ABSTRACT: PAR2 antagonists have potential for treating inflammatory, respiratory, gastrointestinal, neurological, and metabolic disorders, but few antagonists are known. Derivatives of GB88 (3) suggest that all four of its components bind at distinct PAR2 sites with the isoxazole, cyclohexylalanine, and isoleucine determining affinity and selectivity, while the C-terminal substituent determines agonist/antagonist function. Here we report structurally similar PAR2 ligands with opposing functions (agonist vs antagonist) upon binding to PAR2. A biased ligand AY117 (65) was found to antagonize calcium release induced by PAR2 agonists trypsin and hexapeptide 2f-LIGRLO-NH2 (IC50 2.2 and 0.7 ?M, HT29 cells), but it was a selective PAR2 agonist in inhibiting cAMP stimulation and activating ERK1/2 phosphorylation. It showed anti-inflammatory properties both in vitro and in vivo.
Project description: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.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.C391 is a potent and specific peptidomimetic antagonist, blocking multiple signalling pathways (Gq -dependent Ca(2+) , MAPK) induced following peptidomimetic or proteinase activation of human PAR2. In a PAR2-dependent behavioural assay in mice, C391 dose-dependently (75??g maximum effect) blocked the thermal hyperalgesia, mediated by mast cell degranulation.C391 is the first low MW antagonist to block both PAR2 Ca(2+) 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.
Project description:Human embryonic kidney cell (HEK293) were treated with PAR2 peptide agonist 2f-LIGRLO-NH2 (1.5h, 3h, 6h and 12h) or trypsin (6h). Comparison of genes similarly regulated by both treatments allowed better characterization of PAR2 induced response as both agonists had been reported as non-specific for PAR2. Six conditioned experiments with 3 replicates each. Individual grown and harvested.
Project description:Human embryonic kidney cell (HEK293) were treated with PAR2 peptide agonist 2f-LIGRLO-NH2 (1.5h, 3h, 6h and 12h) or trypsin (6h). Comparison of genes similarly regulated by both treatments allowed better characterization of PAR2 induced response as both agonists had been reported as non-specific for PAR2. Overall design: Six conditioned experiments with 3 replicates each. Individual grown and harvested.
Project description:Protease-Activated Receptor-2 (PAR2) has been implicated through genetic knockout mice with cytokine regulation and arthritis development. Many studies have associated PAR2 with inflammatory conditions (arthritis, airways inflammation, IBD) and key events in tumor progression (angiogenesis, metastasis), but they have relied heavily on the use of single agonists to identify physiological roles for PAR2. However such probes are now known not to be highly selective for PAR2, and thus precisely what PAR2 does and what mechanisms of downstream regulation are truly affected remain obscure. Effects of PAR2 activation on gene expression in Human Embryonic Kidney cells (HEK293), a commonly studied cell line in PAR2 research, were investigated here by comparing 19,000 human genes for intersecting up- or down-regulation by both trypsin (an endogenous protease that activates PAR2) and a PAR2 activating hexapeptide (2f-LIGRLO-NH(2)). Among 2,500 human genes regulated similarly by both agonists, there were clear associations between PAR2 activation and cellular metabolism (1,000 genes), the cell cycle, the MAPK pathway, HDAC and sirtuin enzymes, inflammatory cytokines, and anti-complement function. PAR-2 activation up-regulated four genes more than 5 fold (DUSP6, WWOX, AREG, SERPINB2) and down-regulated another six genes more than 3 fold (TXNIP, RARG, ITGB4, CTSD, MSC and TM4SF15). Both PAR2 and PAR1 activation resulted in up-regulated expression of several genes (CD44, FOSL1, TNFRSF12A, RAB3A, COPEB, CORO1C, THBS1, SDC4) known to be important in cancer. This is the first widespread profiling of specific activation of PAR2 and provides a valuable platform for better understanding key mechanistic roles of PAR2 in human physiology. Results clearly support the development of both antagonists and agonists of human PAR2 as potential disease modifying therapeutic agents.
Project description:<h4>Background and purpose</h4>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.<h4>Experimental approach</h4>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.<h4>Key results</h4>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.<h4>Conclusions and implications</h4>The novel PAR2 agonist and antagonist modulate intracellular Ca(2+) and rat paw oedema, providing novel molecular tools for examining PAR2-mediated diseases.
