Allosteric Receptor Modulation uncovers an FFAR2 antagonist as a positive orthosteric modulator/agonist in disguise

Abstract

Two earlier described Free Fatty Acid Receptor 2 (FFAR2)-specific antagonists (CATPB and GLPG0974) have different receptor-interaction characteristics at the molecular/functional level. The inhibitory effect of the two antagonists, on the novel receptor-cross-talk activation signals generated by the ATP-receptor, show that both antagonists inhibit the effect of the positive allosteric FFAR2 modulators (PAMs) AZ1729 and Cmp58. No neutrophil activation was induced by AZ1729 or Cmp58 alone, but together they were co-agonistic PAMs and activated the superoxide generating NADPH-oxidase in neutrophils. This response was inhibited by CATPB but not by GLPG0974; in contrast, GLPG0974 acted as a positive modulator that increased the potency but not the efficacy of the response. At the signaling level, GLPG0974 changed the biased signaling induced by the co-agonistic PAMs, to include a rise in the cytosolic concentration of free calcium ions (Ca2+). This effect was reciprocal, i.e., GLPG0974 triggers a rise in intracellular Ca2+, demonstrating that GLPG0974 may act as an FFAR2 agonist. In summary, by studying the effects of the FFAR2 ligand GLPG0974 on neutrophils activation induced by the co-agonists AZ1729 and Cmp58, we reveal that GLPG0974 in addition to be an antagonist, displays also agonistic and positive FFAR2 modulating functions that affects the NADPH-oxidase activity and the receptor down-stream signaling induced by the two co-agonistic PAMs.