Discovery of 2,3-Dihydro[1,4]dioxino[2,3-g]benzofuran Derivatives as Protease Activated Receptor 4 (PAR4) Antagonists with Potent Antiplatelet Aggregation Activity and Low Bleeding Tendency.

Patients with arterial embolic disease have benefited greatly from antiplatelet therapy. However, hemorrhage risk of antiplatelet agents cannot be ignored. Herein, we describe the discovery of 2,3-dihydro[1,4]dioxino[2,3-g]benzofuran compounds as novel PAR4 antagonists. Notably, the isomers 36 and 37 with the chemotype of phenoxyl methylene substituted on the 2,3-dihydro-1,4-dioxine ring exhibited potent in vitro antiplatelet activity (IC50 = 26.13 nM for 36 and 14.26 nM for 37) and significantly improved metabolic stability in human liver microsomes (T1/2 = 97.6 min for 36 and 11.1 min for BMS-986120). 36 also displayed good oral PK profiles (mice: T1/2 = 7.32 h and F = 45.11%). Both of them showed overall potent ex vivo antiplatelet activity at concentrations of 6 and 12 mg/kg, with no impact on the coagulation system and low bleeding liability. Our work will facilitate development of novel PAR4 antagonists as a safer therapeutic option for arterial embolism.

Protease-Activated Receptor 1-Mediated Damage of Podocytes in Diabetic Nephropathy.

There is clinical evidence that increased urinary serine proteases are associated with the disease severity in the setting of diabetic nephropathy (DN). Elevation of serine proteases may mediate [Ca2+]i dynamics in podocytes through the protease-activated receptors (PARs) pathway, including associated activation of nonspecific cation channels. Cultured human podocytes and freshly isolated glomeruli were used for fluorescence and immunohistochemistry stainings, calcium imaging, Western blot analysis, scanning ion conductance microscopy, and patch clamp analysis. Goto-Kakizaki, Wistar, type 2 DN (T2DN), and a novel PAR1 knockout on T2DN rat background rats were used to test the importance of PAR1-mediated signaling in DN settings. We found that PAR1 activation increases [Ca2+]i via TRPC6 channels. Both human cultured podocytes exposed to high glucose and podocytes from freshly isolated glomeruli of T2DN rats had increased PAR1-mediated [Ca2+]i compared with controls. Imaging experiments revealed that PAR1 activation plays a role in podocyte morphological changes. T2DN rats exhibited a significantly higher response to thrombin and urokinase. Moreover, the plasma concentration of thrombin in T2DN rats was significantly elevated compared with Wistar rats. T2DNPar1-/- rats were embryonically lethal. T2DNPar1+/- rats had a significant decrease in glomerular damage associated with DN lesions. Overall, these data provide evidence that, during the development of DN, elevated levels of serine proteases promote an excessive [Ca2+]i influx in podocytes through PAR1-TRPC6 signaling, ultimately leading to podocyte apoptosis, the development of albuminuria, and glomeruli damage. ARTICLE HIGHLIGHTS: Increased urinary serine proteases are associated with diabetic nephropathy. During the development of diabetic nephropathy in type 2 diabetes, the elevation of serine proteases could overstimulate protease-activated receptor 1 (PAR1). PAR1 signaling is involved in the development of DN via TRPC6-mediated intracellular calcium signaling. This study provides fundamental knowledge that can be used to develop efficient therapeutic approaches targeting serine proteases or corresponding PAR pathways to prevent or slow the progression of diabetes-associated kidney diseases.

MALT1 Is a Targetable Driver of Epithelial-to-Mesenchymal Transition in Claudin-Low, Triple-Negative Breast Cancer.