Project description:We hypothesized that proteinase-activated receptor-2 (PAR2)-mediated vasorelaxation in murine aorta tissue can be due in part to the release of adipocyte-derived relaxing factors (ADRFs).Aortic rings from obese TallyHo and C57Bl6 intact or PAR2-null mice either without or with perivascular adipose tissue (PVAT) were contracted with phenylephrine and relaxation responses to PAR2-selective activating peptides (PAR2-APs: SLIGRL-NH(2) and 2-furoyl-LIGRLO-NH(2) ), trypsin and to PAR2-inactive peptides (LRGILS-NH(2) , 2-furoyl-OLRGIL-NH(2) and LSIGRL-NH(2) ) were measured. Relaxation was monitored in the absence or presence of inhibitors that either alone or in combination were previously shown to inhibit ADRF-mediated responses: L-NAME (NOS), indomethacin (COX), ODQ (guanylate cyclase), catalase (H(2) O(2) ) and the K(+) channel-targeted reagents, apamin, charybdotoxin, 4-aminopyridine and glibenclamide.Endothelium-intact PVAT-free preparations did not respond to PAR2-inactive peptides (LRGILS-NH(2) , LSIGRL-NH(2) , 2-furoyl-OLRGIL-NH(2) ), whereas active PAR2-APs (SLIGRL-NH(2) ; 2-furoyl-LIGRLO-NH(2) ) caused an L-NAME-inhibited relaxation. However, in PVAT-containing preparations treated with L-NAME/ODQ/indomethacin together, both PAR2-APs and trypsin caused relaxant responses in PAR2-intact, but not PAR2-null-derived tissues. The PAR2-induced PVAT-dependent relaxation (SLIGRL-NH(2) ) persisted in the presence of apamin plus charybdotoxin, 4-aminopyridine and glibenclamide, but was blocked by catalase, implicating a role for H(2) O(2) . Surprisingly, the PAR2-inactive peptides, LRGILS-NH(2) and 2-furoyl-OLRGIL-NH(2) (but not LSIGRL-NH(2) ), caused relaxation in PVAT-containing preparations from both PAR2-null and PAR2-intact (C57Bl, TallyHo) mice. The LRGILS-NH(2) -induced relaxation was distinct from the PAR2 response, being blocked by 4-aminopyridine, but not catalase.Distinct ADRFs that may modulate vascular tone in pathophysiological settings can be released from murine PVAT by both PAR2-dependent and PAR2-independent mechanisms.
Project description:A novel class of bivalent ligands targeting putative protease-activated receptor (PAR) heteromers has been prepared based upon reported antagonists for the subtypes PAR1 and PAR2. Modified versions of the PAR1 antagonist RWJ-58259 containing alkyne adapters were connected via cycloaddition reactions to azide-capped polyethylene glycol (PEG) spacers attached to imidazopyridazine-based PAR2 antagonists. Initial studies of the PAR1-PAR2 antagonists indicated that they inhibited G alpha q-mediated calcium mobilization in endothelial and cancer cells driven by both PAR1 and PAR2 agonists. Compounds of this novel class hold promise for the prevention of restenosis, cancer cell metastasis, and other proliferative disorders.
Project description:Protease-activated receptor-2 (PAR2) has been implicated in multiple pathophysiologies but drug discovery is challenging due to low small molecule tractability and a complex activation mechanism. Here we report the pharmacological profiling of a potent new agonist, suggested by molecular modelling to bind in the putative orthosteric site, and two novel PAR2 antagonists with distinctly different mechanisms of inhibition. We identify coupling between different PAR2 binding sites. One antagonist is a competitive inhibitor that binds to the orthosteric site, while a second antagonist is a negative allosteric modulator that binds at a remote site. The allosteric modulator shows probe dependence, more effectively inhibiting peptide than protease activation of PAR2 signalling. Importantly, both antagonists are active in vivo, inhibiting PAR2 agonist-induced acute paw inflammation in rats and preventing activation of mast cells and neutrophils. These results highlight two distinct mechanisms of inhibition that potentially could be targeted for future development of drugs that modulate PAR2.
Project description:BACKGROUND:Sexual dimorphism in biological responses is a critical knowledge for therapeutic proposals. However, gender differences in intestinal stem cell physiology have been poorly studied. Given the important role of the protease-activated receptor PAR2 in the control of colon epithelial primitive cells and cell cycle genes, we have performed a sex-based comparison of its expression and of the effects of PAR2 activation or knockout on cell proliferation and survival functions. METHODS:Epithelial primitive cells isolated from colons from male and female mice were cultured as colonoids, and their number and size were measured. PAR2 activation was triggered by the addition of SLIGRL agonist peptide in the culture medium. PAR2-deficient mice were used to study the impact of PAR2 expression on colon epithelial cell culture and gene expression. RESULTS:Colonoids from female mice were more abundant and larger compared to males, and these differences were further increased after PAR2 activation by specific PAR2 agonist peptide. The proliferation of male epithelial cells was lower compared to females but was specifically increased in PAR2 knockout male cells. PAR2 expression was higher in male colon cells compared to females and controlled the gene expression and activation of key negative signals of the primitive cell proliferation. This PAR2-dependent brake on the proliferation of male colon primitive cells was correlated with stress resistance. CONCLUSIONS:Altogether, these data demonstrate that there is a sexual dimorphism in the PAR2-dependent regulation of primitive cells of the colon crypt.
Project description:Novel peptidomimetic pharmacophores to PAR(2) were designed based on the known activating peptide SLIGRL-NH(2). A set of 15 analogues was evaluated with a model cell line (16HBE14o-) that highly expresses PAR(2). Cells exposed to the PAR(2) activating peptide with N-terminal 2-furoyl modification (2-furoyl-LIGRLO-NH(2)) initiated increases in intracellular calcium concentration ([Ca(2+)](i) EC(50) = 0.84 ?M) and in vitro physiological responses as measured by the xCELLigence real time cell analyzer (RTCA EC(50) = 138 nM). We discovered two selective PAR(2) agonists with comparable potency: compound 1 (2-aminothiazol-4-yl; Ca(2+) EC(50) = 1.77 ?M, RTCA EC(50) = 142 nM) and compound 2 (6-aminonicotinyl; Ca(2+) EC(50) = 2.60 ?M, RTCA EC(50) = 311 nM). Unlike the previously described agonist, these novel agonists are devoid of the metabolically unstable 2-furoyl modification and thus provide potential advantages for PAR(2) peptide design for in vitro and in vivo studies. The novel compounds described herein also serve as a starting point for structure-activity relationship (SAR) design and are, for the first time, evaluated via a unique high throughput in vitro physiological assay. Together these will lead to discovery of more potent agonists and antagonists of PAR(2).