Cysteinyl Leukotriene Receptor Research Articles

Discovery of a Potent and Orally Active Dual GPBAR1/CysLT1R Modulator for the Treatment of Metabolic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are two highly prevalent human diseases caused by excessive fat deposition in the liver. Although multiple approaches have been suggested, NAFLD/NASH remains an unmet clinical need. Here, we report the discovery of a novel class of hybrid molecules designed to function as cysteinyl leukotriene receptor 1 (CysLT1R) antagonists and G protein bile acid receptor 1 (GPBAR1/TGR5) agonists for the treatment of NAFLD/NASH. The most potent of these compounds generated by harnessing the scaffold of the previously described CystLT1R antagonists showed efficacy in reversing liver histopathology features in a preclinical model of NASH, reshaping the liver transcriptome and the lipid and energy metabolism in the liver and adipose tissues. In summary, the present study described a novel orally active dual CysLT1R antagonist/GPBAR1 agonist that effectively protects against the development of NAFLD/NASH, showing promise for further development.

Cysteinyl Leukotriene Receptor Antagonists Associated With a Decreased Incidence of Cancer: A Retrospective Cohort Study.

AimCysteinyl leukotrienes receptor antagonists (LTRAs) are promising chemoprevention options to target cysteinyl leukotriene signaling in cancer. However, only a number of randomized clinical trials (RCTs) or observational studies have been conducted to date; thus, the effect of LTRAs on patients is yet to be elucidated. Using insurance claim data, we aimed to evaluate whether LTRAs have cancer preventive effects by observing patients who took LTRAs.MethodPatients diagnosed with asthma, allergic rhinitis, chronic cough, and have no history of cancer were followed-up from 2005 to 2017. Cox proportional hazard regression analysis was conducted to estimate the hazard ratios (HRs) for cancer risk of LTRA users.ResultWe followed-up (median: 5.6 years) 188,906 matched patients (94,453 LTRA users and 94,453 non-users). LTRA use was associated with a decreased risk of cancer (adjusted HR [aHR] = 0.85, 95% confidence interval [CI] = 0.83–0.87). The cancer risk showed a tendency to decrease rapidly when LTRAs were used in high dose (aHR = 0.56, 95% CI = 0.40–0.79) or for longer durations of more than 3 years (aHR = 0.68, 95% CI = 0.60–0.76) and 5 years (aHR = 0.33, 95% CI = 0.26–0.42). The greater preventive effects of LTRAs were also observed in patients with specific risk factors related to sex, age, smoking, and the presence of comorbidities.ConclusionIn this study, we found that LTRA use was associated with a decreased risk of cancer. The high dose and long duration of the use of LTRAs correlated with a lower cancer risk. Since LTRAs are not yet used for the prevention or treatment of cancer, our findings could be used for developing a new chemo-regimen or designing feasible RCTs.

Control of coronary vascular resistance by eicosanoids via a novel GPCR.

Arachidonic acid metabolites epoxyeicosatrienoates (EETs) and hydroxyeicosatetraenoates (HETEs) are important regulators of myocardial blood flow and coronary vascular resistance (CVR), but their mechanisms of action are not fully understood. We applied a chemoproteomics strategy using a clickable photoaffinity probe to identify G protein-coupled receptor 39 (GPR39) as a microvascular smooth muscle cell (mVSMC) receptor selective for two endogenous eicosanoids, 15-HETE and 14,15-EET, which act on the receptor to oppose each other's activity. The former increases mVSMC intracellular calcium via GPR39 and augments coronary microvascular resistance, and the latter inhibits these actions. Furthermore, we find that the efficacy of both ligands is potentiated by zinc acting as an allosteric modulator. Measurements of coronary perfusion pressure (CPP) in GPR39-null hearts using the Langendorff preparation indicate the receptor senses these eicosanoids to regulate microvascular tone. These results implicate GPR39 as an eicosanoid receptor and key regulator of myocardial tissue perfusion. Our findings will have a major impact on understanding the roles of eicosanoids in cardiovascular physiology and disease and provide an opportunity for the development of novel GPR39-targeting therapies for cardiovascular disease.

