Characterisation of small molecule ligands 4CMTB and 2CTAP as modulators of human FFA2 receptor signalling

Zoe V Schofield,Ernest Tee,Daniel Croker,Trent M Woodruff,Philip M Hansbro,Avril A B Robertson,Reena Halai,David Edwards,Nicholas L Massey,Matthew A Cooper,Chantal Donovan

doi:10.1038/s41598-018-36242-1

Zoe V Schofield, Ernest Tee + Show 9 more

Open Access

https://doi.org/10.1038/s41598-018-36242-1

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Abstract

Short chain fatty acids (SCFAs) are protective against inflammatory diseases. Free fatty acid receptor 2 (FFA2), is a target of SCFAs however, their selectivity for FFA2 over other FFA receptors is limited. This study aimed to functionally characterise 2-(4-chlorophenyl)-3-methyl-N-(thiazole-2-yl)butanamide (4CMTB) and 4-((4-(2-chlorophenyl)thiazole-2-yl)amino)-4oxo-3-phenylbutanoic acid (2CTAP), and their enantiomers, in modulating FFA2 activity. The racemic mixture (R/S) and its constituents (R-) and (S-) 4CMTB or 2CTAP were used to stimulate human (h)FFA2 in the absence or presence of acetate. Calcium ions (Ca2+), phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2) and cyclic adenosine monophosphate (cAMP) were measured. R/S-4CMTB is a functionally selective ago-allosteric ligand that enhances Ca2+ response to acetate. Both R/S-4CMTB and S-4CMTB are more potent activators of pERK1/2 and inhibitors of forskolin-induced cAMP than acetate. S-4CMTB increased neutrophil infiltration in intestinal ischemia reperfusion injury (IRI). 2CTAP inhibited constitutive Ca2+ levels, antagonised acetate-induced pERK1/2 and prevented damage following IRI. This study characterises enantiomers of functionally selective ligands for FFA2 in cells stably expressing hFFA2. It highlights the novel roles of selective FFA2 enantiomers 4CMTB and 2CTAP on Ca2+, pERK1/2 and cAMP and their roles as allosteric modulators which, may assist in efforts to design novel therapeutic agents for FFA2-driven inflammatory diseases.

Highlights

Short chain fatty acids (SCFAs) impart protective effects in inflammatory diseases such as colitis, asthma and arthritis[1]
We report novel roles for the selective Free fatty acid receptor 2 (FFA2) enantiomers, 4CMTB and 2CTAP, on Ca2+, phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2) and cyclic adenosine monophosphate (cAMP) responses and as allosteric modulators and, highlight their activity in an in vivo intestinal ischemia reperfusion injury (IRI) model
To characterise the functional selectivity of 4CMTB and 2CTAP for FFA2, Ca2+, pERK and cAMP assays were carried out, with comparison to the known FFA2 agonist, acetate, in a CHO cell line stably transfected with human FFA2 receptor (hFFA2)

Summary

Introduction

Short chain fatty acids (SCFAs) impart protective effects in inflammatory diseases such as colitis, asthma and arthritis[1] They act on many cell types, including neutrophils, principally by acting on free fatty acid receptors (FFARs). The highest expression of FFA2 is found in innate immune cells, especially in neutrophils, eosinophils, monocytes and B-lymphocytes[2,6,7] This suggests that the receptor may have a role in innate immunity and inflammation. This study fully characterises two FFA2 ligands; R-, S-, R/S- 2-(4-chlorophenyl)-3-methyl-N-(thiazole-2-yl) butanimide (4CMTB) and 4-((4-(2-chlorophenyl)thiazole-2-yl)amino)-4oxo-3-phenylbutanoic acid (2CTAP), and probes their signalling profiles in CHO cells stably expressing the human FFA2 receptor (hFFA2). We report novel roles for the selective FFA2 enantiomers, 4CMTB and 2CTAP, on Ca2+, pERK1/2 and cAMP responses and as allosteric modulators and, highlight their activity in an in vivo intestinal ischemia reperfusion injury (IRI) model

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