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

Background and purpose: Proteinase‐activated receptor 2 (PAR2) is a G‐protein coupled receptor associated with many pathophysiological functions. To date, the development of PAR2 antagonists has been limited. Here, we identify a number of novel peptide‐mimetic PAR2 antagonists and demonstrate inhibitory effects on PAR2‐mediated intracellular signalling pathways and vascular responses. Experimental approach: The peptide‐mimetic compound library based on the structures of PAR2 agonist peptides were screened for inhibition of PAR2‐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 Ca2+ mobilisation in primary human keratinocytes. Both K‐12940 and K‐14585 exhibited competitive inhibition for the binding of a high‐affinity radiolabelled PAR2‐ligand, [3H]‐2‐furoyl‐LIGRL‐NH2, to human PAR2 with Ki 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‐NH2 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 PAR2 competitively, resulting in inhibition of PAR2‐mediated signalling and physiological responses both in vitro and in vivo. These peptide‐mimetic PAR2 antagonists could be useful in evaluating PAR2‐mediated biological events and might lead to a new generation of therapeutically useful antagonists.