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Inflammatory bowel diseases (IBD), including Crohn's disease and ulcerative colitis, are chronic disorders characterized by dysregulated immune response and persistent inflammation. Recent studies suggest that bile acid receptors, particularly GPBAR1, and the transcription factor RORγt play critical roles in modulating intestinal inflammation. This study evaluates the therapeutic potential of PBT002, a dual GPBAR1 agonist and RORγt inverse agonist, in IBD models. The effects of PBT002 were assessed through in vitro and in vivo experiments. Macrophages and T lymphocytes obtained from the buffy coat were exposed to PBT002 to evaluate its immunomodulatory activity. The beneficial effects in vivo were evaluated in mouse models of colitis induced by TNBS, DSS or DSS + IL-23 using also a Gpbar1 knock-out male mice. PBT002 exhibited an EC50 of 1.2 µM for GPBAR1 and an IC50 of 2.8 µM for RORγt. In in vitro, PBT002 modulated macrophage polarization towards an anti-inflammatory M2 phenotype and reduced Th17 cell markers while increasing Treg markers. In the TNBS-induced colitis model, PBT002 reduced weight loss, CDAI, and colon damage, while it modulated cytokine gene expression towards an anti-inflammatory profile. In GPBAR1-/-, the anti-inflammatory effects of PBT002 were attenuated, indicating partial GPBAR1 dependence. RNA sequencing revealed significant modulation of inflammatory pathways by PBT002. In DSS+IL-23 induced colitis, PBT002 mitigated disease exacerbation, reducing pro-inflammatory cytokine levels and immune cell infiltration. In conclusion, PBT002, a GPBAR1 agonist and RORγt inverse agonist, modulates both the innate and adaptive immune responses to reduce inflammation and disease severity in models of IBD.

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Bile acids are steroids formed at the interface of host metabolism and intestinal microbiota. While primary bile acids are generated in the liver from cholesterol metabolism, secondary bile acids represent the products of microbial enzymes. Close to 100 different enzymatic modifications of bile acids structures occur in the human intestine and clinically guided metagenomic and metabolomic analyses have led to the identification of an extraordinary number of novel metabolites. These chemical mediators make an essential contribution to the composition and function of the postbiota, participating to the bidirectional communications of the intestinal microbiota with the host and contributing to the architecture of intestinal-liver and -brain and -endocrine axes. Bile acids exert their function by binding to a group of cell membrane and nuclear receptors collectively known as bile acid-regulated receptors (BARRs), expressed in monocytes, tissue-resident macrophages, CD4+ T effector cells, including Th17, T regulatory cells, dendritic cells and type 3 of intestinal lymphoid cells and NKT cells, highlighting their role in immune regulation. In this review we report on how bile acids and their metabolitesmodulate the immune system in inflammations and cancers and could be exploiting for developing novel therapeutic approaches in these disorders.

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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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Many growth factors and cytokines signal by binding to the extracellular domains of their receptors and driving association and transphosphorylation of the receptor intracellular tyrosine kinase domains, initiating downstream signaling cascades. To enable systematic exploration of how receptor valency and geometry affect signaling outcomes, we designed cyclic homo-oligomers with up to 8 subunits using repeat protein building blocks that can be modularly extended. By incorporating a de novo-designed fibroblast growth factor receptor (FGFR)-binding module into these scaffolds, we generated a series of synthetic signaling ligands that exhibit potent valency- and geometry-dependent Ca2+ release and mitogen-activated protein kinase (MAPK) pathway activation. The high specificity of the designed agonists reveals distinct roles for two FGFR splice variants in driving arterial endothelium and perivascular cell fates during early vascular development. Our designed modular assemblies should be broadly useful for unraveling the complexities of signaling in key developmental transitions and for developing future therapeutic applications.

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The leukemia inhibitory factor (LIF) is member of interleukin (IL)-6 family of cytokines involved immune regulation, morphogenesis and oncogenesis. In cancer tissues, LIF binds a heterodimeric receptor (LIFR), formed by a LIFRß subunit and glycoprotein(gp)130, promoting epithelial mesenchymal transition and cell growth. Bile acids are cholesterol metabolites generated at the interface of host metabolism and the intestinal microbiota. Here we demonstrated that bile acids serve as endogenous antagonist to LIFR in oncogenesis. The tissue characterization of bile acids content in non-cancer and cancer biopsy pairs from gastric adenocarcinomas (GC) demonstrated that bile acids accumulate within cancer tissues, with glyco-deoxycholic acid (GDCA) functioning as negative regulator of LIFR expression. In patient-derived organoids (hPDOs) from GC patients, GDCA reverses LIF-induced stemness and proliferation. In summary, we have identified the secondary bile acids as the first endogenous antagonist to LIFR supporting a development of bile acid-based therapies in LIF-mediated oncogenesis.

