Development and evaluation of small peptidomimetic ligands to protease-activated receptor-2 (PAR2) through the use of lipid tethering

• Published in: PloS one Vol. 9; no. 6; p. e99140
• Main Authors: Boitano, Scott, Hoffman, Justin, Tillu, Dipti V, Asiedu, Marina N, Zhang, Zhenyu, Sherwood, Cara L, Wang, Yan, Dong, Xinzhong, Price, Theodore J, Vagner, Josef
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 13.06.2014; Public Library of Science (PLoS)
• Subjects: Activation; Analogs; Animals; Biology and Life Sciences; Calcium Signaling - drug effects; Calcium signalling; Cells, Cultured; CHO Cells; Collaboration; Cricetulus; Electronic mail systems; G protein-coupled receptors; Hypersensitivity; Kinases; Ligands; Male; Medicine and Health Sciences; Mice; Mice, Inbred ICR; Oligopeptides - chemical synthesis; Oligopeptides - pharmacology; Pain; Par-2 protein; Peg3 protein; Peptides; Peptidomimetics; Pharmacology; Physical Sciences; Physiological aspects; Physiology; Polyethylene; Polyethylene glycol; Protease; Proteases; Proteins; Proteolysis; Proto-Oncogene Mas; R&D; Receptor, PAR-2 - agonists; Receptor, PAR-2 - genetics; Research & development; Signal transduction; Structure-Activity Relationship; Substitutes; Tethering; United States > US; Arizona
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0099140

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.