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

1. Amanda E MacKenzie1, 
2. Gianluigi Caltabiano2, 
3. Toby C Kent3,
4. Laura Jenkins1, 
5. Jennifer E McCallum1, 
6. Brian D Hudson1,
7. Stuart A Nicklin1, 
8. Lindsay Fawcett3, 
9. Rachel Lane3, 
10. Steven J Charlton3, and 
11. Graeme Milligan1,*

+Author Affiliations

1. ¹ University of Glasgow;
2. ² Universitat Autonoma de Barcelona;
3. ³ Novartis

1. ↵* Corresponding author; email: graeme.milligan@glasgow.ac.uk

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 anti-asthma 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. As both lodoxamide and bufrolin are symmetric di-acids their potential mode of binding was explored via mutagenesis based on swapping non-conserved arginine residues within proximity of a key conserved arginine at position 3.36 between the rat and human orthologs. Computational modelling and ligand docking predicted the contributions of different arginine residues, other than at 3.36, in human GPR35 for these two ligands and was 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 SNP. This prediction was confirmed experimentally.

This Article

1. Published online before print October 10, 2013, doi:10.1124/mol.113.089482Molecular PharmacologyOctober 10, 2013mol.113.089482

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• Orphan GPCRs
 
• GRKs, barrestins
 
• Structure-activity relationships and modeling
• Mutagenesis/Chimeric approaches
 
• Leukocytes/Mast cells

• The American Society for Pharmacology and Experimental Therapeutics