Identification of novel species-selective agonists of the G-protein-coupled receptor GPR35 that promote recruitment of β-arrestin-2 and activate Gα13

Abstract

The poorly characterized G-protein-coupled receptor GPR35 has been suggested as a potential exploratory target for the treatment of both metabolic disorders and hypertension. It has also been indicated to play an important role in immune modulation. A major impediment to validation of these concepts and further study of the role of this receptor has been a paucity of pharmacological tools that interact with GPR35. Using a receptor-β-arrestin-2 interaction assay with both human and rat orthologues of GPR35, we identified a number of compounds possessing agonist activity. These included the previously described ligand zaprinast. Although a number of active compounds, including cromolyn disodium and dicumarol, displayed similar potency at both orthologues of GPR35, a number of ligands, including pamoate and niflumic acid, had detectable activity only at human GPR35 whereas others, including zaprinast and luteolin, were markedly selective for the rat orthologue. Previous studies have demonstrated activation of Gα13 by GPR35. A Saccharomyces cerevisiae-based assay employing a chimaeric Gpa1-Gα13 G-protein confirmed that all of the compounds active at human GPR35 in the β-arrestin-2 interaction assay were also able to promote cell growth via Gα13. Each of these ligands also promoted binding of [35S]GTP[S] (guanosine 5'-[γ-[35S]thio]triphosphate) to an epitope-tagged form of Gα13 in a GPR35-dependent manner. The ligands identified in these studies will be useful in interrogating the biological actions of GPR35, but appreciation of the species selectivity of ligands at this receptor will be vital to correctly attribute function.

Distinct Phosphorylation Clusters Determine the Signaling Outcome of Free Fatty Acid Receptor 4/G Protein–Coupled Receptor 120

“…mFFA4-Arrestin 3 Interaction Assay. Arrestin 3 recruitment to mFFA4 was assessed using a bioluminescence resonance energy transfer (BRET) assay (Jenkins et al, 2010;Butcher et al, 2014;Hudson et al, 2014;MacKenzie et al, 2014). Briefly, human embryonic kidney 293T (HEK293T) cells were cotransfected with arrestin 3-Renilla luciferase and eYFP-tagged receptor plasmids in a 1:4 ratio using polyethyleneimine.…”

The Antiallergic Mast Cell Stabilizers Lodoxamide and Bufrolin as the First High and Equipotent Agonists of Human and Rat GPR35

“…Although GPR35 is indicated to be a receptor responsive to the endogenously produced tryptophan metabolite kynurenic acid (Wang et al, 2006), lack of convergence on this issue has resulted in a number of efforts to identify surrogate agonist ligands that might be used to help further define the roles of this receptor. Although a number of such ligands have been identified (Taniguchi et al, 2006;Jenkins et al, 2010;Zhao et al, 2010;Deng et al, 2012;Funke et al, 2013;Neetoo-Isseljee et al, 2013), many of these are either of modest potency and/or display markedly different potency at human and rodent orthologs of GPR35 (Jenkins et al, 2010;Funke et al, 2013;Neetoo-Isseljee et al, 2013). This has posed challenges both in efforts to define the orthosteric binding pocket of the receptor and to use rodents and cell lines derived from such animals to further explore the function of GPR35.…”

“…The antiasthma and antiallergic agents cromolyn disodium (Jenkins et al, 2010;Yang et al, 2010) and nedocromil sodium (Yang et al, 2010) were recently shown to act as moderately potent agonists of GPR35. These ligands are both di-acids with marked mirror image symmetry (Fig.…”

“…Zaprinast (Taniguchi et al, 2006;Jenkins et al, 2010Jenkins et al, , 2011 has become the standard surrogate agonist of GPR35 and, akin to zaprinast, a number of mast cell stabilizers also contain an acid bioisostere. Because zaprinast is an example of a ligand that displays marked variation in potency between rodent and human GPR35 (Taniguchi et al, 2006;Jenkins et al, 2010Jenkins et al, , 2011, this also encouraged us to also explore the contributions of a series of nonmaintained arginine residues within regions of the receptor species orthologs that are predicted to define the ligand binding pocket. Cross-species alterations of such residues provided novel insights into the binding pocket.…”

Efficacy and Safety of Morniflumate for the Treatment of Symptoms Associated with Soft Tissue Inflammation

“…50 Niflumic acid has also been found to be an activator of GPR (G-protein coupled receptor) 35, a protein implicated in a number of conditions including inflammatory pain, asthma, heart disease, cancer, and metabolic diseases, and regarded as a promising therapeutic target. 51 Preclinical studies have shown that morniflumate has marked anti-inflammatory activity, comparable with niflumic acid.…”