Protease activated receptors (PARs) are a category of G-protein coupled receptors (GPCRs) implicated in the progression of a wide range of diseases, including thrombosis, inflammatory disorders, and proliferative diseases. Signal transduction via PARs proceeds via an unusual activation mechanism. Instead of being activated through direct interaction with an extracellular signal like most GPCRs, they are self-activated following cleavage of their extracellular N-terminus by serine proteases to generate a new receptor N-terminus that acts as an intramolecular ligand by folding back onto itself and triggering receptor activation. Short synthetic peptides corresponding to this newly exposed N-terminal tethered ligand can activate three of the four known PARs in the absence of proteases, and such PAR activating peptides (PAR-APs) have served as templates for agonist/antagonist development. In fact much of the evidence for involvement of PARs in diseases has relied upon use of PAR-APs, often of low potency and uncertain selectivity. This review summarizes current structures of PAR agonists and antagonists, the need for more selective and more potent PAR ligands that activate or antagonize this intriguing class of receptors, and outlines the background relevant to PAR activation, assay methods, and physiological properties anticipated for PAR ligands.
"Small peptides or peptidomimetics that mimic the ligand binding properties of the tethered ligand exposed by proteolysis of the N-terminus of the receptor have been used to directly activate PARs , , , . Activating peptides (e.g., SLIGKV-NH2 and SLIGRL-NH2) and peptidomimetics (e.g., 2-furoyl-LIGRLO-NH2
 and 2at-LIGRL-NH2
) have provided useful tools for establishment of structure-activity relationships (SAR) and rational drug design because they limit off-target effects that are often a complication of natural protease activation. "
[Show abstract][Hide abstract] 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 PAR2 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-PEG3-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 PAR2 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.
PLoS ONE 06/2014; 9(6):e99140. DOI:10.1371/journal.pone.0099140 · 3.23 Impact Factor
"J Y Suen et al. et al., 2006; Suen et al., 2010; Yau et al., 2013), so the prospect of using rationally designed PAR2-binding ligands to inhibit only one PAR2-mediated signalling pathway associated with a particular diseased state is an exciting opportunity that may also be possible for other GPCRs. There have been seemingly paradoxical reports of PAR2 being both pro-and antiinflammatory (see Barry et al., 2006; Yau et al., 2013), depending upon the context of cell or animal experiments conducted or disease models studied. The new findings here provide a possible explanation for such opposing actions, suggesting that it may be possible for some ligands to activate certain PAR2-mediated pathways while blocking others, enabling PAR2-mediated disease intervention without affecting beneficial or protective PAR2-mediated physiological responses. "
[Show abstract][Hide abstract] ABSTRACT: Proteinase activated receptor 2 (PAR2 ) is a G protein-coupled receptor associated with inflammation, metabolism and disease. Clues to understanding how to block PAR2 signaling associated with disease without inhibiting PAR2 activation in normal physiology can potentially arise from studies of biased signaling.
PAR2 ligand GB88 was profiled for PAR2 agonist and antagonist properties by multiple functional assays associated with intracellular G-protein coupled signaling in vitro in three cell types and with PAR2 -induced rat paw oedema in vivo.
In HT29 cells, GB88 was a PAR2 antagonist in PAR2-induced Ca(2+) mobilization and PKC phosphorylation, but a PAR2 agonist in attenuating forskolin-induced cAMP accumulation, increasing ERK1/2 phosphorylation, RhoA activation, MYPT phosphorylation and actin filament rearrangement. In CHO-hPAR2 cells, GB88 inhibited Ca(2+) release, but activated Gi/o and increased ERK1/2 phosphorylation. In human kidney tubule cells, it inhibited cytokine secretion (IL6, IL8, GMCSF, TNFα) mediated by PAR2 . A rat paw oedema induced by PAR2 agonists was also inhibited by orally administered GB88 and compared with effects of locally administered inhibitors of G protein-coupled pathways.
GB88 is a unique biased antagonist of PAR2 that selectively inhibits PAR2 /Gq/11 /Ca(2+) /PKC signaling, leading to anti-inflammatory activity in vivo, while being an agonist in activating three other PAR2 activated pathways (cAMP, ERK, Rho) in human cells. These findings highlight opportunities to design drugs to block specific PAR2 -linked signaling pathways in disease, without blocking beneficial PAR2 signaling in normal physiology, and to dissect PAR2-associated mechanisms of disease in vivo.
British Journal of Pharmacology 05/2014; 171(17). DOI:10.1111/bph.12757 · 4.84 Impact Factor
"GPCRs are the most prevalent signalling proteins on the cell surface, mediating a wide variety of physiological responses that can be targeted by drugs (Blakeney et al., 2007). PARs are a unique class of GPCRs that are activated through irreversible modification by extracellular proteases (Bohm et al., 1996; MacFarlane et al., 2001; Coughlin and Camerer, 2003; Steinhoff et al., 2005; Barry et al., 2006). PAR2 is specifically activated by a range of serine proteases, such as trypsin, but not by the PAR1 agonist thrombin. "
[Show abstract][Hide abstract] ABSTRACT: Many cells express proteinase activated receptor 2 (PAR2) on their plasma membrane. PAR2 is activated by proteolytic enzymes, such as trypsin and tryptase that cleave the receptor N-terminus, inititating signalling to intracellular G proteins. Studies on PAR2 have relied heavily upon activating effects of proteases and peptide agonists that lack stability and bioavailability in vivo.
A novel small molecule agonist GB110 and an antagonist GB88 were characterized in vitro against trypsin, peptide agonists, PAR2 antibody, PAR1 agonists and flow cytometry,in seven cell lines using intracellular Ca(2+) mobilization and examined in vivo against PAR2- and PAR1-induced rat paw oedema.
GB110 is a potent non-peptidic agonist activating PAR2-mediated Ca(2+) release in HT29 cells (EC(50) ∼200 nM) and six other human cell lines, inducing PAR2 internalization. GB88 is a unique PAR2 antagonist, inhibiting PAR2 activated Ca(2+) release (IC(50) ∼2 µM) induced by native (trypsin) or synthetic peptide and non-peptide agonists. GB88 was a competitive and surmountable antagonist of agonist 2f-LIGRLO-NH(2), a competitive but insurmountable antagonist of agonist GB110, and a non-competitive insurmountable antagonist of trypsin. GB88 was orally active and anti-inflammatory in vivo, inhibiting acute rat paw oedema elicited by agonist GB110 and proteolytic or peptide agonists of PAR2 but not by corresponding agonists of PAR1 or PAR4.
The novel PAR2 agonist and antagonist modulate intracellular Ca(2+) and rat paw oedema, providing novel molecular tools for examining PAR2-mediated diseases.
British Journal of Pharmacology 08/2011; 165(5):1413-23. DOI:10.1111/j.1476-5381.2011.01610.x · 4.84 Impact Factor
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