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BAR502, a bile acid analogue, is active as dual FXR/GPBAR1 agonist and represents a promising lead for the treatment of cholestasis and NASH. In this paper we report the synthesis and the biological evaluation of a library of hybrid compounds prepared by combining, through high-yield condensation reaction, some fibrates with BAR502.The activity of the new conjugates was evaluated towards FXR, GPBAR1 and PPARα receptors, employing transactivation or cofactor recruitment assays. Compound 1 resulted as the most promising of the series and was subjected to further pharmacological investigation, together with stability evaluation and cell permeation assessment. We have proved by LCMS analysis that compound 1 is hydrolyzed in mice releasing clofibric acid and BAR505, the oxidized metabolite of BAR502, endowed with retained dual FXR/GPBAR1 activity.
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[This corrects the article DOI: 10.1021/acsomega.2c07907.].
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While patients with nonalcoholic fatty liver disease (NAFLD) are at increased risk to develop clinically meaningful cardiovascular diseases (CVD), there are no approved drug designed to target the liver and CVD component of NAFLD. GPBAR1, also known as TGR5, is a G protein coupled receptor for secondary bile acids. In this study we have investigated the effect of GPBAR1 activation by BAR501, a selective GPBAR1 agonist, in Apolipoprotein E deficient (ApoE-/-) mice fed a high fat diet and fructose (Western diet), a validated model of NAFLD-associated atherosclerosis. Using aortic samples from patients who underwent surgery for abdominal aneurism, and ex vivo experiments with endothelial cells and human macrophages, we were able to co-localize the expression of GPBAR1 in CD14+ and PECAM1+ cells. Similar findings were observed in the aortic plaques from ApoE-/- mice. Treating ApoE-/- mice with BAR501, 30 mg/kg for 14 weeks, attenuated the body weight gain while ameliorated the insulin sensitivity by increasing the plasma concentrations of GLP-1 and FGF15. Activation of GPBAR1 reduced the aorta thickness and severity of atherosclerotic lesions and decreased the amount of plaques macrophages. Treating ApoE-/- mice reshaped the aortic transcriptome promoting the expression of anti-inflammatory genes, including IL-10, as also confirmed by tSNE analysis of spleen-derived macrophages. Feeding ApoE-/- mice with BAR501 redirected the bile acid synthesis and the composition of the intestinal microbiota. In conclusion, GPBAR1 agonism attenuates systemic inflammation and improve metabolic profile in a genetic/dietetic model of atherosclerosis. BAR501 might be of utility in the treatment for NAFLD-related CVD.
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Aterosclerosis , Enfermedades Cardiovasculares , Enfermedad del Hígado Graso no Alcohólico , Animales , Humanos , Ratones , Apolipoproteínas E , Aterosclerosis/tratamiento farmacológico , Enfermedades Cardiovasculares/complicaciones , Modelos Animales de Enfermedad , Células Endoteliales , Inflamación/tratamiento farmacológico , Inflamación/complicaciones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/etiología , Receptores Acoplados a Proteínas G/genéticaRESUMEN
BACKGROUND: Patients with nonalcoholic fatty liver disease are at increased risk to develop atherosclerotic cardiovascular diseases. FXR and GPBAR1 are 2 bile acid-activated receptors exploited in the treatment of nonalcoholic fatty liver disease: whether dual GPBAR1/FXR agonists synergize with statins in the treatment of the liver and cardiovascular components of nonalcoholic fatty liver disease is unknown. METHODS AND RESULTS: Investigations of human aortic samples obtained from patients who underwent surgery for aortic aneurysms and Gpbar1-/-, Fxr-/-, and dual Gpbar1-/-Fxr-/- mice demonstrated that GPBAR1 and FXR are expressed in the aortic wall and regulate endothelial cell/macrophage interactions. The expression of GPBAR1 in the human endothelium correlated with the expression of inflammatory biomarkers. Mice lacking Fxr and Gpbar1-/-/Fxr-/- display hypotension and aortic inflammation, along with altered intestinal permeability that deteriorates with age, and severe dysbiosis, along with dysregulated bile acid synthesis. Vasomotor activities of aortic rings were altered by Gpbar1 and Fxr gene ablation. In apolipoprotein E-/- and wild-type mice, BAR502, a dual GPBAR1/FXR agonist, alone or in combination with atorvastatin, reduced cholesterol and low-density lipoprotein plasma levels, mitigated the development of liver steatosis and aortic plaque formation, and shifted the polarization of circulating leukocytes toward an anti-inflammatory phenotype. BAR502/atorvastatin reversed intestinal dysbiosis and dysregulated bile acid synthesis, promoting a shift of bile acid pool composition toward FXR antagonists and GPBAR1 agonists. CONCLUSIONS: FXR and GPBAR1 maintain intestinal, liver, and cardiovascular homeostasis, and their therapeutic targeting with a dual GPBAR1/FXR ligand and atorvastatin holds potential in the treatment of liver and cardiovascular components of nonalcoholic fatty liver disease.