Adipocyte Gq signaling is a regulator of glucose and lipid homeostasis in mice

Obesity is the major driver of the global epidemic in type 2 diabetes (T2D). In individuals with obesity, impaired insulin action leads to increased lipolysis in adipocytes, resulting in elevated plasma free fatty acid (FFA) levels that promote peripheral insulin resistance, a hallmark of T2D. Here we show, by using a combined genetic/biochemical/pharmacologic approach, that increased adipocyte lipolysis can be prevented by selective activation of adipocyte Gq signaling in vitro and in vivo (in mice). Activation of this pathway by a Gq-coupled designer receptor or by an agonist acting on an endogenous adipocyte Gq-coupled receptor (CysLT2 receptor) greatly improved glucose and lipid homeostasis in obese mice or in mice with adipocyte insulin receptor deficiency. Our findings identify adipocyte Gq signaling as an essential regulator of whole-body glucose and lipid homeostasis and should inform the development of novel classes of GPCR-based antidiabetic drugs.

Montelukast, cysteinyl leukotriene receptor 1 antagonist, inhibits cardiac fibrosis by activating APJ

Montelukast, cysteinyl leukotriene receptor 1 (CysLT1R) antagonist, is used clinically for patients with asthma, chronic obstructive pulmonary diseases (COPD), and allergic rhinitis. It has been reported that CysLT1R antagonists could reduce the risks of cardiovascular diseases in animal studies. Cardiac fibrosis is one of the major causes of heart failure. But little is known about the role of Montelukast in cardiac fibrosis and its underlying mechanism. In transverse aortic constriction (TAC) mice, Montelukast improved cardiac pumping function and inhibited cardiac fibrosis by down-regulation of the proteins related to the fibrosis, such as connective tissue growth factor (CTGF), Transforming Growth Factor β (TGF-β), and Alpha-smooth muscle actin (α-SMA). Montelukast reduced cell proliferation and collagen production in neonatal cardiac fibroblasts (CFs) with the pretreatment of 20% serum, while down-regulating the expression of TGF-β, CTGF and α-SMA. Molecules docking methods estimated a high affinity of Montelukast to Apelin receptor (APJ) and an effective chemical structure for Montelukast binding APJ. In Chinese hamster ovary (CHO) cells with stable overexpressing APJ, Montelukast inhibited forskolin (1 μM)-mediated cyclic adenosine monophosphate (cAMP) production and extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylation, while these effects were reversed by pertussis toxin (PTX) pretreatment. APJ silence disrupted the effects of Montelukast in CFs pretreatment by serum 20%. So we concluded that Montelukast inhibited cardiac fibrosis due presumably to the coupling to the APJ-mediated Gi signaling pathway, which may be a promising therapeutic target for cardiac fibrosis.

Zafirlukast Induces VHL- and HIF-2α-Dependent Oxidative Cell Death in 786-O Clear Cell Renal Carcinoma Cells.

Mutations in the Von Hippel–Lindau (VHL) gene are the driving force in many forms of clear cell renal cell carcinoma (ccRCC) and promote hypoxia-inducible factor (HIF)-dependent tumor proliferation, metastasis and angiogenesis. Despite the progress that has already been made, ccRCC generally remain resistant to conventional therapies and ccRCC patients suffer from metastasis and acquired resistance, highlighting the need for novel therapeutic options. Cysteinyl leukotriene receptor 1 (CysLTR1) antagonists, like zafirlukast, are administered in bronchial asthma to control eicosanoid signaling. Intriguingly, long-term use of zafirlukast decreases cancer risk and leukotriene receptor antagonists inhibit tumor growth, but the mechanisms still remain unexplored. Therefore, we aim to understand the mechanisms of zafirlukast-mediated cell death in ccRCC cells. We show that zafirlukast induces VHL-dependent and TNFα-independent non-apoptotic and non-necroptotic cell death in ccRCC cells. Cell death triggered by zafirlukast could be rescued with antioxidants and the PARP-1 inhibitor Olaparib, and additionally relies on HIF-2α. Finally, MG-132-mediated proteasome inhibition sensitized VHL wild-type cells to zafirlukast-induced cell death and inhibition of HIF-2α rescued zafirlukast- and MG-132-triggered cell death. Together, these results highlight the importance of VHL, HIF and proteasomal degradation in zafirlukast-induced oxidative cell death with potentially novel therapeutic implications for ccRCC.

