Development and Evaluation of Small Peptidomimetic Ligands to Protease-Activated Receptor-2 (PAR(2)) through the Use of Lipid Tethering

Protease-activated receptor-2 (PAR(2)) 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 PAR(2) with in vitro physiological and Ca(2+) signaling assays to determine minimal components necessary for potent, specific and full PAR(2) activation. A known PAR(2) activating compound containing a hexadecyl (Hdc) lipid via three polyethylene glycol (PEG) linkers (2at-LIGRL-PEG (3)-Hdc) provided a potent agonist starting point (physiological EC(50) = 1.4 nM; 95% CI: 1.2–2.3 nM). In a set of truncated analogs, 2at-LIGR-PEG (3)-Hdc retained potency (EC(50) = 2.1 nM; 1.3–3.4 nM) with improved selectivity for PAR(2) over Mas1 related G-protein coupled receptor type C11, a GPCR that can be activated by the PAR(2) peptide agonist, SLIGRL-NH(2). 2at-LIG-PEG (3)-Hdc was the smallest full PAR(2) agonist, albeit with a reduced EC(50) (46 nM; 20–100 nM). 2at-LI-PEG (3)-Hdc retained specific activity for PAR(2) with reduced EC(50) (310 nM; 260–360 nM) but displayed partial PAR(2) activation in both physiological and Ca(2+) signaling assays. Further truncation (2at-L-PEG (3)-Hdc and 2at-PEG (3)-Hdc) eliminated in vitro activity. When used in vivo, full and partial PAR(2) in vitro agonists evoked mechanical hypersensitivity at a 15 pmole dose while 2at-L-PEG (3)-Hdc lacked efficacy. Minimum peptidomimetic PAR(2) agonists were developed with known heterocycle substitutes for Ser(1) (isoxazole or aminothiazoyl) and cyclohexylalanine (Cha) as a substitute for Leu(2). Both heterocycle-tetrapeptide and heterocycle-dipeptides displayed PAR(2) specificity, however, only the heterocycle-tetrapeptides displayed full PAR(2) agonism. Using the lipid-tethered-peptidomimetic approach we have developed novel structure activity relationships for PAR(2) that allows for selective probing of PAR(2) function across a broad range of physiological systems.