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Ácidos y Sales Biliares , Inhibidores de Hidroximetilglutaril-CoA Reductasas , Enfermedad del Hígado Graso no Alcohólico , Animales , Humanos , Ratones , Atorvastatina/farmacología , Atorvastatina/uso terapéutico , Ácidos y Sales Biliares/metabolismo , Disbiosis/complicaciones , Disbiosis/metabolismo , Proteínas de Unión al GTP/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Hígado/metabolismo , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Compounds featuring a 1,2,4-oxadiazole core have been recently identified as a new chemotype of farnesoid X receptor (FXR) antagonists. With the aim to expand this class of compounds and to understand the building blocks necessary to maintain the antagonistic activity, we describe herein the synthesis, the pharmacological evaluation, and the in vitro pharmacokinetic properties of a novel series of 1,2,4-oxadiazole derivatives decorated on the nitrogen of the piperidine ring with different N-alkyl and N-aryl side chains. In vitro pharmacological evaluation showed compounds 5 and 11 as the first examples of nonsteroidal dual FXR/Pregnane X receptor (PXR) modulators. In HepG2 cells, these compounds modulated PXR- and FXR-regulated genes, resulting in interesting leads in the treatment of inflammatory disorders. Moreover, molecular docking studies supported the experimental results, disclosing the ligand binding mode and allowing rationalization of the activities of compounds 5 and 11.
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Receptores de Esteroides , Receptor X de Pregnano , Receptores de Esteroides/metabolismo , Receptores Citoplasmáticos y Nucleares , Simulación del Acoplamiento Molecular , Biblioteca de GenesRESUMEN
Retinoic acid receptor-related orphan receptor γ-t (RORγt) and GPBAR1, a transmembrane G-protein-coupled receptor for bile acids, are attractive drug targets to develop clinically relevant small modulators as potent therapeutics for autoimmune diseases. Herein, we designed, synthesized, and evaluated several new bile acid-derived ligands with potent dual activity. Furthermore, we performed molecular docking and MD calculations of the best dual modulators in the two targets to identify the binding modes as well as to better understand the molecular basis of the inverse agonism of RORγt by bile acid derivatives. Among these compounds, 7 was identified as a GPBAR1 agonist (EC50 5.9 µM) and RORγt inverse agonist (IC50 0.107 µM), with excellent pharmacokinetic properties. Finally, the most promising ligand displayed robust anti-inflammatory activity in vitro and in vivo in a mouse model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis.
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Non-alcoholic steatosis (NAFLD) and steatohepatitis (NASH) are two highly prevalent human disorders for which therapy remains suboptimal. Bile acids are signaling molecules acting on two main receptors the Farnesoid-x-receptor (FXR) and G protein coupled receptor GPB AR1. Clinical trials have shown that FXR agonism might result in side effects along with lack of efficacy in restoring liver histopathology. For these reasons a multi-targets therapy combined FXR agonists with agent targeting additional molecular mechanisms might have improved efficacy over selective FXR agonists. In the present study we have compared the effects of BAR502, a dual FXR/GPBAR1 ligand) alone or in combination with ursodeoxycholic acid (UDCA) in a model of NAFLD/NASH induced by feeding mice with a Western diet for 10 weeks. The results demonstrated that while BAR502 and UDCA partially protected against liver damage caused by Western diet, the combination of the two, reversed the pro-atherogenic lipid profile and completely reversed the histopathology damage, attenuating liver steatosis, ballooning, inflammation and fibrosis. Additionally, while both agents increased insulin sensitivity and bile acid signaling, the combination of the two, modulated up top 85 genes in comparison of mice feed a Western diet, strongly reducing expression of inflammatory markers such as chemokines and cytokines. Additionally, the combination of the two agents redirected the bile acid metabolism toward bile acid species that are GPBAR1 agonist while reduced liver bile acid content and increased fecal excretion. Together, these data, highlight the potential role for a combinatorial therapy based on BAR502 and UDCA in treating of NAFLD.
