Potent and Metabolically Stable Agonists for Protease-Activated Receptor-2:Evaluation of Activity in Multiple Assay Systems in Vitro and in Vivo

摘要

To develop potent and metabolically stable agonists for pro-tease-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 cyto-solic Ca~(2+) 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 powerfulpeptide,relative to SLIGKV-OH,was about 100 in the cultured cell Ca~(2+) 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 C57BU6 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.