The novel PAR2 ligand C391 blocks multiple PAR2 signalling pathways in vitro and in vivo

Scott A Boitano, Justin Hoffman, Andrea N. Flynn, Marina N. Asiedu, Dipti V. Tillu, Zhenyu Zhang, Cara L. Sherwood, Candy M. Rivas, Kathryn A. Defea, Josef Vagner, Theodore J. Price

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Background and Purpose Proteinase-activated receptor-2 (PAR2) is a GPCR linked to diverse pathologies, including acute and chronic pain. PAR2 is one of the four PARs that are activated by proteolytic cleavage of the extracellular amino terminus, resulting in an exposed, tethered peptide agonist. Several peptide and peptidomimetic agonists, with high potency and efficacy, have been developed to probe the functions of PAR2, in vitro and in vivo. However, few similarly potent and effective antagonists have been described. Experimental Approach We modified the peptidomimetic PAR2 agonist, 2-furoyl-LIGRLO-NH<inf>2</inf>, to create a novel PAR2 peptidomimetic ligand, C391. C391 was evaluated for PAR2 agonist/antagonist activity to PAR2 across G<inf>q</inf> signalling pathways using the naturally expressing PAR2 cell line 16HBE14o-. For antagonist studies, a highly potent and specific peptidomimetic agonist (2-aminothiazo-4-yl-LIGRL-NH<inf>2</inf>) and proteinase agonist (trypsin) were used to activate PAR2. C391 was also evaluated in vivo for reduction of thermal hyperalgesia, mediated by mast cell degranulation, in mice. Key Results C391 is a potent and specific peptidomimetic antagonist, blocking multiple signalling pathways (G<inf>q</inf>-dependent Ca<sup>2+</sup>, MAPK) induced following peptidomimetic or proteinase activation of human PAR2. In a PAR2-dependent behavioural assay in mice, C391 dose-dependently (75 μg maximum effect) blocked the thermal hyperalgesia, mediated by mast cell degranulation. Conclusions and Implications C391 is the first low MW antagonist to block both PAR2 Ca<sup>2+</sup> and MAPK signalling pathways activated by peptidomimetics and/or proteinase activation. C391 represents a new molecular structure for PAR2 antagonism and can serve as a basis for further development for this important therapeutic target.

Original languageEnglish (US)
Pages (from-to)4535-4545
Number of pages11
JournalBritish Journal of Pharmacology
Volume172
Issue number18
DOIs
StatePublished - Sep 1 2015

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PAR-2 Receptor
Peptidomimetics
Ligands
Cell Degranulation
Peptide Hydrolases
Hyperalgesia
Mast Cells
In Vitro Techniques
Peptides
Acute Pain
Molecular Structure
Chronic Pain
Trypsin

ASJC Scopus subject areas

  • Pharmacology

Cite this

Boitano, S. A., Hoffman, J., Flynn, A. N., Asiedu, M. N., Tillu, D. V., Zhang, Z., ... Price, T. J. (2015). The novel PAR2 ligand C391 blocks multiple PAR2 signalling pathways in vitro and in vivo. British Journal of Pharmacology, 172(18), 4535-4545. https://doi.org/10.1111/bph.13238

The novel PAR2 ligand C391 blocks multiple PAR2 signalling pathways in vitro and in vivo. / Boitano, Scott A; Hoffman, Justin; Flynn, Andrea N.; Asiedu, Marina N.; Tillu, Dipti V.; Zhang, Zhenyu; Sherwood, Cara L.; Rivas, Candy M.; Defea, Kathryn A.; Vagner, Josef; Price, Theodore J.

In: British Journal of Pharmacology, Vol. 172, No. 18, 01.09.2015, p. 4535-4545.