Phenotypic evaluation of constitutive GPCR/G-protein signaling in zebrafish embryos and larvae

Signal transduction initiation by G-protein-coupled receptors (GPCRs) normally begins upon extracellular ligand binding. Some oncogenic GPCR mutants are capable of inducing G-protein signaling without ligand stimulation, thus behaving as constitutively active receptors. Evaluation of disease-causing capacity of constitutively active mutations in animal models requires months of time-consuming experiments, which hampers research progress. Here, using zebrafish embryos transiently expressing with constitutively active mutations via mRNA microinjection, we describe G-protein-subtype-specific phenotypes that can be evaluated over several days. Exogenous expression of the cysteinyl leukotriene receptor type II (CysLT2R) with an oncogenic L1293.43Q mutation by mRNA injection into a fertilized embryo induced developmental arrest during epiboly and eventual embryonic lethality, which were suppressed by treatment with the Gq inhibitor, YM-254890. Embryos with a constitutively active Gαq mutant exhibited an analogous phenotype. Interestingly, expression of constitutively active Gαs, Gαi, and Gα13 mutants induced distinct phenotypes. These phenotypes may thus serve as useful indicators for rapid in vivo evaluation of signaling activity of GPCR and G-protein mutants.

Discovery of dual-target ligands binding to beta2-adrenoceptor and cysteinyl-leukotriene receptor for the potential treatment of asthma from natural products derived DNA-encoded library

The design, synthesis, and discovery of dual-target compounds are considered as a promising strategy to develop new drugs with improved safety and efficacy compared with single-target drugs. This necessitates development of the methodologies that enable us to rapidly and accurately achieve the dual-target leads. Applying rosmarinic acid, 18β-glycyrrhetinic acid, rhein, and ferulic acid as template building blocks, we introduced the self-assembling DNA encoded technique to build the library containing 1,000 compounds. These compounds were screened by receptor chromatography with immobilized beta2-adrenoceptor (β2-AR) and cysteinyl-leukotriene receptor (CysLT), whereby we obtained a derivative of 18β-glycyrrhetinic acid (XC267) that specifically binds to the two receptors. In vitro assessment demonstrated the desired binding affinity of 6.57 × 104 M−1 to β2-AR, 2.82 × 104 M−1 to CysLT, and the dissociation rate constant of 7.52 s−1 to β2-AR, 17.2 s−1 to CysLT. Pharmacological examination with ovalbumin-induced mice demonstrated that XC267 significantly reduced the levels of IL-4, IL-13, and IgE after oral administration of 10 mg/kg. By Western blot analysis, we observed an up-regulated expression of β2-AR and a blocked level of CysLT with a dose-dependent manner in pulmonary bronchial. Our results suggest XC627 is a potential candidate to treat asthma by simultaneously regulating the signaling pathway of the two receptors.

