PAR1 Is a Candidate Target for the Treatment of Peritoneal Dissemination in Gastric Cancer.

BACKGROUND/AIM: Peritoneal dissemination (PD) is a frequent cause of death in gastric cancer (GC), and there is evidence of an association between protease-activated receptor-1 (PAR1) and the development of PD. This study hypothesized that PD in GC might be influenced by PAR1. MATERIALS AND METHODS: The cytotoxic effect of paclitaxel (PTX) on PAR1-transfected MKN45 (MKN45/PAR1) cells was analyzed using the MTT assay, and IC50 values were determined. In female athymic nude mice, MKN45/PAR1 cells were suspended in 0.05 ml phosphate-buffered saline (PBS) medium and inoculated into the stomach mid-wall. In each group, intraperitoneal injections of PBS, PTX, SCH79797 (PAR1-antagonist), or PTX plus SCH79797 were administered on days 8, 15, and 22 following tumor inoculation. At 56 days after tumor inoculation, mice were examined for both abdominal tumor nodule status and size and weight of the tumors. RESULTS: The IC50 of PTX for MKN45/PAR1 cells was 0.0697 µM and that of SCH79797 was 0.0145 µM. Mean survival of the MKN45/PAR1 mice in the PBS group was 28.75 days, whereas survival times for the mice treated with SCH79797, PTX, or a combination of PTX and SCH79797 were 31.2, 49.2, and 48.5 days, respectively. Tumor weight was smaller in the group receiving PTX and SCH79797 intraperitoneally compared with that in the PBS group (1,086±127.2 mg vs. 33.2±19.9 mg; p<0.001). CONCLUSION: The PAR1 antagonist was found to inhibit PD in a PAR1-expressing GC cell line. PAR1 may serve as a promising therapeutic target for managing PD in gastric cancer, as it plays a crucial role in its progression.

Therapeutic Effect of Proteinase-Activated Receptor-1 Antagonist on Colitis-Associated Carcinogenesis.

BACKGROUND & AIMS: Inflammatory bowel disease is associated with carcinogenesis, which limits the prognosis of the patients. The local expression of proteinases and proteinase-activated receptor 1 (PAR1) increases in inflammatory bowel disease. The present study investigated the therapeutic effects of PAR1 antagonism on colitis-associated carcinogenesis. METHODS: A colitis-associated carcinogenesis model was prepared in mice by treatment with azoxymethane (AOM) and dextran sulfate sodium (DSS). PAR1 antagonist E5555 was administered in long- and short-term protocol, starting on the day of AOM injection and 1 week after completing AOM/DSS treatment, respectively. The fecal samples were collected for metagenome analysis of gut microbiota. The intestinal myofibroblasts of the Crohn's disease patients were used to elucidate underlying cellular mechanisms. Caco-2 cells were used to investigate a possible source of PAR1 agonist proteinases. RESULTS: AOM/DSS model showed weight loss, diarrhea, tumor development, inflammation, fibrosis, and increased production of inflammatory cytokines. The ß-diversity, but not α-diversity, of microbiota significantly differed between AOM/DSS and control mice. E5555 alleviated these pathological changes and altered the microbiota ß-diversity in AOM/DSS mice. The thrombin expression was up-regulated in tumor and non-tumor areas, whereas PAR1 mRNA expression was higher in tumor areas compared with non-tumor areas. E5555 inhibited thrombin-triggered elevation of cytosolic Ca2+ concentration and ERK1/2 phosphorylation, as well as IL6-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation in intestinal myofibroblasts. Caco-2 cell-conditioned medium contained immunoreactive thrombin, which cleaved the recombinant protein containing the extracellular domain of PAR1 at the thrombin cleavage site. CONCLUSIONS: PAR1 antagonism is proposed to be a novel therapeutic strategy for treatment of inflammatory bowel disease and its associated carcinogenesis.

Proteinase-activated Receptor-1 Antagonist Attenuates Brain Injury via Regulation of FGL2 and TLR4 after Intracerebral Hemorrhage in Mice.

