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

Abstract

Lack of high potency agonists has restricted analysis of the G protein-coupled receptor GPR35. Moreover, marked variation in potency and/or affinity of current ligands between human and rodent orthologs of GPR35 has limited their productive use in rodent models of physiology. Based on the reported modest potency of the antiasthma and antiallergic ligands cromolyn disodium and nedocromil sodium, we identified the related compounds lodoxamide and bufrolin as high potency agonists of human GPR35. Unlike previously identified high potency agonists that are highly selective for human GPR35, both lodoxamide and bufrolin displayed equivalent potency at rat GPR35. Further synthetic antiallergic ligands, either sharing features of the standard surrogate agonist zaprinast, or with lodoxamide and bufrolin, were also shown to display agonism at either human or rat GPR35. Because both lodoxamide and bufrolin are symmetric di-acids, their potential mode of binding was explored via mutagenesis based on swapping between the rat and human ortholog nonconserved arginine residues within proximity of a key conserved arginine at position 3.36. Computational modeling and ligand docking predicted the contributions of different arginine residues, other than at 3.36, in human GPR35 for these two ligands and were consistent with selective loss of potency of either bufrolin or lodoxamide at distinct arginine mutants. The computational models also suggested that bufrolin and lodoxamide would display reduced potency at a low-frequency human GPR35 single nucleotide polymorphism. This prediction was confirmed experimentally.

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 identity of all new constructs generated was verified by nucleotide sequencing. Bovine arrestin 3 was fused to Renilla luciferase as previously described MacKenzie et al, 2014).…”

Functional G-protein-coupled receptor 35 is expressed by neurons in the CA1 field of the hippocampus

“…This effect has been explained by the inhibitory action of amlexanox on S100A13, a Ca 2+ -binding cargo protein [28]. However, based on the report that amlexanox is a potent GPR35 agonist [10] and on our present observation that this ligand suppresses the firing frequency of CA1 SRIs, it is tempting to speculate that GPR35 plays an important role in ischemia-related mechanisms in the hippocampus. In fact, a recent study found that GPR35 is overexpressed in cardiomyocyte cell membranes after hypoxia [29].…”

“…Their apparent potencies vary with the assay and the animal species from which the receptors originate [9]. For instance, the apparent potency (EC50) for agonists to activate rat GPR35 increases in the order amlexanox (23 nM), zaprinast (98 nM), dicumarol (2 mM), cromolyn sodium (4.4 mM), and pamoic acid (>1000 mM) [9,10]. In contrast, the EC50 for pamoic acid to activate human GPR35 is <100 nM and that of zaprinast is 1 mM [11].…”

News in Cellular Allergology: A Review of the Human Mast Cell and Basophil Granulocyte Literature from January 2013 to May 2015

“…The anti-allergic mast cell stabilisers lodoxamide and bufrolin target GPR35, similar to cromolyn and nedocromil sodium, are more promising drug candidates as they are several orders of magnitude more potent [28]. A number of other factors inhibiting mast cell activation are listed in table 1.…”