Prospective Phase II Trial of Montelukast to Treat Bronchiolitis Obliterans Syndrome after Hematopoietic Cell Transplantation and Investigation into Bronchiolitis Obliterans Syndrome Pathogenesis

Bronchiolitis obliterans syndrome (BOS) is a severe manifestation of chronic graft-versus-host disease (cGVHD) following hematopoietic cell transplantation (HCT). Montelukast interrupts cysteinyl leukotriene (CysLT) activity and may diminish the activation and homing of cells to bronchioles and subsequent fibrosis. We performed a prospective phase II trial to test whether montelukast altered lung decline for patients with BOS after HCT. In this single-arm, open-label, multi-institutional study, the primary endpoints were stability or improvement (<15% decline) in forced expiratory volume in 1 second (FEV1) and a <1-point decline in the slope of FEV1 after 6 months of treatment. Secondary endpoints included symptom and functional responses and immune correlates investigating the role of leukotrienes in BOS progression. The study enrolled 25 patients with moderate to severe lung disease after 3 months of stable cGVHD therapy. Montelukast was well tolerated, and no patient required escalation of BOS-directed therapy. At the primary endpoint, all 23 evaluable patients met the criteria for treatment success using FEV1% predicted, and all but 1 patient had stable or improved FEV1 slope. In those with a >5% improvement in FEV1, clinically meaningful improvements were seen in the Lee scores of breathing, energy, and mood. Improvements in the Human Activity Profile and 6-minute-walk test were observed in those with a <5% decline in FEV1. Overall survival was 87% at 2 years. Immune correlates showed elevated leukotriene receptor levels on blood eosinophils and monocytes versus healthy controls, elevated urine leukotrienes in 45% of the cohort, and CysLT receptors in bronchoalveolar lavage subsets and a predominance of Th2 cells, all pretreatment. These data suggest that montelukast may safely halt the progression of BOS after HCT, and that leukotrienes may play a role in the biology of BOS.

Montelukast suppresses the development of irritable bowel syndrome phenotype possibly through modulating NF-κB signaling in an experimental model.

Irritable bowel syndrome (IBS) is a functionalgut disorder with multi-factorial pathophysiology that causes recurring pain or discomfort in the abdomen, as well as altered bowel habits. Montelukast, a well-known cysteinyl leukotriene receptor 1 (CysLT1R) antagonist, is widely used for the anti-inflammatory management of asthma. The present study aimed to evaluate the effects of pharmacological inhibition of CysLT1R on acetic acid-induced diarrhea-predominant IBS (D-IBS) in rats. Behavioral pain responses to noxious mechanical stimulation were decreased in the montelukast-treated rats as compared to the model animals following colorectal distension (CRD)-induced visceral hypersensitivity. Stool frequency decreased dose-dependently by montelukast in IBS rats exposed to restraint stress. A significantly shorter immobility time was also observed in IBS rats who received montelukast vs IBS group in the forced swimming test (depression-like behavior). Furthermore, there were significant decreases in the NF-κB protein expression, inflammatory cytokine (TNF-α, and IL-1ß) levels, and histopathological inflammatory injuries concomitant with increased anti-inflammatory cytokine, IL-10, in montelukast-treated rats compared with the IBS group. Cysteinyl leukotriene production and CysLT1R mRNA expression showed no remarkable differences among the experimental groups. The present results suggest the possible beneficial effects of montelukast in the management of D-IBS symptoms. The molecular mechanismunderlying such effects, at least to some extent, might be through modulating CysLT1R-mediated NF-κB signaling. Yet, more studies are required to demonstrate the clinical potential of this drug for IBS therapy.

Discovery and Development of the Quininib Series of Ocular Drugs.

The quininib series is a novel collection of small-molecule drugs with antiangiogenic, antivascular permeability, anti-inflammatory, and antiproliferative activity. Quininib was initially identified as a drug hit during a random chemical library screen for determinants of developmental ocular angiogenesis in zebrafish. To enhance drug efficacy, novel quininib analogs were designed by applying medicinal chemistry approaches. The resulting quininib drug series has efficacy in in vitro and ex vivo models of angiogenesis utilizing human cell lines and tissues. In vivo, quininib drugs reduce pathological angiogenesis and retinal vascular permeability in rodent models. Quininib acts as a cysteinyl leukotriene (CysLT) receptor antagonist, revealing new roles of these G-protein-coupled receptors in developmental angiogenesis of the eye and unexpectedly in uveal melanoma (UM). The quininib series highlighted the potential of CysLT receptors as therapeutic targets for retinal vasculopathies (e.g., neovascular age-related macular degeneration, diabetic retinopathy, and diabetic macular edema) and ocular cancers (e.g., UM).

