To develop potent and metabolically stable agonists for protease-activated receptor-2 (PAR-2), we prepared 2-furoylated (2f) derivatives of native PAR-2-activating peptides, 2f-LIGKV-OH, 2f-LIGRL-OH, 2f-LIGKV-NH 2 , and 2f-LIGRL-NH 2 , and systematically evaluated their activity in PAR-2-responsive cell lines and tissues. In both HCT-15 cells and NCTC2544 cells overexpressing PAR-2, all furoylated peptides increased cytosolic Ca 2ϩ levels with a greater potency than the corresponding native peptides, although a similar maximum response was recorded. The absolute potency of each peptide was greater in NCTC2544, possibly due to a higher level of receptor expression. Furthermore, the difference in potency between the 2-furoylated peptides and the native peptides was enhanced when evaluated in the rat superior mesenteric artery and further increased when measuring PAR-2-mediated salivation in ddY mice in vivo. The potency of 2f-LIGRL-NH 2 , the most powerful peptide, relative to SLIGKV-OH, was about 100 in the cultured cell Ca 2ϩ signaling assays, 517 in the vasorelaxation assay, and 1100 in the salivation assay. Amastatin, an aminopeptidase inhibitor, augmented salivation caused by native peptides, but not furoylated peptides. The PAR-2-activating peptides, including the furoylated derivatives, also produced salivation in the wild-type C57BL/6 mice, but not the PAR-2-deficient mice. Our data thus demonstrate that substitution of the N-terminal serine with a furoyl group in native PAR-2-activating peptides dramatically enhances the agonistic activity and decreases degradation by aminopeptidase, leading to development of 2f-LIGRL-NH 2 , the most potent peptide. Furthermore, the data from PAR-2-deficient mice provide ultimate evidence for involvement of PAR-2 in salivation and the selective nature of the 2-furoylated peptides.

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 NFkB transcriptional activity and IL-8 production. The vascular effects of the antagonists were assessed using in vitro and in vivo models.

Abstract. PAR-2 is the second member of the family of proteinase-activated receptors activated by trypsin, tryptase, and several other serine proteinases. In order to evaluate the therapeutic potential for PAR-2, we have performed studies on PAR-2-mediated signal transduction and investigated the effects of PAR-2 gene deficiency in disease models. In addition to the Gprotein-coupled receptor-mediated common signal transduction pathways, inositol 1,4,5-trisphosphate production and mobilization of Ca

To determine the binding characteristics of a highly potent agonist for protease‐activated receptor‐2 (PAR2), 2‐furoyl‐Leu‐Ile‐Gly‐Arg‐Leu‐amide (2‐furoyl‐LIGRL‐NH2), whole‐cell binding assays were performed utilising a radioactive ligand, [3H]2‐furoyl‐LIGRL‐NH2. Specific binding of [3H]2‐furoyl‐LIGRL‐NH2 was observed in NCTC2544 cells, dependent upon PAR2 expression, and competitively displaced by the addition of unlabeled PAR2 agonists. Scatchard analysis of specific saturation binding suggested a single binding site, with Kd of 122±26.1 nM and a corresponding Bmax of 180±6 f mol in 3.0 × 105 cells. The relative binding affinities of a series of modified PAR2 agonist peptides obtained from competition studies paralleled their relative EC50 values for Ca2+ mobilisation assays, indicating improved binding affinities by substitution with 2‐furoyl at the N‐terminus serine. Pretreatment of cells with trypsin reduced specific binding of [3H]2‐furoyl‐LIGRL‐NH2, demonstrating direct competition between the synthetic agonist peptide and the proteolytically revealed tethered ligand for the binding site of the receptor. In HCT‐15 cells endogenously expressing PAR2, the binding of [3H]2‐furoyl‐LIGRL‐NH2 was displaced by addition of unlabeled ligands, Ser‐Leu‐Ile‐Gly‐Lys‐Val (SLIGKV‐OH) or 2‐furoyl‐LIGRL‐NH2. The relative binding affinity of 2‐furoyl‐LIGRL‐NH2 to SLIGKV‐OH was comparable to its relative EC50 value for Ca2+ mobilisation assays. The binding assay was successfully performed in monolayers of PAR2 expressing NCTC2544 and human umbilical vein endothelial cells (HUVEC), in 96‐ and 24‐well plate formats, respectively. These studies indicate that [3H]2‐furoyl‐LIGRL‐NH2 binds to human PAR2 at its ligand‐binding site. The use of this radioligand will be valuable for characterising chemicals that interact to PAR2. British Journal of Pharmacology (2005) 145, 255–263. doi:

T o y o h i k o Aoyama.* M o t o t s u g u Kabeya. A t s u k o Fukushima. a n d T a k a y u k i ~h i o i r i * F a c u l t y o f P h a r m a c e u t i c a l S c i e n c e s . Nagoya C i t y U n i v e r s i t y . T a n a b e -d a r i . M i z u h a -k u . Nagoya 467. Japan A b s t r a c t -L i t h i u m t r i m e t h y l s i l y l d i a r o m e t h a n e r e a c t s s m o o t h l y w i t h i s o t h i o c y a n a t e s i n d i e t h y l e t h e r t o g i v e Z-amino-1.3.4-G.