Protease-activated receptor-2 (PAR(2)) is a G-protein coupled receptor (GPCR) associated with a variety of pathologies. However, the therapeutic potential of PAR(2) is limited by a lack of potent and specific ligands. Following proteolytic cleavage, PAR(2) is activated through a tethered ligand. Hence, we reasoned that lipidation of peptidomimetic ligands could promote membrane targeting and thus significantly improve potency and constructed a series of synthetic tethered ligands (STLs). STLs contained a peptidomimetic PAR(2) agonist (2-aminothiazol-4-yl-LIGRL-NH(2)) bound to a palmitoyl group (Pam) via polyethylene glycol (PEG) linkers. In a high-throughput physiological assay, these STL agonists displayed EC(50) values as low as 1.47 nM, representing a ∼200 fold improvement over the untethered parent ligand. Similarly, these STL agonists were potent activators of signaling pathways associated with PAR(2): EC(50) for Ca(2+) response as low as 3.95 nM; EC(50) for MAPK response as low as 9.49 nM. Moreover, STLs demonstrated significant improvement in potency in vivo, evoking mechanical allodynia with an EC(50) of 14.4 pmol. STLs failed to elicit responses in PAR(2)(-/-) cells at agonist concentrations of >300-fold their EC(50) values. Our results demonstrate that the STL approach is a powerful tool for increasing ligand potency at PAR(2) and represent opportunities for drug development at other protease activated receptors and across GPCRs.-Flynn, A. N., Hoffman, J., Tillu, D. V., Sherwood, C. L., Zhang, Z., Patek, R., Asiedu, M. N. K., Vagner, J., Price, T. J., Boitano, S. Development of highly potent protease-activated receptor 2 agonists via synthetic lipid tethering.