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Enfermedad del Hígado Graso no Alcohólico , Ácido Ursodesoxicólico , Animales , Ratones , Ácidos y Sales Biliares/metabolismo , Hígado/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Ácido Ursodesoxicólico/farmacologíaRESUMEN
Inflammatory bowel disease (IBD) is a chronic and relapsing disease caused by a dysregulated immune response to host intestinal microbiota that occurs in genetically predisposed individuals. IBD encompasses two major clinical entities: ulcerative colitis (UC), limited to the colonic mucosa, and Crohn's disease (CD), which might affect any segment of the gastrointestinal tract. Despite the prevalence of IBD increasing worldwide, therapy remains suboptimal, largely because of the variability of causative mechanisms, raising the need to develop individualized therapeutic approaches targeted to each individual patient. In this context, patients-derived intestinal organoids represent an effective tool for advancing our understanding of IBD's pathogenesis. Organoid 3D culture systems offer a unique model for dissecting epithelial mechanisms involved IBDs and testing individualized therapy, although the lack of a functional immune system and a microbiota, two driving components of the IBD pathogenesis, represent a major barrier to their exploitation in clinical medicine. In this review, we have examined how to improve the translational utility of intestinal organoids in IBD and how co-cultures of 3D or 2D organoids and immune cells and/or intestinal microbiota might help to overcome these limitations.
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Colitis Ulcerosa , Enfermedad de Crohn , Enfermedades Inflamatorias del Intestino , Humanos , Intestinos/patología , Organoides/patologíaRESUMEN
BACKGROUND AND AIM: Drug-induced liver injury (DILI) is a common disorder that involves both direct liver cell toxicity and immune activation. The bile acid receptor, G-protein-coupled bile acid receptor 1 (GPBAR1; Takeda G-protein-coupled receptor 5 [TGR5]), and cysteinyl leukotriene receptor (CYSLTR) 1 are G-protein-coupled receptors activated by bile acids and leukotrienes, exerting opposite effects on cell-to-cell adhesion, inflammation, and immune cell activation. To investigate whether GPBAR1 and CYSLTR1 mutually interact in the development of DILI, we developed an orally active small molecule, CHIN117, that functions as a GPBAR1 agonist and CYSLTR1 antagonist. APPROACH AND RESULTS: RNA-sequencing analysis of liver explants showed that acetaminophen (APAP) intoxication positively modulates the leukotriene pathway, CYSLTR1, 5-lipoxygenase, and 5-lipoxygenase activating protein, whereas GPBAR1 gene expression was unchanged. In mice, acute liver injury induced by orally dosing APAP (500 mg/kg) was severely exacerbated by Gpbar1 gene ablation and attenuated by anti-Cysltr1 small interfering RNA pretreatment. Therapeutic dosing of wild-type mice with CHIN117 reversed the liver damage caused by APAP and modulated up to 1300 genes, including 38 chemokines and receptors, that were not shared by dosing mice with a selective GPBAR1 agonist or CYSLTR1 antagonist. Coexpression of the two receptors was detected in liver sinusoidal endothelial cells (LSECs), monocytes, and Kupffer cells, whereas combinatorial modulation of CYSLTR1 and GPBAR1 potently reversed LSEC/monocyte interactions. CHIN117 reversed liver damage and liver fibrosis in mice administered CCl 4 . CONCLUSIONS: By genetic and pharmacological approaches, we demonstrated that GPBAR1 and CYSLTR1 mutually interact in the development of DILI. A combinatorial approach designed to activate GPBAR1 while inhibiting CYSLTR1 reverses liver injury in models of DILI.