MALT1 is the effector protein of the CARMA/Bcl10/MALT1 (CBM) signalosome, a multiprotein complex that drives pro-inflammatory signaling pathways downstream of a diverse set of receptors. Although CBM activity is best known for its role in immune cells, emerging evidence suggests that it plays a key role in the pathogenesis of solid tumors, where it can be activated by selected G protein-coupled receptors (GPCR). Here, we demonstrated that overexpression of GPCRs implicated in breast cancer pathogenesis, specifically the receptors for Angiotensin II and thrombin (AT1R and PAR1), drove a strong epithelial-to-mesenchymal transition (EMT) program in breast cancer cells that is characteristic of claudin-low, triple-negative breast cancer (TNBC). In concert, MALT1 was activated in these cells and contributed to the dramatic EMT phenotypic changes through regulation of master EMT transcription factors including Snail and ZEB1. Importantly, blocking MALT1 signaling, through either siRNA-mediated depletion of MALT1 protein or pharmacologic inhibition of its activity, was effective at partially reversing the molecular and phenotypic indicators of EMT. Treatment of mice with mepazine, a pharmacologic MALT1 inhibitor, reduced growth of PAR1+, MDA-MB-231 xenografts and had an even more dramatic effect in reducing the burden of metastatic disease. These findings highlight MALT1 as an attractive therapeutic target for claudin-low TNBCs harboring overexpression of one or more selected GPCRs. IMPLICATIONS: This study nominates a GPCR/MALT1 signaling axis as a pathway that can be pharmaceutically targeted to abrogate EMT and metastatic progression in TNBC, an aggressive form of breast cancer that currently lacks targeted therapies.

Recombinant Activated Protein C (rhAPC) Affects Lipopolysaccharide-Induced Mechanical Compliance Changes and Beat Frequency of mESC-Derived Cardiomyocyte Monolayers.

BACKGROUND: Septic cardiomyopathy increases mortality by 70% to 90% and results in mechanical dysfunction of cells. METHODS: Here, we created a LPS-induced in-vitro sepsis model with mouse embryonic stem cell-derived cardiomyocytes (mESC-CM) using the CellDrum technology which simultaneously measures mechanical compliance and beat frequency of mESCs. Visualization of reactive oxygen species (ROS), actin stress fibers, and mRNA quantification of endothelial protein C receptor (EPCR) and protease-activated receptor 1 (PAR1) before/after LPS incubation were used for method validation. Since activated protein C (APC) has cardioprotective effects, samples were treated with human recombinant APC (rhAPC) with/-out LPS predamage to demonstrate the application in therapeutic studies. RESULTS: Twelve hours LPS treatment (5 µg/mL) increased ROS and decreased actin stress fiber density and significantly downregulated EPCR and PAR1 compared to control samples (0.26, 0.39-fold respectively). rhAPC application (5 µg/mL, 12 h) decreased ROS and recovered actin density, EPCR, and PAR1 levels were significantly upregulated compared to LPS predamaged samples (4.79, 3.49-fold respectively). The beat frequencies were significantly decreased after 6- (86%) and 12 h (73%) of LPS application. Mechanical compliance of monolayers significantly increased in a time-dependent manner, up to eight times upon 12-h LPS incubation compared to controls. rhAPC incubation increased the beat frequency by 127% (6h-LPS) and 123% (12h-LPS) and decreased mechanical compliance by 68% (12h-LPS) compared to LPS predamaged samples. CONCLUSION: LPS-induced contraction dysfunction and the reversal effects of rhAPC were successfully assessed by the mechanical properties of mESC-CMs. The CellDrum technology proved a decent tool to simulate sepsis in-vitro.

Improving Outcomes in Cardiovascular Diseases: A Review on Vorapaxar.

Antiplatelet agents are the standard of practice in the management of atherosclerosis and acute coronary syndrome. In contrast to the available antiplatelet agents, vorapaxar represents a novel mechanism of action. It is an antagonist of the platelet protease-activated receptor-1 and inhibits thrombin-induced and thrombin receptor agonist peptide-induced platelet aggregation. The Thrombin Receptor Antagonist in Secondary Prevention of Atherothrombotic Ischemic Events-Thrombolysis in Myocardial Infarction 50 (TRA 2°P-TIMI 50) trial led to the approval of vorapaxar by the Food and Drug Administration and European Medicines Agency for the reduction of thrombotic cardiovascular events in patients with a history of myocardial infarction (MI) or peripheral arterial disease. TRA 2°P-TIMI 50 trial showed that the use of vorapaxar (2.5 mg once/daily) in addition to standard dual antiplatelet therapy with aspirin and a P2Y12 receptor inhibitor was effective in the secondary prevention of recurrent thrombotic events among patients with previous atherothrombosis, particularly in patients with prior MI; at the expense of an increase in major bleeding. Another recently published Vorapaxar Therapy in Patients With Prior Myocardial Infarction Treated With Newer Generation P2Y12 Receptor Inhibitors Prasugrel and Ticagrelor (VORA-PRATIC) study showed that among post-MI patients treated with potent P2Y12 inhibitors (prasugrel or ticagrelor), vorapaxar reduced platelet-driven global thrombogenicity, an effect that persisted, albeit attenuated, in the absence of aspirin. The current review summarizes an up-to-date literature on pharmacokinetics, pharmacodynamics, and clinical efficacy of vorapaxar and proposes future directions of research.

