Protease-activated Receptor-2 Antagonists Research Articles

Discovery of Protease-activated receptor 2 antagonists derived from phenylalanine for the treatment of breast cancer

Protease-activated receptor 2 (PAR2) has garnered attention as a potential therapeutic target in breast cancer. PAR2 is implicated in the activation of extracellular signal-regulated kinase 1/2 (ERK 1/2) via G protein and beta-arrestin pathways, contributing to the proliferation and metastasis of breast cancer cells. Despite the recognized role of PAR2 in breast cancer progression, clinically effective PAR2 antagonists remain elusive. To address this unmet clinical need, we synthesized and evaluated a series of novel compounds that target the orthosteric site of PAR2. Using in silico docking simulations, we identified compound 9a, an optimized derivative of compound 1a ((S)-N-(1-(benzylamino)-1-oxo-3-phenylpropan-2-yl)benzamide), which exhibited enhanced PAR2 antagonistic activity. Subsequent molecular dynamics simulations comparing 9a with the partial agonist 9d revealed that variations in ligand-induced conformational changes and interactions dictated whether the compound acted as an antagonist or agonist of PAR2. The results of this study suggest that further development of 9a could contribute to the advancement of PAR2 antagonists as potential therapeutic agents for breast cancer.

Protease-activated receptor 2: a promising therapeutic target for women's cancers.

Cancers affecting women, such as breast, uterine, ovarian, endometrial and cervical cancers, have become increasingly prevalent. The growing incidence and death rates associated with these cancers warrant the development of innovative and alternative approaches to current treatments. This article investigates the association of women's cancers with a molecular target known as protease-activated receptor 2 (PAR2), a G-protein coupled receptor that is expressed on the surface of cancer cells. Expression levels of the PAR2 gene were curated from publicly available databases and were found to be significantly overexpressed in tissues from patients with breast, uterine, ovarian, endometrial or cervical cancer compared to normal tissues. PAR2 overexpression has been previously linked to tumor progression and, in some cases, tumor growth. Activation of PAR2 by either endogenous proteases or synthetic agonists triggers certain downstream intracellular signaling pathways that have been associated with tumor progression, cell migration and invasion, angiogenesis and apoptosis of cancer cells. While recent advances have led to the identification of several PAR2 antagonists, none has yet been developed for human use. Additionally, PAR2 inhibition has been shown also to increase the efficacy of chemotherapeutic drugs, allowing them to be potentially used at less toxic doses in combination therapies for cancer. The present work briefly summarizes the current status of PAR2 as a potential therapeutic target for treating women's cancers. Significance Statement This article highlights potential roles for PAR2 in cancers affecting women. Overexpression of the PAR2 gene in women's cancers is associated with various oncogenic processes such as tumor progression, cell migration and invasion, ultimately contributing to poorer patient prognoses. Given the increasing incidence of women's cancers, there is an urgent need to develop novel therapeutic drugs and PAR2 represents a promising target for developing new treatments.

Abstract 4961: Novel biased PAR2 inhibitors with best-in-class properties reduce resistance to both chemotherapy and immunotherapy in oncology models

