Potent lipidated antagonists to protease‐activated receptor 2 (PAR₂)

Protease‐activated receptor‐2 (PAR2) belongs to a four‐member family of G‐Protein coupled receptors (GPCRs) that contain internal ligands exposed following exogenous or endogenous protease cleavage of the extracellular amino terminus. PAR2 is associated with a variety of inflammatory conditions, including asthma and pain. The contributions of PAR2 signaling to disease has been hindered by the lack of potent, efficacious antagonists, and their potential for biased‐ligand signaling. We recently demonstrated that lipid tethering of known PAR2 peptidomimetic agonists based on the primary trypsin cleavage sequence (SLIGRL) increased their potency > 200 fold. In this study, we used lipid tethering (hexadecyl (Hdc) group with polyethylene glycol (PEG) spacers) and heterocycle (2‐aminothiazoyl; 2‐at) substitution of hexapeptide sequence derived from the primary cleavage site of kallikreins 4/16 (SSKGRS) to elucidate novel PAR2 antagonists. Compound 562 (C562), 2‐aminothiazol‐4yl‐SKGRS‐PEG3‐Hdc blocks PAR2 Ca2+ signaling elicited via peptidomimetics (2‐at‐LIGRL‐NH2) or via asthma associated protease activation (Alternaria alternata filtrates) in cultured human bronchial epithelial cells (16HBE14o‐). This compound was a biased‐signaling antagonist in that it had no effect on mitogen activated protein kinase (MAPK) signaling, the other major signaling pathway activated via PAR2. A shortened version of C562, 2‐at‐SKGR‐PEG3‐Hdc (C595), maintained antagonistic activity against peptidomimetic activation in an in vitro physiological signaling assay (xCELLigence). C595 is closely related to the previously described potent and specific PAR2 agonist, 2‐at‐LIGR‐PEG3‐Hdc. Thus, we screened a series of potential PAR2 ligands with a heterocycle serine substitute followed by four amino acids (XXGR) and the PEG3‐Hdc lipid tether. We describe several potent agonists, and one partial agonist (C608, 2‐at‐TIGR‐PEG3‐Hdc) that also acts as a potent, specific and biased signaling antagonist of PAR2. When used in nanomolar concentrations, C608 blocked PAR2‐dependent Ca2+ signaling via protease or peptidomimetics without effects on MAPK signaling. C562, C595 and C608 are novel pharmacological tools that can be used to evaluate the physiological consequences of PAR2 full and biased ligand signaling.Support or Funding InformationThis work was primarily funded by a multi‐PI grant (NS 073664 to SB, JV and TJP) from the National Institutes of Health. Additional support for this work was from the following grants: National Institute of Health training grants (T32 HL 007249 for ANF; T32 ES 007091 for CLS), National Institutes of Health R01NS 065926 (TJP) and R01AI083403 (SB; Michael O. Daines, PI), State of Arizona Technology and Research Initiative Fund Awarded through Bio5 (JV).