title: The novel PAR2 ligand C391 blocks multiple PAR2 signalling pathways in vitro and in vivo.
abstract: OBJECTIVE
  Proteinase-activated receptor-2 (PAR2) is a GPCR linked to diverse pathologies, including acute and chronic pain. PAR2 is one of the four PARs that are activated by proteolytic cleavage of the extracellular amino terminus, resulting in an exposed, tethered peptide agonist. Several peptide and peptidomimetic agonists, with high potency and efficacy, have been developed to probe the functions of PAR2, in vitro and in vivo. However, few similarly potent and effective antagonists have been described.
  
  
  METHODS
  We modified the peptidomimetic PAR2 agonist, 2-furoyl-LIGRLO-NH2, to create a novel PAR2 peptidomimetic ligand, C391. C391 was evaluated for PAR2 agonist/antagonist activity to PAR2 across Gqsignalling pathways using the naturally expressing PAR2 cell line 16HBE14o-. For antagonist studies, a highly potent and specific peptidomimetic agonist (2-aminothiazo-4-yl-LIGRL-NH2) and proteinase agonist (trypsin) were used to activate PAR2. C391 was also evaluated in vivo for reduction of thermal hyperalgesia, mediated by mast cell degranulation, in mice.
  
  
  RESULTS
  C391 is a potent and specific peptidomimetic antagonist, blocking multiple signalling pathways (Gq-dependent Ca2+, MAPK) induced following peptidomimetic or proteinase activation of human PAR2. In a PAR2-dependent behavioural assay in mice, C391 dose-dependently (75 μg maximum effect) blocked the thermal hyperalgesia, mediated by mast cell degranulation.
  
  
  CONCLUSIONS
  C391 is the first low MW antagonist to block both PAR2 Ca2+and MAPK signalling pathways activated by peptidomimetics and/or proteinase activation. C391 represents a new molecular structure for PAR2 antagonism and can serve as a basis for further development for this important therapeutic target.
authors.affiliation: Arizona Respiratory Center and Department of Physiology, University of Arizona, Tucson, AZ, USA.|The BIO5 Collaborative Research Institute, University of Arizona, Tucson, AZ, USA.
authors.forename: Scott
authors.initials: S
authors.lastname: Boitano
authors.affiliation: Arizona Respiratory Center and Department of Physiology, University of Arizona, Tucson, AZ, USA.|The BIO5 Collaborative Research Institute, University of Arizona, Tucson, AZ, USA.
authors.forename: Justin
authors.initials: J
authors.lastname: Hoffman
authors.affiliation: Arizona Respiratory Center and Department of Physiology, University of Arizona, Tucson, AZ, USA.|The BIO5 Collaborative Research Institute, University of Arizona, Tucson, AZ, USA.
authors.forename: Andrea N
authors.initials: AN
authors.lastname: Flynn
authors.affiliation: Department of Pharmacology, University of Arizona, Tucson, AZ, USA.
authors.forename: Marina N
authors.initials: MN
authors.lastname: Asiedu
authors.affiliation: Department of Pharmacology, University of Arizona, Tucson, AZ, USA.
authors.forename: Dipti V
authors.initials: DV
authors.lastname: Tillu
authors.affiliation: The BIO5 Collaborative Research Institute, University of Arizona, Tucson, AZ, USA.
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Arizona Respiratory Center and Department of Physiology, University of Arizona, Tucson, AZ, USA.|The BIO5 Collaborative Research Institute, University of Arizona, Tucson, AZ, USA.
authors.forename: Cara L
authors.initials: CL
authors.lastname: Sherwood
authors.affiliation: Arizona Respiratory Center and Department of Physiology, University of Arizona, Tucson, AZ, USA.|The BIO5 Collaborative Research Institute, University of Arizona, Tucson, AZ, USA.|Graduate Interdisciplinary Program in Physiological Sciences, University of Arizona, Tucson, AZ, USA.
authors.forename: Candy M
authors.initials: CM
authors.lastname: Rivas
authors.affiliation: Biomedical Sciences Division, University of California, Riverside, CA, USA.
authors.forename: Kathryn A
authors.initials: KA
authors.lastname: DeFea
authors.affiliation: The BIO5 Collaborative Research Institute, University of Arizona, Tucson, AZ, USA.
authors.forename: Josef
authors.initials: J
authors.lastname: Vagner
authors.affiliation: The BIO5 Collaborative Research Institute, University of Arizona, Tucson, AZ, USA.|Department of Pharmacology, University of Arizona, Tucson, AZ, USA.|School of Behavioral and Brain Sciences, University of Texas at Dallas, Dallas, TX, USA.
authors.forename: Theodore J
authors.initials: TJ
authors.lastname: Price