Development and evaluation of small peptidomimetic ligands to Protease-Activated Receptor-2 (PAR2) through the use of lipid tethering

Scott A Boitano, Justin Hoffman, Dipti V. Tillu, Marina N. Asiedu, Zhenyu Zhang, Cara L. Sherwood, Yan Wang, Xinzhong Dong, Theodore J. Price, Josef Vagner

Research output: Contribution to journalArticle

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Abstract

Protease-activated receptor-2 (PAR2) is a G-Protein Coupled Receptor (GPCR) activated by proteolytic cleavage to expose an attached, tethered ligand (SLIGRL). We evaluated the ability for lipid-tethered- peptidomimetics to activate PAR2 with in vitro physiological and Ca2+ signaling assays to determine minimal components necessary for potent, specific and full PAR2 activation. A known PAR2 activating compound containing a hexadecyl (Hdc) lipid via three polyethylene glycol (PEG) linkers (2at-LIGRL-PEG3-Hdc) provided a potent agonist starting point (physiological EC50 = 1.4 nM; 95% CI: 1.2-2.3 nM). In a set of truncated analogs, 2at-LIGR-PEG3-Hdc retained potency (EC50 = 2.1 nM; 1.3-3.4 nM) with improved selectivity for PAR 2 over Mas1 related G-protein coupled receptor type C11, a GPCR that can be activated by the PAR2 peptide agonist, SLIGRL-NH2. 2at-LIG-PEG3-Hdc was the smallest full PAR2 agonist, albeit with a reduced EC50 (46 nM; 20-100 nM). 2at-LI-PEG 3-Hdc retained specific activity for PAR2 with reduced EC50 (310 nM; 260-360 nM) but displayed partial PAR2 activation in both physiological and Ca2+ signaling assays. Further truncation (2at-L-PEG3-Hdc and 2at-PEG3-Hdc) eliminated in vitro activity. When used in vivo, full and partial PAR2 in vitro agonists evoked mechanical hypersensitivity at a 15 pmole dose while 2at-L-PEG3-Hdc lacked efficacy. Minimum peptidomimetic PAR 2 agonists were developed with known heterocycle substitutes for Ser1 (isoxazole or aminothiazoyl) and cyclohexylalanine (Cha) as a substitute for Leu2. Both heterocycle-tetrapeptide and heterocycle-dipeptides displayed PAR2 specificity, however, only the heterocycle-tetrapeptides displayed full PAR2 agonism. Using the lipid-tethered-peptidomimetic approach we have developed novel structure activity relationships for PAR2 that allows for selective probing of PAR2 function across a broad range of physiological systems.

Original languageEnglish (US)
Article numbere99140
JournalPLoS One
Volume9
Issue number6
DOIs
StatePublished - Jun 13 2014

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PAR-2 Receptor
Peptidomimetics
proteinases
Ligands
Lipids
receptors
lipids
agonists
G-Protein-Coupled Receptors
seryl-leucyl-isoleucyl-glycyl-arginyl-leucine
polyethylene glycol
ligands
Assays
Chemical activation
Isoxazoles
calcium
dipeptides
Dipeptides
assays
structure-activity relationships

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Development and evaluation of small peptidomimetic ligands to Protease-Activated Receptor-2 (PAR2) through the use of lipid tethering. / Boitano, Scott A; Hoffman, Justin; Tillu, Dipti V.; Asiedu, Marina N.; Zhang, Zhenyu; Sherwood, Cara L.; Wang, Yan; Dong, Xinzhong; Price, Theodore J.; Vagner, Josef.

In: PLoS One, Vol. 9, No. 6, e99140, 13.06.2014.

Research output: Contribution to journalArticle

Boitano, Scott A ; Hoffman, Justin ; Tillu, Dipti V. ; Asiedu, Marina N. ; Zhang, Zhenyu ; Sherwood, Cara L. ; Wang, Yan ; Dong, Xinzhong ; Price, Theodore J. ; Vagner, Josef. / Development and evaluation of small peptidomimetic ligands to Protease-Activated Receptor-2 (PAR2) through the use of lipid tethering. In: PLoS One. 2014 ; Vol. 9, No. 6.
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