Pharmacological properties of acid N ‐thiazolylamide FFA 2 agonists

FFA 2 is a receptor for short‐chain fatty acids. Propionate ( C3 ) and 4‐chloro‐ α ‐(1‐methylethyl)‐ N ‐2‐thiazolyl‐benzeneacetamide (4‐ CMTB ), the prototypical synthetic FFA 2 agonist, evoke calcium mobilization in neutrophils and inhibit lipolysis in adipocytes via this G‐protein‐coupled receptor. 4‐ CMTB contains an N ‐thiazolylamide motif but no acid group, and 4‐ CMTB and C3 bind to different sites on FFA 2 and show allosteric cooperativity. Recently, FFA 2 agonists have been described that contain both N ‐thiazolylamide and carboxylate groups, reminiscent of bitopic ligands. These are thought to engage the carboxylate‐binding site on FFA 2, but preliminary evidence suggests they do not bind to the same site as 4‐ CMTB even though both contain N ‐thiazolylamide. Here, we describe the characterization of four FFA 2 ligands containing both N ‐thiazolylamide and carboxylate. (R)‐3‐benzyl‐4‐((4‐(2‐chlorophenyl)thiazol‐2‐yl)(methyl)amino)‐4‐oxobutanoic acid (compound 14 ) exhibits allosteric agonism with 4‐ CMTB but not C3 . Three other compounds agonize FFA 2 in [ 35 S] GTP γ S‐incorporation or cAMP assays but behave as inverse agonists in yeast‐based gene‐reporter assays, showing orthosteric antagonism of C3 responses but allosteric antagonism of 4‐ CMTB responses. Thus, the bitopic‐like FFA 2 ligands engage the orthosteric site but do not compete at the site of 4‐ CMTB binding on an FFA 2 receptor molecule. Compound 14 activates FFA 2 on human neutrophils and mouse adipocytes, but appears not to inhibit lipolysis upon treatment of human primary adipocytes in spite of the presence of a functional FFA 2 receptor in these cells. Hence, these new ligands may reveal differences in coupling of FFA 2 between human and rodent adipose tissues.