Potent agonists of the protease activated receptor2 (PAR₂)

Novel peptidomimetic pharmacophores to PAR₂ were designed based on the known activating peptide SLIGRL-NH₂. A set of 15 analogues was evaluated with a model cell line (16HBE14o-) that highly expresses PAR ₂. Cells exposed to the PAR₂ activating peptide with N-terminal 2-furoyl modification (2-furoyl-LIGRLO-NH₂) initiated increases in intracellular calcium concentration ([Ca²⁺]_i EC₅₀ = 0.84 μM) and in vitro physiological responses as measured by the xCELLigence real time cell analyzer (RTCA EC₅₀ = 138 nM). We discovered two selective PAR₂ agonists with comparable potency: compound 1 (2-aminothiazol-4-yl; Ca²⁺ EC₅₀ = 1.77 μM, RTCA EC₅₀ = 142 nM) and compound 2 (6-aminonicotinyl; Ca²⁺ EC₅₀ = 2.60 μM, RTCA EC₅₀ = 311 nM). Unlike the previously described agonist, these novel agonists are devoid of the metabolically unstable 2-furoyl modification and thus provide potential advantages for PAR₂ peptide design for in vitro and in vivo studies. The novel compounds described herein also serve as a starting point for structure-activity relationship (SAR) design and are, for the first time, evaluated via a unique high throughput in vitro physiological assay. Together these will lead to discovery of more potent agonists and antagonists of PAR ₂.