Potent and metabolically stable agonists for protease-activated receptor-2: evaluation of activity in multiple assay systems in vitro and in vivo

Atsufumi Kawabata, Toru Kanke, Daiki Yonezawa, Tsuyoshi Ishiki, Masako Saka, Mototsugu Kabeya, Fumiko Sekiguchi, Satoko Kubo, Ryotaro Kuroda, Masahiro Iwaki, Kousaku Katsura, Robin Plevin

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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-NH2, and 2f-LIGRL-NH2, 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 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 powerful peptide, relative to SLIGKV-OH, was about 100 in the cultured cell Ca2+ 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-NH2, 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.

Original languageEnglish
Pages (from-to)1098-1107
Number of pages10
JournalJournal of Pharmacology and Experimental Therapeutics
Issue number3
Publication statusPublished - 1 Jun 2004


  • protease-activated receptors
  • amino acid substitution
  • protease activated receptor 2 agonist
  • amino terminal sequence

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