Research output: Contribution to journalArticle

Boitano, SA, Hoffman, J, Flynn, AN, Asiedu, MN, Tillu, DV, Zhang, Z, Sherwood, CL, Rivas, CM, Defea, KA, Vagner, J & Price, TJ 2015, 'The novel PAR2 ligand C391 blocks multiple PAR2 signalling pathways in vitro and in vivo', British Journal of Pharmacology, vol. 172, no. 18, pp. 4535-4545. https://doi.org/10.1111/bph.13238
Boitano, Scott A ; Hoffman, Justin ; Flynn, Andrea N. ; Asiedu, Marina N. ; Tillu, Dipti V. ; Zhang, Zhenyu ; Sherwood, Cara L. ; Rivas, Candy M. ; Defea, Kathryn A. ; Vagner, Josef ; Price, Theodore J. / The novel PAR2 ligand C391 blocks multiple PAR2 signalling pathways in vitro and in vivo. In: British Journal of Pharmacology. 2015 ; Vol. 172, No. 18. pp. 4535-4545.
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abstract = "Background and Purpose Proteinase-activated receptor-2 (PAR2) is a GPCR linked to diverse pathologies, including acute and chronic pain. PAR2 is one of the four PARs that are activated by proteolytic cleavage of the extracellular amino terminus, resulting in an exposed, tethered peptide agonist. Several peptide and peptidomimetic agonists, with high potency and efficacy, have been developed to probe the functions of PAR2, in vitro and in vivo. However, few similarly potent and effective antagonists have been described. Experimental Approach We modified the peptidomimetic PAR2 agonist, 2-furoyl-LIGRLO-NH2, to create a novel PAR2 peptidomimetic ligand, C391. C391 was evaluated for PAR2 agonist/antagonist activity to PAR2 across Gq signalling pathways using the naturally expressing PAR2 cell line 16HBE14o-. For antagonist studies, a highly potent and specific peptidomimetic agonist (2-aminothiazo-4-yl-LIGRL-NH2) and proteinase agonist (trypsin) were used to activate PAR2. C391 was also evaluated in vivo for reduction of thermal hyperalgesia, mediated by mast cell degranulation, in mice. Key Results C391 is a potent and specific peptidomimetic antagonist, blocking multiple signalling pathways (Gq-dependent Ca2+, MAPK) induced following peptidomimetic or proteinase activation of human PAR2. In a PAR2-dependent behavioural assay in mice, C391 dose-dependently (75 μg maximum effect) blocked the thermal hyperalgesia, mediated by mast cell degranulation. Conclusions and Implications C391 is the first low MW antagonist to block both PAR2 Ca2+ and MAPK signalling pathways activated by peptidomimetics and/or proteinase activation. C391 represents a new molecular structure for PAR2 antagonism and can serve as a basis for further development for this important therapeutic target.",
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AU - Tillu, Dipti V.

AU - Zhang, Zhenyu

AU - Sherwood, Cara L.

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N2 - Background and Purpose Proteinase-activated receptor-2 (PAR2) is a GPCR linked to diverse pathologies, including acute and chronic pain. PAR2 is one of the four PARs that are activated by proteolytic cleavage of the extracellular amino terminus, resulting in an exposed, tethered peptide agonist. Several peptide and peptidomimetic agonists, with high potency and efficacy, have been developed to probe the functions of PAR2, in vitro and in vivo. However, few similarly potent and effective antagonists have been described. Experimental Approach We modified the peptidomimetic PAR2 agonist, 2-furoyl-LIGRLO-NH2, to create a novel PAR2 peptidomimetic ligand, C391. C391 was evaluated for PAR2 agonist/antagonist activity to PAR2 across Gq signalling pathways using the naturally expressing PAR2 cell line 16HBE14o-. For antagonist studies, a highly potent and specific peptidomimetic agonist (2-aminothiazo-4-yl-LIGRL-NH2) and proteinase agonist (trypsin) were used to activate PAR2. C391 was also evaluated in vivo for reduction of thermal hyperalgesia, mediated by mast cell degranulation, in mice. Key Results C391 is a potent and specific peptidomimetic antagonist, blocking multiple signalling pathways (Gq-dependent Ca2+, MAPK) induced following peptidomimetic or proteinase activation of human PAR2. In a PAR2-dependent behavioural assay in mice, C391 dose-dependently (75 μg maximum effect) blocked the thermal hyperalgesia, mediated by mast cell degranulation. Conclusions and Implications C391 is the first low MW antagonist to block both PAR2 Ca2+ and MAPK signalling pathways activated by peptidomimetics and/or proteinase activation. C391 represents a new molecular structure for PAR2 antagonism and can serve as a basis for further development for this important therapeutic target.

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