Potent Small Agonists of Protease Activated Receptor 2

Abstract

Many proteases cut the PAR2 N-terminus resulting in conformational changes that activate cells. Synthetic peptides corresponding to newly exposed N-terminal sequences of PAR2 also activate the receptor at micromolar concentrations. PAR2-selective small molecules reported here induce PAR2-mediated intracellular calcium signaling at nanomolar concentrations (EC50 = 15-100 nM, iCa(2+), CHO-hPAR2 cells). These are the most potent and efficient small molecule ligands to activate PAR2-mediated calcium release and chemotaxis, including for human breast and prostate cancer cells.

Keywords: Protease activated receptor 2; agonist; cyclohexylglycine; structure−activity relationships.

Figure 1

Known synthetic PAR2 agonists.^,,−

Figure 2

Predicted binding mode for agonist 14 in a PAR2 homology model derived from nociceptin/orphanin FQ receptor (pdb code 4EA3). (A) Agonist AY77 (14) is predicted to form multiple hydrogen bonds and hydrophobic interactions with PAR2. (B) Surface view of PAR2 model showing Isox, Cha, and Chg components occupying three distinct binding pockets. (C) iCa²⁺ responses induced by agonist 14 were significantly reduced in PAR2 mutants Y156A, Y326A, D228A, L307A, and Y82A compared to wild-type (WT), suggesting that these residues interact with 14. Each data point is mean ± SEM of 3–5 independent experiments. (D) The C-terminal amide NH₂ of 14 forms hydrogen bonds with Y156 and D228. The cyclohexylglycine may make a hydrophobic interaction with L307 and the amide carbonyl of Cha may form a water mediated H-bond with Y326. Isoxazole may form a H-bond with Y82.

Figure 3

(a) Agonist 14 was stable in rat plasma after 4 h (pH 7.4, 37 °C, initial concentration 1 μM) compared to peptides 1 and 6. The plasma samples were analyzed by LCMS (n = 3). (b) Agonists 1 and 14 (AY77) activate Ca²⁺ release via PAR2. Experiments were performed in CHO-hPAR2 cells (solid lines) versus CHO cells (PAR2^–/–, dotted lines), n ≥ 3. Each data point represents mean ± SEM.

Figure 4

PAR2-activated chemotaxis in MDA-MB-231 breast cancer cells. Compounds 1 and 14 (100 nM) induced chemotaxis in a transwell migration assay. Preincubating cells with PAR2 antibody SAM11 (1 μg/mL) 30 min prior to agonist addition resulted in inhibition of chemotaxis. The chemotactic index is the ratio of agonist to random migration (24 h) through a 5 μm membrane, mean ± SEM, ***p < 0.001.