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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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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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PURPOSE: The gastric adenocarcinoma (GC) represents the third cause of cancer-related mortality worldwide, and available therapeutic options remain sub-optimal. The Fibroblast growth factor receptors (FGFRs) are oncogenic transmembrane tyrosine kinase receptors. FGFR inhibitors have been approved for the treatment of various cancers and a STAT3-dependent regulation of FGFR4 has been documented in the H.pylori infected intestinal GC. Therefore, the modulation of FGFR4 might be useful for the treatment of GC. METHODS: To investigate wich factors could modulate FGFR4 signalling in GC, we employed RNA-seq analysis on GC patients biopsies, human patients derived organoids (PDOs) and cancer cell lines. RESULTS: We report that FGFR4 expression/function is regulated by the leukemia inhibitory factor (LIF) an IL-6 related oncogenic cytokine, in JAK1/STAT3 dependent manner. The transcriptomic analysis revealed a direct correlation between the expression of LIFR and FGFR4 in the tissue of an exploratory cohort of 31 GC and confirmed these findings by two external validation cohorts of GC. A LIFR inhibitor (LIR-201) abrogates STAT3 phosphorylation induced by LIF as well as recruitment of pSTAT3 to the promoter of FGFR4. Furthermore, inhibition of FGFR4 by roblitinib or siRNA abrogates STAT3 phosphorylation and oncogentic effects of LIF in GC cells, indicating that FGFR4 is a downstream target of LIF/LIFR complex. Treating cells with LIR-201 abrogates oncogenic potential of FGF19, the physiological ligand of FGFR4. CONCLUSIONS: Together these data unreveal a previously unregnized regulatory mechanism of FGFR4 by LIF/LIFR and demonstrate that LIF and FGF19 converge on the regulation of oncogenic STAT3 in GC cells.

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After myocardial infarction (MI), a significant portion of heart muscle is replaced with scar tissue, progressively leading to heart failure. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CM) offer a promising option for improving cardiac function after MI. However, hPSC-CM transplantation can lead to engraftment arrhythmia (EA). EA is a transient phenomenon arising shortly after transplantation then spontaneously resolving after a few weeks. The underlying mechanism of EA is unknown. We hypothesize that EA may be explained partially by time-varying, spatially heterogeneous, graft-host electrical coupling. Here, we created computational slice models derived from histological images that reflect different configuration of grafts in the infarcted ventricle. We ran simulations with varying degrees of connection imposed upon the graft-host perimeter to assess how heterogeneous electrical coupling affected EA with non-conductive scar, slow-conducting scar and scar replaced by host myocardium. We also quantified the effect of variation in intrinsic graft conductivity. Susceptibility to EA initially increased and subsequently decreased with increasing graft-host coupling, suggesting the waxing and waning of EA is regulated by progressive increases in graft-host coupling. Different spatial distributions of graft, host and scar yielded markedly different susceptibility curves. Computationally replacing non-conductive scar with host myocardium or slow-conducting scar, and increasing intrinsic graft conductivity both demonstrated potential means to blunt EA vulnerability. These data show how graft location, especially relative to scar, along with its dynamic electrical coupling to host, can influence EA burden; moreover, they offer a rational base for further studies aimed to define the optimal delivery of hPSC-CM injection. KEY POINTS: Human pluripotent stem cell-derived cardiomyocytes (hPSC-CM) hold great cardiac regenerative potential but can also cause engraftment arrhythmias (EA). Spatiotemporal evolution in the pattern of electrical coupling between injected hPSC-CMs and surrounding host myocardium may explain the dynamics of EA observed in large animal models. We conducted simulations in histology-derived 2D slice computational models to assess the effects of heterogeneous graft-host electrical coupling on EA propensity, with or without scar tissue. Our findings suggest spatiotemporally heterogeneous graft-host coupling can create an electrophysiological milieu that favours graft-initiated host excitation, a surrogate metric of EA susceptibility. Removing scar from our models reduced but did not abolish the propensity for this phenomenon. Conversely, reduced intra-graft electrical connectedness increased the incidence of graft-initiated host excitation. The computational framework created for this study can be used to generate new hypotheses, targeted delivery of hPSC-CMs.

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Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) offer a promising cell-based therapy for myocardial infarction. However, the presence of transitory ventricular arrhythmias, termed engraftment arrhythmias (EAs), hampers clinical applications. We hypothesized that EA results from pacemaker-like activity of hPSC-CMs associated with their developmental immaturity. We characterized ion channel expression patterns during maturation of transplanted hPSC-CMs and used pharmacology and genome editing to identify those responsible for automaticity in vitro. Multiple engineered cell lines were then transplanted in vivo into uninjured porcine hearts. Abolishing depolarization-associated genes HCN4, CACNA1H, and SLC8A1, along with overexpressing hyperpolarization-associated KCNJ2, creates hPSC-CMs that lack automaticity but contract when externally stimulated. When transplanted in vivo, these cells engrafted and coupled electromechanically with host cardiomyocytes without causing sustained EAs. This study supports the hypothesis that the immature electrophysiological prolife of hPSC-CMs mechanistically underlies EA. Thus, targeting automaticity should improve the safety profile of hPSC-CMs for cardiac remuscularization.