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Enfermedad Hepática Inducida por Sustancias y Drogas , Hepatopatías , Ratones , Animales , Ácidos y Sales Biliares/metabolismo , Araquidonato 5-Lipooxigenasa/metabolismo , Células Endoteliales/metabolismo , Acetaminofén/toxicidad , Receptores Acoplados a Proteínas G/metabolismo , Hepatopatías/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Leucotrienos/metabolismo , Proteínas de Unión al GTP/metabolismoRESUMEN
Pancreatic cancer is a leading cause of cancer mortality and is projected to become the second-most common cause of cancer mortality in the next decade. While gene-wide association studies and next generation sequencing analyses have identified molecular patterns and transcriptome profiles with prognostic relevance, therapeutic opportunities remain limited. Among the genes that are upregulated in pancreatic ductal adenocarcinomas (PDAC), the leukaemia inhibitory factor (LIF), a cytokine belonging to IL-6 family, has emerged as potential therapeutic candidate. LIF is aberrantly secreted by tumour cells and promotes tumour progression in pancreatic and other solid tumours through aberrant activation of the LIF receptor (LIFR) and downstream signalling that involves the JAK1/STAT3 pathway. Since there are no LIFR antagonists available for clinical use, we developed an in silico strategy to identify potential LIFR antagonists and drug repositioning with regard to LIFR antagonists. The results of these studies allowed the identification of mifepristone, a progesterone/glucocorticoid antagonist, clinically used in medical abortion, as a potent LIFR antagonist. Computational studies revealed that mifepristone binding partially overlapped the LIFR binding site. LIF and LIFR are expressed by human PDAC tissues and PDAC cell lines, including MIA-PaCa-2 and PANC-1 cells. Exposure of these cell lines to mifepristone reverses cell proliferation, migration and epithelial mesenchymal transition induced by LIF in a concentration-dependent manner. Mifepristone inhibits LIFR signalling and reverses STAT3 phosphorylation induced by LIF. Together, these data support the repositioning of mifepristone as a potential therapeutic agent in the treatment of PDAC.
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Adenocarcinoma , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Embarazo , Femenino , Humanos , Receptores OSM-LIF/genética , Mifepristona/farmacología , Mifepristona/uso terapéutico , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Reposicionamiento de Medicamentos , Carcinoma Ductal Pancreático/patología , Antagonistas de Hormonas/farmacología , Neoplasias PancreáticasRESUMEN
Non-alcoholic steatosis (NAFLD) and steatohepatitis (NASH) are two highly prevalent human disorders for which therapy remains suboptimal. Bile acids play an essential role in regulating liver metabolism, and several bile acids-based therapy are currently investigated for their potential therapeutic efficacy in NAFLD/NASH. Bile acids exert their functions, at least in part, by modulating two main receptors the Farnesoid-x-receptor (FXR) and the G protein-coupled receptor, GPBAR1. In the present study we have compared the pharmacological effects of two bile acids, the ursodeoxycholic acid (UDCA) and its derivative norUDCA, in a model of NAFLD/NASH induced by feeding mice with a Western diet for 12 weeks. The results of these studies demonstrated that both UDCA and norUDCA protected against development of steatosis and fibrosis, but did not reduce the hepatocytes ballooning nor the development of a pro-atherogenic lipid profile. Both agents reduced liver lipogenesis and ameliorated insulin sensitivity and adipocytes signaling as shown by increased expression of adiponectin. Mechanistically, UDCA acts as weak GPBAR1 agonist, while norUDCA exerted no effect on both GPBAR1 and FXR. In vivo administration of UDCA resets bile acid synthesis and promotes a shift toward bile acids species that are GPBAR1 agonists, UDCA, TUDCA and hyodeoxycholic acid, and increases GLP1 expression in the ileum. In contrast norUDCA is poorly metabolized exerting a minimal impact on GPBAR1 signaling. Together, these data, highlight the potential role of UDCA and norUDCA in treating of NAFLD, though these beneficial effects are supported by different mechanisms.
