High-Throughput Identification and Characterization of Novel, Species-selective GPR35 Agonists

Abstract

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 b-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 b-arrestin recruitment and G proteindependent 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 b-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.

GABABreceptor allosteric modulators exhibit pathway-dependent and species-selective activity

“…; Neetoo‐Isseljee et al. ) as well as for allosteric modulators of the mGluR1 receptor, another family C GPCR (Knoflach et al. ).…”

Receptor selectivity between the G proteins Gα 12 and Gα 13 is defined by a single leucine‐to‐isoleucine variation

“…Compound 1 {4‐[(Z)‐[(2Z)‐2‐(2‐fluorobenzylidene)‐4‐oxo‐1,3‐thiazolidin‐5‐ylidene]methyl] benzoic acid} is described in Neetoo‐Isseljee et al . (25). Bufrolin [5,6‐dimethyl‐2‐nitro‐1H‐indene‐1,3(2H)‐dione] is described in Mackenzie et al .…”

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

“…4B). We and others have reported that many compounds first identified as ligands at human GPR35, including zaprinast, display markedly different potency and/or activity at rodent orthologs (Taniguchi et al, 2006;Jenkins et al, 2010Jenkins et al, , 2012Funke et al, 2013;Neetoo-Isseljee et al, 2013). We next examined the activity of a variety of mast cell stabilizers at the rat ortholog of GPR35 in the BRET-based b-arrestin-2 interaction assay.…”

“…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.…”