Development and evaluation of small peptidomimetic ligands to Protease-Activated Receptor-2 (PAR₂) through the use of lipid tethering

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 PAR ₂ 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-PEG ₃-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 PAR ₂ 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.