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Enfermedad del Hígado Graso no Alcohólico , Ácido Ursodesoxicólico , Animales , Ácidos y Sales Biliares , Humanos , Ratones , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Receptores Acoplados a Proteínas G , Roedores , Ácido Ursodesoxicólico/farmacología , Ácido Ursodesoxicólico/uso terapéuticoRESUMEN
Gastric cancer (GC) is the third cause of cancer-related mortality worldwide. Nevertheless, because GC screening programs are not cost-effective, most patients receive diagnosis in the advanced stages, when surgical options are limited. Peritoneal dissemination occurs in approximately one-third of patients with GC at the diagnosis and is a strong predictor of poor outcome. Despite the clinical relevance, biological and molecular mechanisms underlying the development of peritoneal metastasis in GC remain poorly defined. Here, we report results of a high-throughput sequencing of transcriptome expression in paired samples of non-neoplastic and neoplastic gastric samples from 31 patients with GC with or without peritoneal carcinomatosis. The RNA-seq analysis led to the discovery of a group of highly upregulated or downregulated genes, including the leukemia inhibitory factor receptor (LIFR) and one cut domain family member 2 (ONECUT2) that were differentially modulated in patients with peritoneal disease in comparison with patients without peritoneal involvement. Both LIFR and ONECUT2 predicted survival at univariate statistical analysis. LIFR and its major ligand LIF belong to the interleukin-6 (IL-6) cytokine family and have a central role in immune system regulation, carcinogenesis, and dissemination in several human cancers. To confirm the mechanistic role of the LIF/LIFR pathway in promoting GC progression, GC cell lines were challenged in vitro with LIF and a LIFR inhibitor. Among several GC cell lines, MKN45 cells displayed the higher expression of the receptor, and their exposure to LIF promotes a concentration-dependent proliferation and epithelial-mesenchymal transition (EMT), as shown by modulation of relative expression of E-cadherin/vimentin along with JAK and STAT3 phosphorylation and acquisition of a migratory phenotype. Furthermore, exposure to LIF promoted the adhesion of MKN45 cells to the peritoneum in an ex vivo assay. These effects were reversed by the pharmacological blockade of LIFR signaling. Together, these data suggest that LIFR might have a major role in promoting disease progression and peritoneal dissemination in patients with GC and that development of LIF/LIFR inhibitors might have a role in the treatment of GC.
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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.
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BACKGROUND & AIMS: ACE2, a carboxypeptidase that generates Ang-(1-7) from Ang II, is highly expressed in the lung, small intestine and colon. GPBAR1, is a G protein bile acid receptor that promotes the release of the insulinotropic factor glucagon-like peptide (GLP)-1 and attenuates intestinal inflammation. METHODS: We investigated the expression of ACE2, GLP-1 and GPBAR1 in two cohorts of Crohn's disease (CD) patients and three mouse models of colitis and Gpbar1-/- mice. Activation of GPBAR1 in these models and in vitro was achieved by BAR501, a selective GPBAR1 agonist. RESULTS: In IBD patients, ACE2 mRNA expression was regulated in a site-specific manner in response to inflammation. While expression of ileal ACE2 mRNA was reduced, the colon expression was induced. Colon expression of ACE2 mRNA in IBD correlated with expression of TNF-α and GPBAR1. A positive correlation occurred between GCG and GPBAR1 in human samples and animal models of colitis. In these models, ACE2 mRNA expression was further upregulated by GPABR1 agonism and reversed by exendin-3, a GLP-1 receptor antagonist. In in vitro studies, liraglutide, a GLP-1 analogue, increased the expression of ACE2 in colon epithelial cells/macrophages co-cultures. CONCLUSIONS: ACE2 mRNA expression in the colon of IBD patients and rodent models of colitis is regulated in a TNF-α- and GLP-1-dependent manner. We have identified a GPBAR1/GLP-1 mechanism as a positive modulator of ACE2.
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Enzima Convertidora de Angiotensina 2 , Colitis , Enfermedad de Crohn , Péptido 1 Similar al Glucagón , Receptores Acoplados a Proteínas G , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Ácidos y Sales Biliares , Péptido 1 Similar al Glucagón/metabolismo , Humanos , Inflamación , Ratones , ARN Mensajero/genética , Receptores Acoplados a Proteínas G/metabolismo , Factor de Necrosis Tumoral alfaRESUMEN
Advancements in stem cell research have enabled the establishment of three-dimensional (3D) primary cell cultures, known as organoids. These culture systems follow the organization of an in vivo organ, as they enclose the different epithelial cell lines of which it is normally composed. Generation of these 3D cultures has bridged the gap between in vitro models, made up by two-dimensional (2D) cancer cell lines cultures, and in vivo animal models, that have major differences with human diseases. Organoids are increasingly used as a model to study colonization of gastric mucosa by infectious agents and to better understand host-microbe interactions and the molecular events that lead to infection, pathogen-epithelial cells interactions and mechanisms of gastric mucosal injury. In this review we will focus on the role of organoids as a tool to investigate molecular interactions of Helicobacter (H.) pylori and Epstein Barr Virus (EBV) and gastric mucosa and how these infections, that affect ≈ 45% of the world population, might progress to gastric cancer, a highly prevalent cancer and the third leading cause of cancer death.