Abstract A large-scale meta-analysis has recently identified Protease-activated receptor 2 (PAR2) gene expression to be significantly associated with resistance to immune checkpoint blockade (ICB) in cancer patients and preclinical models. PAR2 and its ligands (proteases) are indeed upregulated in different cancer types and are expressed by various cells in the tumor microenvironment. In cancer cells, the PAR2 receptor controls cell migration, proliferation, survival, and expression of inflammatory cytokines. In immune cells, it influences the infiltration and phenotype of macrophages and T cells. Therefore, PAR2 represents a promising therapeutic target in oncology and immuno-oncology. A novel series of potent and selective PAR2 inhibitors has been developed at Domain Therapeutics. In vitro experiments demonstrated unique properties of our PAR2 small molecule antagonists when compared to those of competitors. The antagonists inhibit pathogenic signaling pathways (i.e. Gz, G13, Gq, G14 and G15 protein activation as well as intracellular calcium) but not βarrestin2 recruitment, potentially reducing the risks of drug resistance. Furthermore, they maintain high potency and insurmountability in conditions that mimic the tumor microenvironment (high concentration of proteases and acidic pH). Finally, their pharmacokinetic properties are compatible with a once-a-day oral administration and demonstrate no signs of in vivo toxicity except at high doses (>500 mg/kg). Proof-of-concept experiments showed that PAR2 antagonists prevented PAR2-mediated resistance to Gefitinib in vitro and increased the potency of anti-PD1 therapy in vivo in pre-clinical syngeneic mouse models. Immunohistochemistry analyses from cancer patient biopsies confirmed that high expression levels of PAR2 in cancer and stromal cells within the tumor microenvironment significantly associates with the patient overall survival. In conclusion, new potent and selective negative allosteric modulators of PAR2 have been developed, they demonstrate strong potency by alleviating the resistance to both chemo- and immunotherapy in cancer models. These findings confirm the high value of PAR2 as a therapeutic target and demonstrates the relevance of small molecule inhibitors targeting this receptor to treat cancer. Citation Format: Thibaut Brugat, Francesco Bergami, Baptiste Rugeri, Aurélie Janvier, Edith Steinberg, Luc Baron, Mandy Recolet, Xavier Wirth, Meriem Semache, Antoine Mousson, Camille Dietsch, Quentin Ruet, Orphée Blanchard, Célia Jacoberger-Foissac, Isabelle Cousineau, Maleck Kadiri, Anne-Laure Blayo, Christel Franchet, Stanislas Mayer, John Stagg, Nathalie Lenne, Stephan Schann. Novel biased PAR2 inhibitors with best-in-class properties reduce resistance to both chemotherapy and immunotherapy in oncology models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4961.

C781, a β-Arrestin Biased Antagonist at Protease-Activated Receptor-2 (PAR2), Displays in vivo Efficacy Against Protease-Induced Pain in Mice

Given the limited options and often harmful side effects of current analgesics and the suffering caused by the opioid crisis, new classes of pain therapeutics are needed. Protease-activated receptors (PARs), particularly PAR2, are implicated in a variety of pathologies, including pain. Since the discovery of the role of PAR2 in pain, development of potent and specific antagonists has been slow. In this study, we describe the in vivo characterization of a novel small molecule/peptidomimetic hybrid compound, C781, as a β-arrestin-biased PAR2 antagonist. In vivo behavioral studies were done in mice using von Frey filaments and the Mouse Grimace Scale. Pharmacokinetic studies were done to assess pharmacokinetic/pharmacodynamic relationship in vivo. We used both prevention and reversal paradigms with protease treatment to determine whether C781 could attenuate protease-evoked pain. C781 effectively prevented and reversed mechanical and spontaneous nociceptive behaviors in response to small molecule PAR2 agonists, mast cell activators, and neutrophil elastase. The ED50 of C781 (intraperitoneal dosing) for inhibition of PAR2 agonist (20.9 ng 2-AT)-evoked nociception was 6.3 mg/kg. C781 was not efficacious in the carrageenan inflammation model. Pharmacokinetic studies indicated limited long-term systemic bioavailability for C781 suggesting that optimizing pharmacokinetic properties could improve in vivo efficacy. Our work demonstrates in vivo efficacy of a biased PAR2 antagonist that selectively inhibits β-arrestin/MAPK signaling downstream of PAR2. Given the importance of this signaling pathway in PAR2-evoked nociception, C781 exemplifies a key pharmacophore for PAR2 that can be optimized for clinical development. PerspectiveOur work provides evidence that PAR2 antagonists that only block certain aspects of signaling by the receptor can be effective for blocking protease-evoked pain in mice. This is important because it creates a rationale for developing safer PAR2-targeting approaches for pain treatment.

Histone 2B Facilitates Plasminogen-Enhanced Endothelial Migration through Protease-Activated Receptor 1 (PAR1) and Protease-Activated Receptor 2 (PAR2).

Plasminogen and its multiple receptors have been implicated in the responses of many different cell types. Among these receptors, histone 2B (H2B) has been shown to play a prominent role in macrophage responses. The contribution of H2B to plasminogen-induced endothelial migration, an event relevant to wound healing and angiogenesis, is unknown. Plasminogen enhanced the migration of endothelial cells, which was inhibited by both Protease-Activated Receptor-1 (PAR1) and 2 (PAR2) antagonists. H2B was detected on viable endothelial cells of venous and arterial origin, and an antibody to H2B that blocks plasminogen binding also inhibited the plasminogen-dependent migration by these cells. The antibody blockade was as effective as PAR1 or PAR2 antagonists in inhibiting endothelial cell migration. In pull-down experiments, H2B formed a complex with both PAR1 and PAR2 but not β3 integrin, another receptor implicated in endothelial migration in the presence of plasminogen. H2B was found to be associated with clathrin adapator protein, AP2µ (clathrin AP2µ) and β-arrestin2, which are central to the internationalization/signaling machinery of the PARs. These associations with PAR1-clathrin adaptor AP2µ- and PAR2-β-arrestin2-dependent internalization/signaling pathways provide a mechanism to link plasminogen to responses such as wound healing and angiogenesis.