Eicosanoids in the pancreatic tumor microenvironment - a multicellular, multifaceted progression.

Eicosanoids, oxidized fatty acids that serve as cell-signaling molecules, have been broadly implicated in tumorigenesis. Here, we aimed to identify eicosanoids associated with pancreatic tumorigenesis and the cell types responsible for their synthesis. We profiled normal pancreas and pancreatic ductal adenocarcinoma (PDAC) in mouse models and patient samples using mass spectrometry. We interrogated RNA sequencing datasets for eicosanoid synthase or receptor expression. Findings were confirmed by immunostaining. In murine models, we identified elevated levels of PGD2, prostacyclin, and thromboxanes in neoplasia while PGE2, 12-HHTre, HETEs, and HDoHEs are elevated specifically in tumors. Analysis of scRNA-seq datasets suggests that PGE2 and prostacyclins are derived from fibroblasts, PGD2 and thromboxanes from myeloid cells, and PGD2 and 5-HETE from tuft cells. In patient samples, we identified a transition from PGD2 to PGE2-producing enzymes in the epithelium during the transition to PDAC, fibroblast/tumor expression of PTGIS, and myeloid/tumor cell expression of TBXAS1. Our analyses identify key changes in eicosanoid species during pancreatic tumorigenesis and the cell types that contribute to their synthesis. Thromboxane and prostacyclin expression is conserved between animal models and human disease and may represent new druggable targets.

Effects of treatment with montelukast alone, budesonide/formoterol alone and a combination of both in cough variant asthma

BackgroundWhether cysteinyl-leukotriene receptor antagonists (LTRAs) have a similar antitussive effect to inhaled corticosteroids and long-acting β2-agonist (ICS/LABA), and that LTRA plus ICS/LABA is superior to LTRAs alone or ICS/LABA alone in treating cough variant asthma (CVA) remain unclear. This study aimed to investigate and compare the efficacy of montelukast alone, budesonide/formoterol alone and the combination of both in the treatment of CVA.MethodsNinety-nine CVA patients were assigned randomly in a 1:1:1 ratio to receive montelukast (M group: 10 mg, once daily), budesonide/formoterol (BF group: 160/4.5 μg, one puff, twice daily), or montelukast plus budesonide/formoterol (MBF group) for 8 weeks. The primary outcomes were changes in the cough visual analogue scale (VAS) score, daytime cough symptom score (CSS) and night-time CSS, and the secondary outcomes comprised changes in cough reflex sensitivity (CRS), the percentage of sputum eosinophils (sputum Eos%) and fractional exhaled nitric oxide (FeNO). CRS was presented with the lowest concentration of capsaicin that induced at least 5 coughs (C5). The repeated measure was used in data analysis.ResultsThe median cough VAS score (median from 6.0 to 2.0 in the M group, 5.0 to 1.0 in the BF group and 6.0 to 1.0 in the MBF group, all p < 0.001), daytime CSS (all p < 0.01) and night-time CSS (all p < 0.001) decreased significantly in all three groups after treatment for 8 weeks. Meanwhile, the LogC5 and sputum Eos% improved significantly in all three groups after 8 weeks treatment (all p < 0.05). No significant differences were found in the changes of the VAS score, daytime and night-time CSSs, LogC5 and sputum Eos% among the three groups from baseline to week 8 (all p > 0.05). The BF and MBF groups also showed significant decreases in FeNO after 8 weeks treatment (p = 0.001 and p = 0.008, respectively), while no significant change was found in the M group (p = 0.457). Treatment with MBF for 8 weeks significantly improved the FEV1/FVC as well as the MMEF% pred and decreased the blood Eos% (all p < 0.05).ConclusionsMontelukast alone, budesonide/formoterol alone and a combination of both were effective in improving cough symptom, decreasing cough reflex sensitivity and alleviating eosinophilic airway inflammation in patients with CVA, and the antitussive effect and anti-eosinophilic airway inflammation were similar.Trial registration ClinicalTrials.gov, number NCT01404013.