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Farnesoid-x-receptor (FXR) agonists, currently trialed in patients with non-alcoholic steatosis (NAFLD), worsen the pro-atherogenic lipid profile and might require a comedication with statin. Here we report that mice feed a high fat/high cholesterol diet (HFD) are protected from developing a pro-atherogenic lipid profile because their ability to dispose cholesterol through bile acids. This protective mechanism is mediated by suppression of FXR signaling in the liver by muricholic acids (MCAs) generated in mice from chenodeoxycholic acid (CDCA). In contrast to CDCA, MCAs are FXR antagonists and promote a CYP7A1-dependent increase of bile acids synthesis. In mice feed a HFD, the treatment with obeticholic acid, a clinical stage FXR agonist, failed to improve the liver histopathology while reduced Cyp7a1 and Cyp8b1 genes expression and bile acids synthesis and excretion. In contrast, treating mice with atorvastatin mitigated liver and vascular injury caused by the HFD while increased the bile acids synthesis and excretion. Atorvastatin increased the percentage of 7α-dehydroxylase expressing bacteria in the intestine promoting the formation of deoxycholic acid and litocholic acid, two GPBAR1 agonists, along with the expression of GPBAR1-regulated genes in the white adipose tissue and colon. In conclusion, present results highlight the central role of bile acids in regulating lipid and cholesterol metabolism in response to atorvastatin and provide explanations for limited efficacy of FXR agonists in the treatment of NAFLD.
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Atorvastatina/farmacología , Hígado Graso/tratamiento farmacológico , Hígado/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacos , Enfermedades Vasculares/tratamiento farmacológico , Animales , Bacterias/metabolismo , Ácidos y Sales Biliares/metabolismo , Colesterol 7-alfa-Hidroxilasa/metabolismo , Colesterol en la Dieta/efectos adversos , Colesterol en la Dieta/farmacología , Hígado Graso/inducido químicamente , Hígado Graso/metabolismo , Hígado Graso/microbiología , Microbioma Gastrointestinal/efectos de los fármacos , Masculino , Ratones , Esteroide 12-alfa-Hidroxilasa/metabolismo , Enfermedades Vasculares/inducido químicamente , Enfermedades Vasculares/metabolismo , Enfermedades Vasculares/microbiologíaRESUMEN
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 REV5901âthe first reported dual compoundâwith 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.
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Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Receptores Acoplados a Proteínas G/efectos de los fármacos , Receptores de Leucotrienos/efectos de los fármacos , Animales , Colitis/tratamiento farmacológico , Humanos , Leucotrieno D4/farmacología , Macrófagos/efectos de los fármacos , Ratones , Simulación del Acoplamiento Molecular , Unión Proteica , Células RAW 264.7 , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Leucotrienos/metabolismo , Relación Estructura-ActividadRESUMEN
Nonnutritive sweeteners (NNSs) are widely employed as dietary substitutes for classical sugars thanks to their safety profile and low toxicity. In this study, a re-evaluation of the biological effects of steviol (1), the main metabolite from Stevia rebaudiana glycosides, was performed using the Inverse Virtual Screening (IVS) target fishing computational approach. Starting from well-known pharmacological properties of Stevia rebaudiana glycosides, this computational tool was employed for predicting the putative interacting targets of 1 and, afterwards, of its five synthetic ester derivatives 2-6, accounting a large panel of proteins involved in cancer and inflammation events. Applying this methodology, the farnesoid X receptor (FXR) was identified as the putative target partner of 1-6. The predicted ligand-protein interactions were corroborated by transactivation assays, specifically disclosing the agonistic activity of 1 and the antagonistic activities of 2-6 on FXR. The reported results highlight the feasibility of IVS as a fast and potent tool for predicting the interacting targets of query compounds, addressing the re-evaluation of their bioactivity. In light of the obtained results, the presumably safe profile of known compounds, such as the case of steviol (1), is critically discussed.
