Novel antagonists for proteinase-activated receptor 2: Inhibition of cellular and vascular responses in vitro and in vivo

Abstract

Background and purpose: Proteinase-activated receptor 2 (PAR 2) is a G-protein coupled receptor associated with many pathophysiological functions. To date, the development of PAR 2 antagonists has been limited. Here, we identify a number of novel peptide-mimetic PAR 2 antagonists and demonstrate inhibitory effects on PAR 2-mediated intracellular signalling pathways and vascular responses. Experimental approach: The peptide-mimetic compound library based on the structures of PAR 2 agonist peptides were screened for inhibition of PAR 2-induced calcium mobilisation in human keratinocytes. Representative compounds were further evaluated by radioligand binding and inhibition of NFκB transcriptional activity and IL-8 production. The vascular effects of the antagonists were assessed using in vitro and in vivo models. Key results: Two compounds, K-12940 and K-14585, significantly reduced SLIGKV-induced Ca 2+ mobilisation in primary human keratinocytes. Both K-12940 and K-14585 exhibited competitive inhibition for the binding of a high-affinity radiolabelled PAR 2-ligand, [ 3H]-2-furoyl-LIGRL-NH 2, to human PAR 2 with K i values of 1.94 and 0.627 μM respectively. NFκB reporter activity and IL-8 production were also significantly reduced. Furthermore, relaxation of rat-isolated aorta induced by SLIGRL-NH 2 was inhibited competitively by K-14585. K-14585 also significantly lowered plasma extravasation in the dorsal skin of guinea pigs and reduced salivation in mice. Conclusions and implications: K-12940 and K-14585 antagonized PAR 2 competitively, resulting in inhibition of PAR 2-mediated signalling and physiological responses both in vitro and in vivo. These peptide-mimetic PAR 2 antagonists could be useful in evaluating PAR 2-mediated biological events and might lead to a new generation of therapeutically useful antagonists. © 2009 The British Pharmacological Society.