Leukotriene C4 synthase is a novel PPARγ target gene, and leukotriene C4 and D4 activate adipogenesis through cysteinyl LT1 receptors in adipocytes

Leukotriene (LT) C4 synthase (LTC4S) catalyzes the conversion from LTA4 to LTC4, which is a proinflammatory lipid mediator in asthma and other inflammatory diseases. LTC4 is metabolized to LTD4 and LTE4, all of which are known as cysteinyl (Cys) LTs and exert physiological functions through CysLT receptors. LTC4S is expressed in adipocytes. However, the function of CysLTs and the regulatory mechanism in adipocytes remain unclear. In this study, we investigated the expression of LTC4S and production of CysLTs in murine adipocyte 3T3-L1 cells and their underlying regulatory mechanisms. Expression of LTC4S and production of LTC4 and CysLTs increased during adipogenesis, whereas siRNA-mediated suppression of LTC4S expression repressed adipogenesis by reducing adipogenic gene expression. The CysLT1 receptor, one of the two LTC4 receptors, was expressed in adipocytes. LTC4 and LTD4 increased the intracellular triglyceride levels and adipogenic gene expression, and their enhancement was suppressed by co-treatment with pranlukast, a CysLT1 receptor antagonist. Moreover, the expression profiles of LTC4S gene/protein during adipogenesis resembled those of peroxisome proliferator-activated receptor (PPAR) γ. LTC4S expression was further upregulated by treatment with troglitazone, a PPARγ agonist. Promoter-luciferase and chromatin immunoprecipitation assays showed that PPARγ directly bound to the PPAR response element of the LTC4S gene promoter in adipocytes. These results indicate that the LTC4S gene expression was enhanced by PPARγ, and LTC4 and LTD4 activated adipogenesis through CysLT1 receptors in 3T3-L1 cells. Thus, LTC4S and CysLT1 receptors are novel potential targets for the treatment of obesity.

Tuft cell-produced cysteinyl leukotrienes and IL-25 synergistically initiate lung type 2 inflammation.

Aeroallergen sensing by airway epithelial cells triggers pathogenic immune responses leading to type 2 inflammation, the hallmark of chronic airway diseases such as asthma. Tuft cells are rare epithelial cells and the dominant source of interleukin-25 (IL-25), an epithelial cytokine, and cysteinyl leukotrienes (CysLTs), lipid mediators of vascular permeability and chemotaxis. How these two mediators derived from the same cell might cooperatively promote type 2 inflammation in the airways has not been clarified. Here, we showed that inhalation of the parent leukotriene C4 (LTC4) in combination with a subthreshold dose of IL-25 led to activation of two innate immune cells: inflammatory type 2 innate lymphoid cell (ILC2) for proliferation and cytokine production, and dendritic cells (DCs). This cooperative effect led to a much greater recruitment of eosinophils and CD4+ T cell expansion indicative of synergy. Whereas lung eosinophilia was dominantly mediated through the classical CysLT receptor CysLT1R, type 2 cytokines and activation of innate immune cells required signaling through CysLT1R and partially CysLT2R. Tuft cell–specific deletion of Ltc4s, the terminal enzyme required for CysLT production, reduced lung inflammation and the systemic immune response after inhalation of the mold aeroallergen Alternaria; this effect was further enhanced by concomitant blockade of IL-25. Our findings identified a potent synergy of CysLTs and IL-25 downstream of aeroallergen-trigged activation of airway tuft cells leading to a highly polarized type 2 immune response and further implicate airway tuft cells as powerful modulators of type 2 immunity in the lungs.