PAR2-Induced Tissue Factor Synthesis by Primary Cultures of Human Kidney Tubular Epithelial Cells Is Modified by Glucose Availability.

Coagulopathies common to patients with diabetes and chronic kidney disease (CKD) are not fully understood. Fibrin deposits in the kidney suggest the local presence of clotting factors including tissue factor (TF). In this study, we investigated the effect of glucose availability on the synthesis of TF by cultured human kidney tubular epithelial cells (HTECs) in response to activation of protease-activated receptor 2 (PAR2). PAR2 activation by peptide 2f-LIGRLO-NH2 (2F, 2 µM) enhanced the synthesis and secretion of active TF (~45 kDa) which was blocked by a PAR2 antagonist (I-191). Treatment with 2F also significantly increased the consumption of glucose from the cell medium and lactate secretion. Culturing HTECs in 25 mM glucose enhanced TF synthesis and secretion over 5 mM glucose, while addition of 5 mM 2-deoxyglucose (2DOG) significantly decreased TF synthesis and reduced its molecular weight (~40 kDa). Blocking glycosylation with tunicamycin also reduced 2F-induced TF synthesis while reducing its molecular weight (~36 kDa). In conclusion, PAR2-induced TF synthesis in HTECs is enhanced by culture in high concentrations of glucose and suppressed by inhibiting either PAR2 activation (I-191), glycolysis (2DOG) or glycosylation (tunicamycin). These results may help explain how elevated concentrations of glucose promote clotting abnormities in diabetic kidney disease. The application of PAR2 antagonists to treat CKD should be investigated further.

Protease-activated receptor-2 ligands reveal orthosteric and allosteric mechanisms of receptor inhibition

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.

The development of proteinase-activated receptor-2 modulators and the challenges involved.

Protease-activated receptor-2 (PAR2) has been extensively studied since its discovery in the mid-1990. Despite the advances in understanding PAR2 pharmacology, it has taken almost 25 years for the first inhibitor to reach clinical trials, and so far, no PAR2 antagonist has been approved for human use. Research has employed classical approaches to develop a wide array of PAR2 agonists and antagonists, consisting of peptides, peptoids and antibodies to name a few, with a surge in patent applications over this period. Recent breakthroughs in PAR2 structure determination has provided a unique insight into proposed PAR2 ligand binding sites. Publication of the first crystal structures of PAR2 resolved in complex with two novel non-peptide small molecule antagonists (AZ8838 and AZ3451) revealed two distinct binding pockets, originally presumed to be allosteric sites, with a PAR2 antibody (Fab3949) used to block tethered ligand engagement with the peptide-binding domain of the receptor. Further studies have proposed orthosteric site occupancy for AZ8838 as a competitive antagonist. One company has taken the first PAR2 antibody (MEDI0618) into phase I clinical trial (NCT04198558). While this first-in-human trial is at the early stages of the assessment of safety, other research into the structural characterisation of PAR2 is still ongoing in an attempt to identify new ways to target receptor activity. This review will focus on the development of novel PAR2 modulators developed to date, with an emphasis placed upon the advances made in the pharmacological targeting of PAR2 activity as a strategy to limit chronic inflammatory disease.

PAR2, Keratinocytes, and Cathepsin S Mediate the Sensory Effects of Ciguatoxins Responsible for Ciguatera Poisoning