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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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Ischemic heart failure is due to irreversible loss of cardiomyocytes. Preclinical studies showed that human pluripotent stem cell (hPSC)-derived cardiomyocytes could remuscularize infarcted hearts and improve cardiac function. However, these cardiomyocytes remained immature. Incorporating hPSC-derived epicardial cells has been shown to improve cardiomyocyte maturation, but the exact mechanisms are unknown. We posited epicardial fibronectin (FN1) as a mediator of epicardial-cardiomyocyte crosstalk and assessed its role in driving hPSC-derived cardiomyocyte maturation in 3D-engineered heart tissues (3D-EHTs). We found that the loss of FN1 with peptide inhibition F(pUR4), CRISPR-Cas9-mediated FN1 knockout, or tetracycline-inducible FN1 knockdown in 3D-EHTs resulted in immature cardiomyocytes with decreased contractile function, and inefficient Ca2+ handling. Conversely, when we supplemented 3D-EHTs with recombinant human FN1, we could recover hPSC-derived cardiomyocyte maturation. Finally, our RNA-sequencing analyses found FN1 within a wider paracrine network of epicardial-cardiomyocyte crosstalk, thus solidifying FN1 as a key driver of hPSC-derived cardiomyocyte maturation in 3D-EHTs.

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Introduction: The leukemia inhibitory factor (LIF), is a cytokine belonging to IL-6 family, whose overexpression correlate with poor prognosis in cancer patients, including pancreatic ductal adenocarcinoma (PDAC). LIF signaling is mediate by its binding to the heterodimeric LIF receptor (LIFR) complex formed by the LIFR receptor and Gp130, leading to JAK1/STAT3 activation. Bile acids are steroid that modulates the expression/activity of membrane and nuclear receptors, including the Farnesoid-X-Receptor (FXR) and G Protein Bile Acid Activated Receptor (GPBAR1). Methods: Herein we have investigated whether ligands to FXR and GPBAR1 modulate LIF/LIFR pathway in PDAC cells and whether these receptors are expressed in human neoplastic tissues. Results: The transcriptome analysis of a cohort of PDCA patients revealed that expression of LIF and LIFR is increased in the neoplastic tissue in comparison to paired non-neoplastic tissues. By in vitro assay we found that both primary and secondary bile acids exert a weak antagonistic effect on LIF/LIFR signaling. In contrast, BAR502 a non-bile acid steroidal dual FXR and GPBAR1 ligand, potently inhibits binding of LIF to LIFR with an IC50 of 3.8 µM. Discussion: BAR502 reverses the pattern LIF-induced in a FXR and GPBAR1 independent manner, suggesting a potential role for BAR502 in the treatment of LIFR overexpressing-PDAC.

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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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BACKGROUND AND PURPOSE: Transient receptor potential melastatin type-8 (TRPM8) is a cold-sensitive cation channel protein belonging to the TRP superfamily of ion channels. Here, we reveal the molecular mechanism of TRPM8 and its clinical relevance in colorectal cancer (CRC). EXPERIMENTAL APPROACH: TRPM8 expression and its correlation with the survival rate of CRC patients was analysed. To identify the key pathways and genes related to TRPM8 high expression, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were conducted in CRC patients. TRPM8 functional role was assessed by using Trpm8-/- mice in models of sporadic and colitis-associated colon cancer. TRPM8 pharmacological targeting by WS12 was evaluated in murine models of CRC. KEY RESULTS: TRPM8 is overexpressed in colon primary tumours and in CD326+ tumour cell fraction. TRPM8 high expression was related to lower survival rate of CRC patients, Wnt-Frizzled signalling hyperactivation and adenomatous polyposis coli down-regulation. In sporadic and colitis-associated models of colon cancer, either absence or pharmacological desensitization of TRPM8 reduced tumour development via inhibition of the oncogenic Wnt/ß-catenin signalling. TRPM8 pharmacological blockade reduced tumour growth in CRC xenograft mice by reducing the transcription of Wnt signalling regulators and the activation of ß-catenin and its target oncogenes such as C-Myc and Cyclin D1. CONCLUSION AND IMPLICATIONS: Human data provide valuable insights to propose TRPM8 as a prognostic marker with a negative predictive value for CRC patient survival. Animal experiments demonstrate TRPM8 involvement in colon cancer pathophysiology and its potential as a drug target for CRC.

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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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