Agonists and antagonists of protease activated receptors (PARs).
ABSTRACT 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.
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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 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. · 3.53 Impact Factor
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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; · 5.07 Impact Factor
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ABSTRACT: Abstract The use of phytochemicals either singly or in combination with other anticancer drugs comes with an advantage of less toxicity and minimal side effects. Signaling Pathways play central role in cell cycle, cell growth, metabolism, etc. Thus, the identification of phytochemicals with promising antagonistic effect on the receptor/s playing key role in single transduction may have better therapeutic application. With this background, phytochemicals were screened against Protease activated receptor 2 (PAR2). PAR2 belong to the superfamily of GPCRs and is an important target for breast cancer. Using in silico methods, this study was able to identify the phytochemicals with promising binding affinity suggesting their therapeutic potential in the treatment of breast cancer. The finding of this study acquires importance as the information on the possible agonist and antagonist of PAR2 is limited due its unique mechanism of activation.Journal of biomolecular Structure & Dynamics 11/2014; · 2.98 Impact Factor