Ciguatera fish poisoning is caused by the consumption of fish contaminated with ciguatoxins (CTXs). The most distressing symptoms are cutaneous sensory disturbances, including cold dysesthesia and itch. CTXs are neurotoxins known to activate voltage-gated sodium channels, but no specific treatment exists. Peptidergic neurons have been critically involved in ciguatera fish poisoning sensory disturbances. Protease-activated receptor-2 (PAR2) is an itch- and pain-related G protein‒coupled receptor whose activation leads to a calcium-dependent neuropeptide release. In this study, we studied the role of voltage-gated sodium channels, PAR2, and the PAR2 agonist cathepsin S in the cytosolic calcium increase and subsequent release of the neuropeptide substance P elicited by Pacific CTX-2 (P-CTX-2) in rat sensory neurons and human epidermal keratinocytes. In sensory neurons, the P-CTX-2‒evoked calcium response was driven by voltage-gated sodium channels and PAR2-dependent mechanisms. In keratinocytes, P-CTX-2 also induced voltage-gated sodium channels and PAR2-dependent marked calcium response. In the cocultured cells, P-CTX-2 significantly increased cathepsin S activity, and cathepsin S and PAR2 antagonists almost abolished P-CTX-2‒elicited substance P release. Keratinocytes synergistically favored the induced substance P release. Our results demonstrate that the sensory effects of CTXs involve the cathepsin S-PAR2 pathway and are potentiated by their direct action on nonexcitable keratinocytes through the same pathway.

Cathepsin S acts via protease-activated receptor 2 to activate sensory neurons and induce itch-like behaviour

Chronic itch is a debilitating condition characterised by excessive scratching and is a symptom frequently reported in skin diseases such as atopic dermatitis. It has been proposed that release of the cysteine protease Cathepsin S (CatS) from skin keratinocytes or immune cells resident in or infiltrating the skin could act as a pruritogen in chronic itch conditions. CatS is known to activate protease-activated receptor 2 (PAR2). We therefore hypothesised that enzymatic activation of neuronally expressed PAR2 by CatS was responsible for activation of sensory neurons and transmission of itch signals. Intradermally-injected human recombinant (hr)-CatS or the PAR2 agonist, SLIGRL-NH2 behaved as pruritogens by causing scratching behaviour in mice. Hr-CatS-induced scratching behaviour was prevented by CatS inhibitors and PAR2 antagonists and reduced by 50% in TRPV1−/− mice compared with wild-type mice, whilst no significant reduction in scratching behaviour was observed in TRPA1−/− mice. Cultured dorsal root ganglion (DRG) cells showed an increase in [Ca2+]i following incubation with hr-CatS, and the percentage of neurons that responded to hr-CatS decreased in the presence of a PAR2 antagonist or in cultures of neurons from TRPV1−/− mice. Taken together, our results indicate CatS acts as a pruritogen via PAR2 activation in TRPV1-expressing sensory neurons.

Protease-activated receptor-2 signaling through β-arrestin-2 mediates Alternaria alkaline serine protease-induced airway inflammation.

Alternaria alternata is a fungal allergen associated with severe asthma and asthma exacerbations. Similarly to other asthma-associated allergens, Alternaria secretes a serine-like trypsin protease(s) that is thought to act through the G protein-coupled receptor protease-activated receptor-2 (PAR2) to induce asthma symptoms. However, specific mechanisms underlying Alternaria-induced PAR2 activation and signaling remain ill-defined. We sought to determine whether Alternaria-induced PAR2 signaling contributed to asthma symptoms via a PAR2/β-arrestin signaling axis, identify the protease activity responsible for PAR2 signaling, and determine whether protease activity was sufficient for Alternaria-induced asthma symptoms in animal models. We initially used in vitro models to demonstrate Alternaria-induced PAR2/β-arrestin-2 signaling. Alternaria filtrates were then used to sensitize and challenge wild-type, PAR2-/- and β-arrestin-2-/- mice in vivo. Intranasal administration of Alternaria filtrate resulted in a protease-dependent increase of airway inflammation and mucin production in wild-type but not PAR2-/- or β-arrestin-2-/- mice. Protease was isolated from Alternaria preparations, and select in vitro and in vivo experiments were repeated to evaluate sufficiency of the isolated Alternaria protease to induce asthma phenotype. Administration of a single isolated serine protease from Alternaria, Alternaria alkaline serine protease (AASP), was sufficient to fully activate PAR2 signaling and induce β-arrestin-2-/--dependent eosinophil and lymphocyte recruitment in vivo. In conclusion, Alternaria filtrates induce airway inflammation and mucus hyperplasia largely via AASP using the PAR2/β-arrestin signaling axis. Thus, β-arrestin-biased PAR2 antagonists represent novel therapeutic targets for treating aeroallergen-induced asthma.

Structural Characterization of Agonist Binding to Protease-Activated Receptor 2 through Mutagenesis and Computational Modeling.

