title: Development and evaluation of small peptidomimetic ligands to protease-activated receptor-2 (PAR2) through the use of lipid tethering.
abstract: Protease-activated receptor-2 (PAR2) is a G-Protein Coupled Receptor (GPCR) activated by proteolytic cleavage to expose an attached, tethered ligand (SLIGRL). We evaluated the ability for lipid-tethered-peptidomimetics to activate PAR2 with in vitro physiological and Ca2+ signaling assays to determine minimal components necessary for potent, specific and full PAR2 activation. A known PAR2 activating compound containing a hexadecyl (Hdc) lipid via three polyethylene glycol (PEG) linkers (2at-LIGRL-PEG3-Hdc) provided a potent agonist starting point (physiological EC50 = 1.4 nM; 95% CI: 1.2-2.3 nM). In a set of truncated analogs, 2at-LIGR-PEG3-Hdc retained potency (EC50 = 2.1 nM; 1.3-3.4 nM) with improved selectivity for PAR2 over Mas1 related G-protein coupled receptor type C11, a GPCR that can be activated by the PAR2 peptide agonist, SLIGRL-NH2. 2at-LIG-PEG3-Hdc was the smallest full PAR2 agonist, albeit with a reduced EC50 (46 nM; 20-100 nM). 2at-LI-PEG3-Hdc retained specific activity for PAR2 with reduced EC50 (310 nM; 260-360 nM) but displayed partial PAR2 activation in both physiological and Ca2+ signaling assays. Further truncation (2at-L-PEG3-Hdc and 2at-PEG3-Hdc) eliminated in vitro activity. When used in vivo, full and partial PAR2 in vitro agonists evoked mechanical hypersensitivity at a 15 pmole dose while 2at-L-PEG3-Hdc lacked efficacy. Minimum peptidomimetic PAR2 agonists were developed with known heterocycle substitutes for Ser1 (isoxazole or aminothiazoyl) and cyclohexylalanine (Cha) as a substitute for Leu2. Both heterocycle-tetrapeptide and heterocycle-dipeptides displayed PAR2 specificity, however, only the heterocycle-tetrapeptides displayed full PAR2 agonism. Using the lipid-tethered-peptidomimetic approach we have developed novel structure activity relationships for PAR2 that allows for selective probing of PAR2 function across a broad range of physiological systems.
authors.affiliation: Arizona Respiratory Center and Department of Physiology, University of Arizona, Tucson, Arizona, United States of America; The BIO5 Collaborative Research Institute, University of Arizona, Tucson, Arizona, United States of America.
authors.forename: Scott
authors.initials: S
authors.lastname: Boitano
authors.affiliation: Arizona Respiratory Center and Department of Physiology, University of Arizona, Tucson, Arizona, United States of America; The BIO5 Collaborative Research Institute, University of Arizona, Tucson, Arizona, United States of America.
authors.forename: Justin
authors.initials: J
authors.lastname: Hoffman
authors.affiliation: Department of Pharmacology, University of Arizona, Tucson, Arizona, United States of America.
authors.forename: Dipti V
authors.initials: DV
authors.lastname: Tillu
authors.affiliation: Department of Pharmacology, University of Arizona, Tucson, Arizona, United States of America.
authors.forename: Marina N
authors.initials: MN
authors.lastname: Asiedu
authors.affiliation: The BIO5 Collaborative Research Institute, University of Arizona, Tucson, Arizona, United States of America.
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Arizona Respiratory Center and Department of Physiology, University of Arizona, Tucson, Arizona, United States of America; The BIO5 Collaborative Research Institute, University of Arizona, Tucson, Arizona, United States of America.
authors.forename: Cara L
authors.initials: CL
authors.lastname: Sherwood
authors.affiliation: State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Neuroscience, Johns Hopkins University, Baltimore, Maryland, United States of America.
authors.forename: Xinzhong
authors.initials: X
authors.lastname: Dong
authors.affiliation: The BIO5 Collaborative Research Institute, University of Arizona, Tucson, Arizona, United States of America; Department of Pharmacology, University of Arizona, Tucson, Arizona, United States of America.
authors.forename: Theodore J
authors.initials: TJ
authors.lastname: Price
authors.affiliation: The BIO5 Collaborative Research Institute, University of Arizona, Tucson, Arizona, United States of America.
authors.forename: Josef
authors.initials: J
authors.lastname: Vagner
mesh_terms: D000818:Animals; D016466:CHO Cells; D020013:Calcium Signaling / Q000187:drug effects*; D002478:Cells, Cultured; D003412:Cricetulus; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D009842:Oligopeptides / Q000138:chemical synthesis* / Q000494:pharmacology*; D057786:Peptidomimetics; D000090063:Proto-Oncogene Mas; D044464:Receptor, PAR-2 / Q000819:agonists* / Q000235:genetics*; D013329:Structure-Activity Relationship
chemical_list: C000602226:2at-LIGRL-PEG3-Hdc; C000715471:Mas1 protein, mouse; D009842:Oligopeptides; D057786:Peptidomimetics; D000090063:Proto-Oncogene Mas; D044464:Receptor, PAR-2; C112356:seryl-leucyl-isoleucyl-glycyl--arginyl-leucinamide