title: Development of highly potent protease-activated receptor 2 agonists via synthetic lipid tethering.
abstract: Protease-activated receptor-2 (PAR₂) is a G-protein coupled receptor (GPCR) associated with a variety of pathologies. However, the therapeutic potential of PAR₂ is limited by a lack of potent and specific ligands. Following proteolytic cleavage, PAR₂ 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 PAR₂ agonist (2-aminothiazol-4-yl-LIGRL-NH₂) bound to a palmitoyl group (Pam) via polyethylene glycol (PEG) linkers. In a high-throughput physiological assay, these STL agonists displayed EC₅₀ 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 PAR₂: EC₅₀ for Ca(2+) response as low as 3.95 nM; EC₅₀ for MAPK response as low as 9.49 nM. Moreover, STLs demonstrated significant improvement in potency in vivo, evoking mechanical allodynia with an EC₅₀ of 14.4 pmol. STLs failed to elicit responses in PAR2(-/-) cells at agonist concentrations of >300-fold their EC₅₀ values. Our results demonstrate that the STL approach is a powerful tool for increasing ligand potency at PAR₂ and represent opportunities for drug development at other protease activated receptors and across GPCRs.
authors.affiliation: Department of Physiology, Arizona Health Sciences Center, Tucson, AZ 85724-5030, USA.
authors.forename: Andrea N
authors.initials: AN
authors.lastname: Flynn
authors.forename: Justin
authors.initials: J
authors.lastname: Hoffman
authors.forename: Dipti V
authors.initials: DV
authors.lastname: Tillu
authors.forename: Cara L
authors.initials: CL
authors.lastname: Sherwood
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Zhang
authors.forename: Renata
authors.initials: R
authors.lastname: Patek
authors.forename: Marina N K
authors.initials: MN
authors.lastname: Asiedu
authors.forename: Josef
authors.initials: J
authors.lastname: Vagner
authors.forename: Theodore J
authors.initials: TJ
authors.lastname: Price
authors.forename: Scott
authors.initials: S
authors.lastname: Boitano
mesh_terms: D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects*; D002460:Cell Line / Q000187:drug effects; D006801:Humans; D006930:Hyperalgesia / Q000188:drug therapy; D008024:Ligands; D050356:Lipid Metabolism / Q000187:drug effects*; D009952:Ornithine / Q000031:analogs & derivatives / Q000494:pharmacology; D010168:Palmitates / Q000494:pharmacology*; D057786:Peptidomimetics / Q000494:pharmacology*; D044464:Receptor, PAR-2 / Q000819:agonists*; D013329:Structure-Activity Relationship
chemical_list: C000595466:2at-LIGRLO(PEG3-Pam)-NH2; D008024:Ligands; D010168:Palmitates; D057786:Peptidomimetics; D044464:Receptor, PAR-2; D009952:Ornithine; D002118:Calcium