Cysteinyl Leukotriene Pathway and Cancer.

Cancer remains a leading cause of death worldwide, despite many advances being made in recent decades. Changes in the tumor microenvironment, including dysregulated immunity, may contribute to carcinogenesis and cancer progression. The cysteinyl leukotriene (CysLT) pathway is involved in several signal pathways, having various functions in different tissues. We summarized major findings of studies about the roles of the CysLT pathway in cancer. Many in vitro studies suggested the roles of CysLTs in cell survival/proliferation via CysLT1 receptor (CysLT1R). CysLT1R antagonism decreased cell vitality and induced cell death in several types of cancer cells, such as colorectal, urological, breast, lung and neurological malignancies. CysLTs were also associated with multidrug resistance of cancer, and CysLT1R antagonism might reverse chemoresistance. Some animal studies demonstrated the beneficial effects of CysLT1R antagonist in inhibiting tumorigenesis and progression of some cancer types, particularly colorectal cancer and lung cancer. The expression of CysLT1R was shown in various cancer tissues, particularly colorectal cancer and urological malignancies, and higher expression was associated with a poorer prognosis. The chemo-preventive effects of CysLT1R antagonists were demonstrated in two large retrospective cohort studies. In summary, the roles of the CysLT pathway in cancer have been delineated, whereas further studies are still warranted.

Chronobiological activity of cysteinyl leukotriene receptor 1 during basal and induced autophagy in the ARPE-19 retinal pigment epithelial cell line.

Autophagy is an important cellular mechanism for maintaining cellular homeostasis, and its impairment correlates highly with age and age-related diseases. Retinal pigment epithelial (RPE) cells of the eye represent a crucial model for studying autophagy, as RPE functions and integrity are highly dependent on an efficient autophagic process. Cysteinyl leukotriene receptor 1 (CysLTR1) acts in immunoregulation and cellular stress responses and is a potential regulator of basal and adaptive autophagy. As basal autophagy is a dynamic process, the aim of this study was to define the role of CysLTR1 in autophagy regulation in a chronobiologic context using the ARPE-19 human RPE cell line.Effects of CysLTR1 inhibition on basal autophagic activity were analyzed at inactive/low and high lysosomal degradation activity with the antagonists zafirlukast (ZTK) and montelukast (MTK) at a dosage of 100 nM for 3 hours. Abundances of the autophagy markers LC3-II and SQSTM1 and LC3B particles were analyzed in the absence and presence of lysosomal inhibitors using western blot analysis and immunofluorescence microscopy.CysLTR1 antagonization revealed a biphasic effect of CysLTR1 on autophagosome formation and lysosomal degradation that depended on the autophagic activity of cells at treatment initiation. ZTK and MTK affected lysosomal degradation, but only ZTK regulated autophagosome formation. In addition, dexamethasone treatment and serum shock induced autophagy, which was repressed by CysLTR1 antagonization. As a newly identified autophagy modulator, CysLTR1 appears to be a key player in the chronobiological regulation of basal autophagy and adaptive autophagy in RPE cells.

Studying the Effects of Vitamin A on the Severity of Allergic Rhinitis and Asthma