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Productos Biológicos/farmacología , Diterpenos de Tipo Kaurano/farmacología , Glicósidos/farmacología , Receptores Citoplasmáticos y Nucleares/agonistas , Stevia/química , Productos Biológicos/química , Productos Biológicos/aislamiento & purificación , Diterpenos de Tipo Kaurano/química , Diterpenos de Tipo Kaurano/aislamiento & purificación , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Glicósidos/química , Glicósidos/aislamiento & purificación , Células Hep G2 , Humanos , Conformación Molecular , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
The severe acute respiratory syndrome (SARS)-CoV-2 is the pathogenetic agent of Corona Virus Induced Disease (COVID)19. The virus enters the human cells after binding to the angiotensin converting enzyme (ACE)2 receptor in target tissues. ACE2 expression is induced in response to inflammation. The colon expression of ACE2 is upregulated in patients with inflammatory bowel disease (IBD), highlighting a potential risk of intestinal inflammation in promoting viral entry in the human body. Because mechanisms that regulate ACE2 expression in the intestine are poorly understood and there is a need of anti-SARS-CoV-2 therapies, we have settled to investigate whether natural flavonoids might regulate the expression of Ace2 in intestinal models of inflammation. The results of these studies demonstrated that pelargonidin activates the Aryl hydrocarbon Receptor (AHR) in vitro and reverses intestinal inflammation caused by chronic exposure to high fat diet or to the intestinal braking-barrier agent TNBS in a AhR-dependent manner. In these two models, development of colon inflammation associated with upregulation of Ace2 mRNA expression. Colon levels of Ace2 mRNA were directly correlated with Tnf-α mRNA levels. Molecular docking studies suggested that pelargonidin binds a fatty acid binding pocket on the receptor binding domain of SARS-CoV-2 Spike protein. In vitro studies demonstrated that pelargonidin significantly reduces the binding of SARS-CoV-2 Spike protein to ACE2 and reduces the SARS-CoV-2 replication in a concentration-dependent manner. In summary, we have provided evidence that a natural flavonoid might hold potential in reducing intestinal inflammation and ACE2 induction in the inflamed colon in a AhR-dependent manner.
Asunto(s)
Enzima Convertidora de Angiotensina 2/biosíntesis , Antocianinas/farmacología , Descubrimiento de Drogas/métodos , Regulación Enzimológica de la Expresión Génica , Receptores de Hidrocarburo de Aril/agonistas , SARS-CoV-2/efectos de los fármacos , Enzima Convertidora de Angiotensina 2/antagonistas & inhibidores , Enzima Convertidora de Angiotensina 2/genética , Animales , Antocianinas/química , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Células Hep G2 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Hidrocarburo de Aril/metabolismo , SARS-CoV-2/metabolismo , Células VeroRESUMEN
Autophagy is a highly conserved catabolic process activated by fasting and caloric restriction. FXR, a receptor for primary bile acids, reverses the activity of cAMP-response element binding protein (CREB) on autophagy-related genes (Atg)s and terminates autophagy in the fed state. GPBAR1, a receptor for secondary bile acids, exerts its genomic effects via cAMP-CREB pathway. By genetic and pharmacological approaches, we have obtained evidence that GPBAR1 functions as a positive modulator of autophagy in liver and white adipose tissue (WAT) in fasting. Mechanistically, we found that Gpbar1-/- mice lack the expression of Cyp2c70 a gene essential for generation of muricholic acids which are FXR antagonists, and have an FXR-biased bile acid pool. Because FXR represses autophagy, Gpbar1-/- mice show a defective regulation of autophagy in fasting. BAR501, a selective GPBAR1 agonist, induces autophagy in fed mice. Defective regulation of autophagy in Gpbar1-/- could be reversed by FXR antagonism, while repression of autophagy by feeding was partially abrogated by FXR gene ablation, and FXR activation repressed Atgs in the fast state. BAR501 reversed the negative regulatory effects of feeding and FXR agonism on autophagy and promoted the recruitment of CREB to a CRE on the LC3 promoter. In mice exposed to chronic high caloric intake, GPBAR1 agonism ameliorated insulin sensitivity and induced Atgs expression in the liver and WAT. In summary, GPBAR1 is required for positive regulation of autophagy in fasting and its ligands reverse the repressive effects exerted on liver and WAT autophagy flow by FXR in fed.