PAR-1 Antagonism to Promote Gut Mucosa Healing in Crohn's Disease Patients: A New Avenue for CVT120165.

A new paradigm has been added for the treatment of inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. In addition to resolving symptoms and inflammatory cell activation, the objective of tissue repair and mucosal healing is also now considered a primary goal. In the search of mediators that would be responsible for delayed mucosal healing, protease-activated receptor-1 (PAR-1) has emerged as a most interesting target. Indeed, in Crohn's disease, the endogenous PAR-1 agonist thrombin is drastically activated. Activation of PAR-1 is known to be associated with epithelial dysfunctions that hamper mucosal homeostasis. This review gathers the scientific evidences of a potential role for PAR-1 in mucosal damage and mucosal dysfunctions associated with chronic intestinal inflammation. The potential clinical benefits of PAR-1 antagonism to promote mucosal repair in CD patients are discussed. Targeted local delivery of a PAR-1 antagonist molecule such as CVT120165, a formulated version of the FDA-approved PAR-1 antagonist vorapaxar, at the mucosa of Crohn's disease patients could be proposed as a new indication for IBD that could be rapidly tested in clinical trials.

The potential clinical benefits and indications of PAR-1 antagonism to treat inflammatory bowel diseases are discussed.

Influence of parstatin on experimental periodontal disease and repair in rats.

BACKGROUND: Parstatin is a 41-amino acid peptide, formed by proteolytic cleavage on activation of the protease activated receptor-1, with antiangiogenic properties. The purpose of this study is to evaluate the influence of synthetic parstatin on experimental periodontal disease and repair in rats. METHODS: Ligature-induced periodontitis was established in rats and the influence of parstatin administration was assessed after 8 and 15 days for periodontal disease and 24 hours and 8 days after repair after ligature removal. RESULTS: Parstatin administration significantly inhibited gingival myeloperoxidase activity, interleukin (IL)-1ß, tumor necrosis factor-α, and IL-6 levels and led to suppression of macrophages and collagen degradation. At periodontal tissues under repair, parstatin increased the gingival levels of endostatin and decreased vascular endothelial growth factor expression and blood vessel number but did not influence histologic healing. In addition, the tomographic linear bone loss was significantly reduced at 15 days of periodontitis when the rats were treated with parstatin compared to their respective phosphate-buffered saline-treated controls. CONCLUSIONS: Parstatin suppresses the periodontal tissue breakdown followed by experimental periodontitis in rats and did not impair periodontal tissue repair, despite its antiangiogenic effect. Parstatin may represent a novel approach to modulate host response in chronic periodontal disease.