Proteinase-activated receptor-1 (PAR1) antagonist plays a protective effect in brain injury. We investigated the potential function and mechanisms of PAR1 antagonist in ICH-induced brain injury. Results showed that PAR1 antagonist protected against neurobehavior deficits, brain edema and blood-brain barrier integrity in ICH mice via the JNK/ERK/p38 MAPK signaling pathway at 24 h after ICH. In addition, ICH resulted in the increase of FGL2 and TLR4 expression over time, and phosphorylated (p-) JNK, ERK, p38 MAPK and IKKα expression. Suppression of FGL2 or TLR4 alleviated brain injury and decreased the expression of p-JNK, p-ERK, p-p38 MAPK and p-IKKα at 24 h after ICH, while overexpression of them showed the opposite result. Moreover, the protective effect of PAR1 antagonist on ICH-induced brain injury was blocked by FGL2 or TLR4 overexpression, and the levels of p-JNK, p-ERK and p-p38 MAPK were increased. Furthermore, PAR1 antagonist combined with TLR4 antagonist markedly alleviated brain injury after ICH at 72 h. Overall, PAR1 antagonist protected against short-term brain injury, and the effect of PAR1 antagonist on ICH-induced brain injury was mediated by FGL2 or TLR4.

Investigating detailed interactions between novel PAR1 antagonist F16357 and the receptor using docking and molecular dynamic simulations.

Currently, Vorapaxar is the only recently FDA-approved antiplatelet drug targeting Protease-activated receptor 1 (PAR1). However, a novel antagonist, F16357, has been shown to prevent painful bladder syndrome, also known as interstitial cystitis (IC). Unfortunately, there is no high resolution structure of the F16357-receptor complex, hindering its optimization as a therapeutic agent. In this study, we used docking and molecular dynamic (MD) simulations to investigate the detailed interactions between F16357 and PAR1 at a molecular level. The recently solved crystal structure of human PAR1 complexed with Vorapaxar was used in our docking of F16357 into the binding pocket of the receptor. To enhance binding pose selection, F16357 was docked first without constraints and then with a positional constraint to invert its orientation to become similar to that of Vorapaxar. The three systems, with crystal Vorapaxar, F16357 and an inverted F16357, were subjected to 3.0µs MD simulations. The MM-GBSA binding energy analysis showed that F16357 binds more strongly in a pose obtained from an unrestrained docking than in the inverted pose from a restrained docking; and Vorapaxar binds more strongly than F17357. This ordering is consistent with the experimental pIC50 values. Our structural data showed subtle changes in the binding pose between Vorapaxar and F16357. Transmembrane helices 1, 2, 5, and 7 were most significantly affected; most notably a large kink at F2795.47 in TM helix 5 of the Vorapaxar complex was completely absent in the F16357 complex. The results of this study facilitate the future development of other therapeutic PAR1 antagonists.

Insight into human protease activated receptor-1 as anticancer target by molecular modelling.

Protease-activated receptor 1 (PAR1) has been established as a promising target in many diseases, including various cancers. Strong evidence also suggests its role in metastasis. It is proved experimentally that PAR1 can induce numerous cell phenotypes, i.e. proliferation and differentiation. A strong link between PAR1 gene overexpression and high levels of ß-catenin was suggested by a study of the PAR1-Gα(13)-DVL axis in ß-catenin stabilization in cancers. An in vitro study was carried out to analyze PAR1 expression by flow cytometry on CD38+138+ plasma cells obtained from patients either at diagnosis (n: 46) (newly diagnosed multiple myeloma (NDMM)) or at relapse (n: 45) (relapsed/refractory multiple myeloma (RRMM)) and compared with the controls. Our previously synthesized benzoxazole (XT2B) and benzamide (XT5) derivatives were tested with in vitro 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, which revealed significant inhibitory activity on PAR1. We provide docking studies using Autodock Vina of these newly tested compounds to compare with the known PAR1 inhibitors in order to examine the binding mechanisms. In addition, the docking results are validated using HYDE binding assessment and a neural network (NN) scoring function.

