assays. The effectiveness and mode of action of two recently reported GPR35 antagonists, methyl-5-[(tert-butylcarbamothioylhydrazinylidene)methyl]-1-(2,4-difluorophenyl)pyrazole-4-carboxylate (CID-2745687) and 2-hydroxy-4-[4-(5Z)-5-[(E)-2-methyl-3-phenylprop-2-enylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]butanoylamino)benzoic acid (ML-145), were investigated. Both CID-2745687 and ML-145 competitively inhibited the effects at human GPR35 of cromolyn disodium and zaprinast, two agonists that share an overlapping binding site. By contrast, although ML-145 also competitively antagonized the effects of pamoate, CID-2745687 acted in a noncompetitive fashion. Neither ML-145 nor CID-2745687 was able to effectively antagonize the agonist effects of either zaprinast or cromolyn disodium at either rodent ortholog of GPR35. These studies demonstrate that marked species selectivity of ligands at GPR35 is not restricted to agonists and considerable care is required to select appropriate ligands to explore the function of GPR35 in nonhuman cells and tissues