Protease-activated receptor 2 (PAR2) is a G-protein-coupled receptor that is activated by proteolytic cleavage of its N-terminus. The unmasked N-terminal peptide then binds to the transmembrane bundle, leading to activation of intracellular signaling pathways associated with inflammation and cancer. Recently determined crystal structures have revealed binding sites of PAR2 antagonists, but the binding mode of the peptide agonist remains unknown. In order to generate a model of PAR2 in complex with peptide SLIGKV, corresponding to the trypsin-exposed tethered ligand, the orthosteric binding site was probed by iterative combinations of receptor mutagenesis, agonist ligand modifications, and data-driven structural modeling. Flexible-receptor docking identified a conserved binding mode for agonists related to the endogenous ligand that was consistent with the experimental data and allowed synthesis of a novel peptide (1-benzyl-1H[1,2,3]triazole-4-yl-LIGKV) with functional potency higher than that of SLIGKV. The final model may be used to understand the structural basis of PAR2 activation and in virtual screens to identify novel agonists and competitive antagonists. The combined experimental and computational approach to characterize agonist binding to PAR2 can be extended to study the many other G protein-coupled receptors that recognize peptides or proteins.

New therapies to relieve pain: The search for more efficient and safer alternatives to opioid pain killers.

EMBO Reports (2018) e46925 Opioids are a largely efficient medication for relieving severe pain and are prescribed for a wide variety of indications including metastatic cancer, arthritis, neuropathy or post‐surgical pain. These drugs target opioid receptors to produce effects similar to morphine and include moderately strong painkillers such as oxycodone and hydrocodone (Vicodin), and strong drugs like fentanyl, which resembles opium‐derived morphine and heroin. The great efficiency of opioids in treating severe pain has also been the major drawback for longer‐term use given their addictive power. This does not matter for short‐term use, for instance to ease post‐operative pain, or terminal cancer when the primary requirement is to relieve suffering. Yet, the addictive side effect has become a huge societal problem; in addition, opioids do not work against all types of chronic pain, including some of the most severe cases such as cancer‐induced bone pain or some cases of neuropathy resulting from nerve damage. These problems have spurred efforts to develop new drugs for treating pain with some success in developing derivatives with reduced side effects. The other major avenue of research is the identification of relevant pain pathways or protein binding sites including those involved in the placebo effect. Their efficiency has spurred physicians to prescribe opioids too readily, while drug companies have heavily advertised their effects and understated the side effects to boost usage. This three‐way collusion between patients, doctors and pharmaceutical companies has stoked the ongoing epidemic of opioid addiction that has afflicted the USA and Canada in particular, but also other developed countries in Europe and Asia. Apart from exacting a heavy social toll through loss of motivation or loss of jobs, impact on relationships, crime and so on, opioid addiction has dangerous physiological effects, notably on the respiratory system, which causes most of the overdose …

Protease-activated receptor-2 in endosomes signals persistent pain of irritable bowel syndrome

Once activated at the surface of cells, G protein-coupled receptors (GPCRs) redistribute to endosomes, where they can continue to signal. Whether GPCRs in endosomes generate signals that contribute to human disease is unknown. We evaluated endosomal signaling of protease-activated receptor-2 (PAR2), which has been proposed to mediate pain in patients with irritable bowel syndrome (IBS). Trypsin, elastase, and cathepsin S, which are activated in the colonic mucosa of patients with IBS and in experimental animals with colitis, caused persistent PAR2-dependent hyperexcitability of nociceptors, sensitization of colonic afferent neurons to mechanical stimuli, and somatic mechanical allodynia. Inhibitors of clathrin- and dynamin-dependent endocytosis and of mitogen-activated protein kinase kinase-1 prevented trypsin-induced hyperexcitability, sensitization, and allodynia. However, they did not affect elastase- or cathepsin S-induced hyperexcitability, sensitization, or allodynia. Trypsin stimulated endocytosis of PAR2, which signaled from endosomes to activate extracellular signal-regulated kinase. Elastase and cathepsin S did not stimulate endocytosis of PAR2, which signaled from the plasma membrane to activate adenylyl cyclase. Biopsies of colonic mucosa from IBS patients released proteases that induced persistent PAR2-dependent hyperexcitability of nociceptors, and PAR2 association with β-arrestins, which mediate endocytosis. Conjugation to cholestanol promoted delivery and retention of antagonists in endosomes containing PAR2 A cholestanol-conjugated PAR2 antagonist prevented persistent trypsin- and IBS protease-induced hyperexcitability of nociceptors. The results reveal that PAR2 signaling from endosomes underlies the persistent hyperexcitability of nociceptors that mediates chronic pain of IBS. Endosomally targeted PAR2 antagonists are potential therapies for IBS pain. GPCRs in endosomes transmit signals that contribute to human diseases.