In vitro pharmacological characterization of vorapaxar, a novel platelet thrombin receptor antagonist.

Vorapaxar is a novel protease-activated receptor-1 (PAR1) antagonist recently approved for the reduction of thrombotic cardiovascular events in patients with a history of myocardial infarction or with peripheral arterial disease. The present study provides a comprehensive in vitro pharmacological characterization of vorapaxar interaction with the PAR1 receptor on human platelets. Similar studies were performed with a metabolite of vorapaxar (M20). Vorapaxar and M20 were competitive PAR1 antagonists that demonstrated concentration-dependent, saturable, specific, and slowly reversible binding to the receptor present on intact human platelets. The affinities of vorapaxar and M20 for the PAR1 receptor were in the low nanomolar range, as determined by saturation-, kinetic- and competitive binding studies. The calculated Kd and Ki values for vorapaxar increased in the presence of plasma, indicating a decrease in the free fraction available for binding to the PAR1 receptor on human platelets. Vorapaxar was also evaluated in functional assays using thrombin or a PAR1 agonist peptide (SFLLRN). Vorapaxar and M20 completely blocked thrombin-stimulated PAR1/ß-arrestin association in recombinant cells and abolished thrombin-stimulated calcium influx in washed human platelets and vascular smooth muscle cells. Moreover, vorapaxar and M20 inhibited PAR1 agonist peptide-mediated platelet aggregation in human platelet rich plasma with a steep concentration response relationship. Vorapaxar exhibited high selectivity for inhibition of PAR1 over other platelet GPCRs. In conclusion, vorapaxar is a potent PAR1 antagonist exhibiting saturable, reversible, selective binding with slow off-rate kinetics and effectively inhibits thrombin's PAR1-mediated actions on human platelets.

Risk and benefit of direct oral anticoagulants or PAR-1 antagonists in addition to antiplatelet therapy in patients with acute coronary syndrome.

BACKGROUND: The overall risk-benefit profile of direct oral anticoagulants (DOACs) or PAR-1 antagonists in addition to antiplatelet therapy for patients with acute coronary syndrome (ACS) has not been clearly established. METHODS: Studies evaluating clinical outcomes of DOACs (including direct Xa inhibitors and direct thrombin inhibitors) or PAR-1 antagonists in addition to standard antiplatelet therapy in patients with recent ACS, published before Nov 2014, were screen. Eleven double blind, placebo-controlled, randomized clinical studies including 46782 patients were identified. RESULTS: The study revealed an up to 3-fold increased risk of hemorrhagic stroke in patients receiving DOACs (OR 3.45, 95% CI 1.62 to 7.37, P=0.001, and I(2)=0%) or PAR-1 antagonists (OR 2.60, 95% CI 1.18 to 5.69, P=0.02, and I(2)=0%) in addition to antiplatelet therapy compared to those with antiplatelet therapy alone. Despite a moderate but significant reduction of composite death/MI/stroke was observed in patients with additional DOACs (OR 0.86, 95% CI 0.78 to 0.94, P=0.002, and I(2)=0%) or PAR-1 antagonists (OR 0.89, 95% CI 0.80 to 0.98, P=0.02, and I(2)=0%), due to the remarkably increased major bleeding risks, overall net clinical outcomes (death/MI/stroke/major bleeding) did not differ between patients with or without additional DOACs (OR 0.99, 95% CI 0.91 to 1.09, P=0.88, and I(2)=0%) or PAR-1 antagonists (OR 0.98, 95% CI 0.91 to 1.05, P=0.55, and I(2)=0%). CONCLUSIONS: In patients with ACS, the addition of DOACs or PAR-1 antagonists to antiplatelet therapy led to a modest but significant reduction in composite efficacy outcome at the cost of a substantial increase in hemorrhagic stroke and major bleeding events.

Himbacine-derived thrombin receptor antagonists: c7-spirocyclic analogues of vorapaxar.