Allergic asthma is a complex lung disease characterized by breathlessness, airway inflammation, and obstruction. Allergy and allergic rhinitis (AR) are the main triggers of asthma. Vitamin A is an important supplementary factor for the physiological activation of the immune system. In the present study, we investigated the effects of vitamin A on the exacerbation of allergic asthma symptoms. BALB/c mice were allocated to four groups. Asthma was created in two groups, and in the other two groups, rhinitis was induced. One of the asthma groups and one of the rhinitis groups orally received vitamin A (20 IU/g for 15 days). The levels of Immunoglobulin (Ig) E, histamine, leukotriene B4 (LTB4), Cysteinyl leukotriene receptor (Cys-LT), interleukin (IL)-4, IL-5, IL-13, and IL-35 as well as eosinophil peroxidase activity, were measured. Also, the histopathology of mice lungs was evaluated. The levels of total IgE, LTB4, Cys-LT, IL-4, IL-5, IL-17, and IL-33, eosinophil peroxidase activity, perivascular and peribronchial inflammation significantly decreased in vitamin A-treated asthma and rhinitis groups compared to non-treated groups. Also, IL-13 and histamine levels, hyperplasia of the goblet cell, and hyper-secretion of the mucus insignificantly decreased in vitamin A-treated asthma and rhinitis groups. Asthma and AR are common diseases that are generally developed due to the dysregulation of the immune system. Vitamin A plays an important role in controlling the immunopathologic mechanisms of allergic diseases. Vitamin A could be a useful supplement in managing AR and asthma by decreasing the severity of inflammatory responses. Therefore, control of vitamin A deficiency is recommended in Allergy.

Leukotriene D4 Upregulates Oxidized Low-Density Lipoprotein Receptor 1 and CD36 to Enhance Oxidized LDL Uptake and Phagocytosis in Macrophages Through Cysteinyl Leukotriene Receptor 1.

Endothelial permeability, leukocyte attachment, and unregulated oxidized LDL (oxLDL) uptake by macrophages leading to the formation of foam cells are all vital in the initiation and progression of atherosclerosis. During inflammation, several inflammatory mediators regulate this process through the expression of distinct oxLDL binding cell surface receptors on macrophages. We have previously shown that Leukotriene D4 (LTD4) promotes endothelial dysfunction, increasing endothelial permeability and enhancing TNFα-mediated attachment of monocytes to endothelium, which hints at its possible role in atherosclerosis. Here we analyzed the effect of LTD4 on macrophage function. Macrophages mainly express CysLT1R and flux calcium in response to LTD4. Further, LTD4 potentiates phagocytosis in macrophages as revealed by the uptake of zymosan particles. Notably, LTD4 augmented macrophage phagocytosis and oxLDL uptake which is sensitive to MK-571 [Montelukast (MK)], a CysLT1R-specific antagonist. Mechanistically, LTD4 upregulated two receptors central to foam cell formation, oxidized low-density lipoprotein receptor-1 (OLR1/LOX-1), and CD36 in a time and dose-dependent manner. Finally, LTD4 enhanced the secretion of chemokines MCP-1 and MIP1β. Our results suggest that LTD4 contributes to atherosclerosis either through driving foam cell formation or recruitment of immune cells or both. CysLT1R antagonists are safely being used in the treatment of asthma, and the findings from the current study suggest that these can be re-purposed for the treatment of atherosclerosis.

Structural Basis for Developing Multitarget Compounds Acting on Cysteinyl Leukotriene Receptor 1 and G-Protein-Coupled Bile Acid Receptor 1.

G-protein-coupled receptors (GPCRs) are the molecular target of 40% of marketed drugs and the most investigated structures to develop novel therapeutics. Different members of the GPCRs superfamily can modulate the same cellular process acting on diverse pathways, thus representing an attractive opportunity to achieve multitarget drugs with synergic pharmacological effects. Here, we present a series of compounds with dual activity toward cysteinyl leukotriene receptor 1 (CysLT1R) and G-protein-coupled bile acid receptor 1 (GPBAR1). They are derivatives of REV5901─the first reported dual compound─with therapeutic potential in the treatment of colitis and other inflammatory processes. We report the binding mode of the most active compounds in the two GPCRs, revealing unprecedented structural basis for future drug design studies, including the presence of a polar group opportunely spaced from an aromatic ring in the ligand to interact with Arg792.60 of CysLT1R and achieve dual activity.