Isoxazole-tethered diarylheptanoid analogs: Discovery of a new drug-like PAR2 antagonist

A new class of isoxazole-tethered diarylheptanoids having characteristic 1,3-syn-diol and 1,3-anti-diol chemophoric moieties, e.g. 4a–d and 5a–c respectively, have been designed and synthesized starting from d-glucose following a stereo-conserved general synthetic strategy. The isoxazole heterocycle was installed using our recently elaborated methodology deploying Magtrieve™ as a selective oxidizing agent. Two of these new analogs 4a and 5a exhibited significantly improved in vitro drug-like properties including solubility, metabolic stability, cell permeability and lack of nonspecific cytotoxicity when compared with curcumin-I. In a HEK293 cell-based intracellular calcium [Ca2+]i release assay, 4a and 5a, when tested at 30 μM, inhibited the trypsin agonist induced protease-activated receptor-2 (PAR2) activity by 80% and 70% respectively. IC50 of 4a (SB70) has been determined as 6 μM which is in the same range of current benchmarks for PAR2 antagonists.

A Potent Antagonist of Protease-Activated Receptor 2 That Inhibits Multiple Signaling Functions in Human Cancer Cells.

Protease-activated receptor 2 (PAR2) is a cell surface protein linked to G-protein dependent and independent intracellular signaling pathways that produce a wide range of physiological responses, including those related to metabolism, inflammation, pain, and cancer. Certain proteases, peptides, and nonpeptides are known to potently activate PAR2. However, no effective potent PAR2 antagonists have been reported yet despite their anticipated therapeutic potential. This study investigates antagonism of key PAR2-dependent signaling properties and functions by the imidazopyridazine compound I-191 (4-(8-(tert-butyl)-6-(4-fluorophenyl)imidazo[1,2-b]pyridazine-2-carbonyl)-3,3-dimethylpiperazin-2-one) in cancer cells. At nanomolar concentrations, I-191 inhibited PAR2 binding of and activation by structurally distinct PAR2 agonists (trypsin, peptide, nonpeptide) in a concentration-dependent manner in cells of the human colon adenocarcinoma grade II cell line (HT29). I-191 potently attenuated multiple PAR2-mediated intracellular signaling pathways leading to Ca2+ release, extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, Ras homologue gene family, member A (RhoA) activation, and inhibition of forskolin-induced cAMP accumulation. The mechanism of action of I-191 was investigated using binding and calcium mobilization studies in HT29 cells where I-191 was shown to be noncompetitive and a negative allosteric modulator of the agonist 2f-LIGRL-NH2 The compound alone did not activate these PAR2-mediated pathways, even at high micromolar concentrations, indicating no bias in these signaling properties. I-191 also potently inhibited PAR2-mediated downstream functional responses, including expression and secretion of inflammatory cytokines and cell apoptosis and migration, in human colon adenocarcinoma grade II cell line (HT29) and human breast adenocarcinoma cells (MDA-MB-231). These findings indicate that I-191 is a potent PAR2 antagonist that inhibits multiple PAR2-induced signaling pathways and functional responses. I-191 may be a valuable tool for characterizing PAR2 functions in cancer and in other cellular, physiological, and disease settings.

Protease–activated receptor 2 exacerbates adenine–induced renal tubulointerstitial injury in mice

Hypercoagulability is associated with chronic kidney disease (CKD). Tissue factor/factor VIIa complex and factor Xa in the coagulation cascade are known to activate protease–activated receptor 2 (PAR2), and to cause inflammation and tissue injury. Although PAR2 is highly expressed in the kidney, it is unclear whether PAR2 plays a pathogenic role in CKD. To test this, we fed the mice lacking Par2 (F2rl1−/−) and wild type (F2rl1+/+) mice with adenine diet to induce tubulointerstitial injury, a hallmark of CKD. Adenine-treated mice showed severe renal dysfunction, tubular atrophy, and fibrosis. Fibrin deposition and the expression of tissue factor and PARs markedly increased in their kidneys. Lack of Par2 attenuated renal histological damage and reduced the expression levels of genes related to inflammation, fibrosis, and oxidative stress. Our data indicate that PAR2 is critically important in the pathogenesis of adenine-induced tubular injury. PAR2 antagonists under development could be useful to treat and prevent CKD.