We have synthesized several C7-spirocyclic analogues of vorapaxar and evaluated their in vitro activities against PAR-1 receptor. Some of these analogues showed activities and rat plasma levels comparable to vorapaxar. Compound 5c from this series showed excellent PAR-1 activity (K i = 5.1 nM). We also present a model of these spirocyclic compounds docked to the PAR-1 receptor based on the X-ray crystal structure of vorapaxar bound to PAR-1 receptor. This model explains some of the structure-activity relationships in this series.

Himbacine-derived thrombin receptor antagonists: c7-aminomethyl and c9a-hydroxy analogues of vorapaxar.

We have synthesized several C7-aminomethyl analogues of vorapaxar that are potent PAR-1 antagonists. Many of these analogues showed excellent in vitro binding affinity and pharmacokinetics profile in rats. Compound 6a from this series showed excellent PAR-1 activity (K i = 5 nM). We have also synthesized a C9a-hydroxy analogue of vorapaxar, which showed very good PAR-1 affinity (K i = 19.5 nM) along with excellent rat pharmacokinetic profile and ex vivo efficacy in the cynomolgus monkey.

Discovery of Octahydroindenes as PAR1 Antagonists.

Octahydroindene was identified as a novel scaffold for protease activated receptor 1 (PAR1) antagonists. Herein, the 2-position (C2) was explored for structure-activity relationship (SAR) studies. Compounds 14, 19, and 23b showed IC50 values of 1.3, 8.6, and 2.7 nM in a PAR1 radioligand binding assay, respectively, and their inhibitory activities on platelet activation were comparable to that of vorapaxar in a platelet rich plasma (PRP) aggregation assay. This series of compounds showed high potency and no significant cytotoxicity; however, the compounds were metabolically unstable in both human and rat liver microsomes. Current research efforts are focused on optimizing the compounds to improve metabolic stability and physicochemical properties as well as potency.

Coagulation factor Xa induces an inflammatory signalling by activation of protease-activated receptors in human atrial tissue.

Activated factor X (FXa) is an important player in the coagulation cascade responsible for thrombin generation, which is activated during atrial fibrillation. Increasing evidence suggests that FXa influences cell signalling in various cell types by activating protease-activated receptors (PARs). It is so far not known if molecular effects of FXa affect atrial signal transduction. To study the effects of FXa, human atrial tissue slices were cultivated with FXa up to 24h. Additionally, rapid pacing was applied at 4Hz to resemble atrial fibrillation. The inhibitory impact of FXa antagonist (Rivaroxaban), protease-activated receptor 1 antagonist (SCH79797), and protease-activated receptor 2 antagonist (GB83) were analysed under experimental conditions. The exposure of atrial tissue to FXa resulted in the 1.7 fold upregulation of PAR2-mRNA, activation of MAP kinases (ERK1/2) and NF-κB signalling. Furthermore FXa increased the expression of adhesion molecule ICAM-1 (1.82 ± 0.20), chemokine IL-8 (1.94 ± 0.20), as well as prothrombotic molecule PAI-1 (1.52 ± 0.17). The combination of rapid pacing and FXa caused significant upregulation of PAR1 (2.82 ± 0.22), PAR2 (2.66 ± 0.40), ICAM-1 (2.13 ± 0.25), IL-8 (2.22 ± 0.24), LOX-1 (2.59 ± 0.35), and PAI-1 (2.65 ± 0.52) at the mRNA level. Rivaroxaban and GB83 prevented upregulation of PARs, ICAM-1, LOX-1, IL-8, and activation of MAP kinases. The elevation in the expression of PAI-1 was hindered in the presence of SCH79797, or Rivaroxaban. The present study indicates that FXa mediates inflammatory signalling in atrial tissue. Importantly, FXa and tachyarrhythmia act synergistically to increase expression of protease-activated receptors and inflammatory mediators. Rivaroxaban prevented effectively FXa-induced molecular effects in human atrial tissue particularly during rapid pacing.