Targeting protease activated receptor-1 with P1pal-12 limits bleomycin-induced pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis is the most devastating fibrotic diffuse parenchymal lung disease which remains refractory to pharmacological therapies. Therefore, novel treatments are urgently required. Protease-activated receptor (PAR)-1 is a G-protein-coupled receptor that mediates critical signalling pathways in pathology and physiology. Bleomycin-induced lung fibrosis has been shown to be diminished in PAR-1-deficient mice. The purpose of this study is to investigate whether pharmacological PAR-1 inhibition is a potential therapeutic option to combat pulmonary fibrosis. METHODS: Pulmonary fibrosis was induced by intranasal instillation of bleomycin into wild-type mice with or without a specific PAR-1 antagonist (ie, P1pal-12, a pepducin that blocks the PAR-1/G-protein interaction). Fibrosis was assessed by hydroxyproline analysis, immunohistochemistry, quantitative PCR and western blot for fibrotic markers expression. RESULTS: We first show that P1pal-12 effectively inhibits PAR-1-induced profibrotic responses in fibroblasts. Next, we show that once daily treatment with 0.5, 2.5 or 10 mg/kg P1pal-12 reduced the severity and extent of fibrotic lesions in a dose-dependent manner. These findings correlated with significant decreases in fibronectin, collagen and α smooth muscle actin expression at the mRNA and protein level in treated mice. To further establish the potential clinical applicability of PAR-1 inhibition, we analysed fibrosis in mice treated with P1pal-12 1 or 7 days after bleomycin instillation. Interestingly, when administered 7 days after the induction of fibrosis, P1pal-12 was as effective in limiting the development of pulmonary fibrosis as when administration was started before bleomycin instillation. CONCLUSIONS: Overall, targeting PAR-1 may be a promising treatment for pulmonary fibrosis.

Parstatin suppresses ocular neovascularization and inflammation.

PURPOSE: Parstatin is a 41-mer peptide formed by proteolytic cleavage on activation of the PAR1 receptor. The authors recently showed that parstatin is a potent inhibitor of angiogenesis. The purpose of the present study was to evaluate the therapeutic effect of parstatin on ocular neovascularization. METHODS: Choroidal neovascularization was generated in mice using laser-induced rupture of Bruch's membrane and was assessed after 14 days after perfusion of FITC-dextran. Oxygen-induced retinal neovascularization was established in neonatal mice by exposing them to 75% O(2) at postnatal day (P)7 for 5 days and then placing them in room air for 5 days. Evaluation was performed on P17 after staining with anti-mouse PECAM-1. The effect of parstatin was tested after intravitreal administration. The effects of subconjunctival-injected parstatin on corneal neovascularization and inflammation in rats were assessed 7 days after chemical burn-induced corneal neovascularization. Retinal leukostasis in mice was assessed after perfusion with FITC-conjugated concanavalin A. RESULTS: Parstatin potently inhibited choroidal neovascularization with an IC(50) of approximately 3 µg and a maximum inhibition of 59% at 10 µg. Parstatin suppressed retinal neovascularization with maximum inhibition of 60% at 3 µg. Ten-microgram and 30-µg doses appeared to be toxic to the neonatal retina. Subconjunctival parstatin inhibited corneal neovascularization, with 200 µg the most effective dose (59% inhibition). In addition, parstatin significantly inhibited corneal inflammation and VEGF-induced retinal leukostasis. In all models tested, scrambled parstatin was without any significant effect. CONCLUSIONS: Parstatin is a potent antiangiogenic agent of ocular neovascularization and may have clinical potential in the treatment of angiogenesis-related ocular disorders.

SCH-530348, a thrombin receptor (PAR-1) antagonist for the prevention and treatment of atherothrombosis.

SCH-530348 is a novel antiplatelet agent undergoing development by Schering-Plough Corp for the treatment and prevention of atherothrombosis. The compound is an orally administered himbacine analog that potently antagonizes the platelet thrombin receptor protease-activated receptor 1 (PAR-1), which leaves the procoagulant function of thrombin intact. In preclinical studies, SCH-530348 demonstrated no effect on bleed time or coagulation parameters. In both cynomolgus monkeys and humans, the compound had high bioavailability and inhibited ex vivo TRAP (thrombin receptor-activating peptide)-stimulated platelet aggregation in a potent and long-lasting manner. In a phase II clinical trial of patients undergoing percutaneous coronary intervention, SCH-530348 added to standard therapy with aspirin and clopidogrel did not increase major or minor thrombolysis in myocardial infarction bleeding, and demonstrated a trend toward decreased major adverse cardiovascular events versus placebo. At the time of publication, three phase III trials were underway to assess the efficacy and safety of SCH-530348 for at least 1 year in up to 35,000 patients with acute coronary syndromes or atherosclerosis. The distinct mechanism of action of SCH-530348 allows for cardiovascular protection without the liability of increased bleeding associated with other antiplatelet therapies. Phase III trials in high-risk patients will determine the use of SCH-530348 in cardiological practice.