Effects of PAR2 antagonist on inflammatory signals and tight junction expression in protease‐activated canine primary epithelial keratinocytes

Effects of <scp>PAR</scp>2 antagonist on inflammatory signals and tight junction expression in protease‐activated canine primary epithelial keratinocytes

Potent lipidated antagonists to protease‐activated receptor 2 (PAR2)

Protease‐activated receptor‐2 (PAR2) belongs to a four‐member family of G‐Protein coupled receptors (GPCRs) that contain internal ligands exposed following exogenous or endogenous protease cleavage of the extracellular amino terminus. PAR2 is associated with a variety of inflammatory conditions, including asthma and pain. The contributions of PAR2 signaling to disease has been hindered by the lack of potent, efficacious antagonists, and their potential for biased‐ligand signaling. We recently demonstrated that lipid tethering of known PAR2 peptidomimetic agonists based on the primary trypsin cleavage sequence (SLIGRL) increased their potency &gt; 200 fold. In this study, we used lipid tethering (hexadecyl (Hdc) group with polyethylene glycol (PEG) spacers) and heterocycle (2‐aminothiazoyl; 2‐at) substitution of hexapeptide sequence derived from the primary cleavage site of kallikreins 4/16 (SSKGRS) to elucidate novel PAR2 antagonists. Compound 562 (C562), 2‐aminothiazol‐4yl‐SKGRS‐PEG3‐Hdc blocks PAR2 Ca2+ signaling elicited via peptidomimetics (2‐at‐LIGRL‐NH2) or via asthma associated protease activation (Alternaria alternata filtrates) in cultured human bronchial epithelial cells (16HBE14o‐). This compound was a biased‐signaling antagonist in that it had no effect on mitogen activated protein kinase (MAPK) signaling, the other major signaling pathway activated via PAR2. A shortened version of C562, 2‐at‐SKGR‐PEG3‐Hdc (C595), maintained antagonistic activity against peptidomimetic activation in an in vitro physiological signaling assay (xCELLigence). C595 is closely related to the previously described potent and specific PAR2 agonist, 2‐at‐LIGR‐PEG3‐Hdc. Thus, we screened a series of potential PAR2 ligands with a heterocycle serine substitute followed by four amino acids (XXGR) and the PEG3‐Hdc lipid tether. We describe several potent agonists, and one partial agonist (C608, 2‐at‐TIGR‐PEG3‐Hdc) that also acts as a potent, specific and biased signaling antagonist of PAR2. When used in nanomolar concentrations, C608 blocked PAR2‐dependent Ca2+ signaling via protease or peptidomimetics without effects on MAPK signaling. C562, C595 and C608 are novel pharmacological tools that can be used to evaluate the physiological consequences of PAR2 full and biased ligand signaling.Support or Funding InformationThis work was primarily funded by a multi‐PI grant (NS 073664 to SB, JV and TJP) from the National Institutes of Health. Additional support for this work was from the following grants: National Institute of Health training grants (T32 HL 007249 for ANF; T32 ES 007091 for CLS), National Institutes of Health R01NS 065926 (TJP) and R01AI083403 (SB; Michael O. Daines, PI), State of Arizona Technology and Research Initiative Fund Awarded through Bio5 (JV).

Benzylamide antagonists of protease activated receptor 2 with anti-inflammatory activity.

Activation of protease activated receptor 2 (PAR2) has been implicated in inflammatory and metabolic disorders and its inhibition may yield novel therapeutics. Here, we report a series of PAR2 antagonists based on C-terminal capping of 5-isoxazolyl-L-cyclohexylalanine-L-isoleucine, with benzylamine analogues being effective new PAR2 antagonists. 5-Isoxazolyl-L-cyclohexylalanine-L-isoleucine-2-methoxybenzylamine (10) inhibited PAR2-, but not PAR1-, induced release of Ca(2+) (IC50 0.5 μM) in human colon cells, IL-6 and TNFα secretion (IC50 1-5 μM) from human kidney cells, and was anti-inflammatory in acute rat paw inflammation (ED50 5 mg/kg sc). These findings show that new benzylamide antagonists of PAR2 have anti-inflammatory activity.