Bile acids serve as endogenous antagonists of the Leukemia inhibitory factor (LIF) receptor in oncogenesis.

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.

Correction to "Discovery of a Novel Class of Dual GPBAR1 Agonists-RORγt Inverse Agonists for the Treatment of IL-17-Mediated Disorders".

[This corrects the article DOI: 10.1021/acsomega.2c07907.].

Expanding the Library of 1,2,4-Oxadiazole Derivatives: Discovery of New Farnesoid X Receptor (FXR) Antagonists/Pregnane X Receptor (PXR) Agonists.

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.

Discovery of a Novel Class of Dual GPBAR1 Agonists-RORγt Inverse Agonists for the Treatment of IL-17-Mediated Disorders.

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.

Phytochemical Analysis of the Methanolic Extract and Essential Oil from Leaves of Industrial Hemp Futura 75 Cultivar: Isolation of a New Cannabinoid Derivative and Biological Profile Using Computational Approaches.

Cannabis sativa L. is a plant belonging to the Cannabaceae family, cultivated for its psychoactive cannabinoid (Δ9-THC) concentration or for its fiber and nutrient content in industrial use. Industrial hemp shows a low Δ9-THC level and is a valuable source of phytochemicals, mainly represented by cannabinoids, flavones, terpenes, and alkaloids, with health-promoting effects. In the present study, we investigated the phytochemical composition of leaves of the industrial hemp cultivar Futura 75, a monoecious cultivar commercially used for food preparations or cosmetic purposes. Leaves are generally discarded, and represent waste products. We analyzed the methanol extract of Futura 75 leaves by HPLC and NMR spectroscopy and the essential oil by GC-MS. In addition, in order to compare the chemical constituents, we prepared the water infusion. One new cannabinoid derivative (1) and seven known components, namely, cannabidiol (2), cannabidiolic acid (3), ß-cannabispirol (4), ß-cannabispirol (5), canniprene (6), cannabiripsol (7), and cannflavin B (8) were identified. The content of CBD was highest in all preparations. In addition, we present the outcomes of a computational study focused on elucidating the role of 2α-hydroxy-Δ3,7-cannabitriol (1), CBD (2), and CBDA (3) in inflammation and thrombogenesis.

Biological Profile of Two Gentiana lutea L. Metabolites Using Computational Approaches and In Vitro Tests.

Natural products have been the main source of bioactive molecules for centuries. We tested the biological profile of two metabolites extracted from Gentiana lutea L. by means of computational techniques and in vitro assays. The two molecules (loganic acid and gentiopicroside) were tested in silico using an innovative technique, named Inverse Virtual Screening (IVS), to highlight putative partners among a panel of proteins involved in inflammation and cancer events. A positive binding with cyclooxygenase-2 (COX-2), alpha-1-antichymotrypsin, and alpha-1-acid glycoprotein emerged from the computational experiments and the outcomes from the promising interaction with COX-2 were confirmed by Western blot, highlighting the reliability of IVS in the field of the natural products.

Hijacking SARS-CoV-2/ACE2 Receptor Interaction by Natural and Semi-synthetic Steroidal Agents Acting on Functional Pockets on the Receptor Binding Domain.

The coronavirus disease 2019 (COVID-19) is a respiratory tract infection caused by the severe acute respiratory syndrome coronavirus (SARS)-CoV-2. In light of the urgent need to identify novel approaches to be used in the emergency phase, we have embarked on an exploratory campaign aimed at repurposing natural substances and clinically available drugs as potential anti-SARS-CoV2-2 agents by targeting viral proteins. Here we report on a strategy based on the virtual screening of druggable pockets located in the central ß-sheet core of the SARS-CoV-2 Spike's protein receptor binding domain (RBD). By combining an in silico approach and molecular in vitro testing we have been able to identify several triterpenoid/steroidal agents that inhibit interaction of the Spike RBD with the carboxypeptidase domain of the Angiotensin Converting Enzyme (ACE2). In detail, we provide evidence that potential binding sites exist in the RBD of the SARS CoV-2 Spike protein and that occupancy of these pockets reduces the ability of the RBD to bind to the ACE2 consensus in vitro. Naturally occurring and clinically available triterpenoids such as glycyrrhetinic and oleanolic acids, as well as primary and secondary bile acids and their amidated derivatives such as glyco-ursodeoxycholic acid and semi-synthetic derivatives such as obeticholic acid reduces the RBD/ACE2 binding. In aggregate, these results might help to define novel approaches to COVID-19 based on SARS-CoV-2 entry inhibitors.

GPBAR1 Activation by C6-Substituted Hyodeoxycholane Analogues Protect against Colitis.

GPBAR1 agonists have been identified as potential leads for the treatment of diseases related to colon inflammation such as Crohn's and ulcerative colitis. In this paper, we report the discovery of a small library of hyodeoxycholane analogues, decorated at C-6 with different substituents, as potent and selective GPBAR1 agonists. In vitro pharmacological assays showed that compound 6 selectively activates GPBAR1 (EC50 = 0.3 µM) and reduces the production of pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α) in THP1 cells. The binding mode of compound 6 in GPBAR1 was elucidated by docking calculations. Moreover, compound 6 protects against TNBS-induced colitis in Gpbar1+/+ rodent model, representing an intriguing lead for the treatment of these inflammatory disorders.

Wound healing activity and phytochemical screening of purified fractions of Sempervivum tectorum L. leaves on HCT 116.

INTRODUCTION: Sempervivum tectorum L. (Crassulaceae), is a succulent perennial plant widespread in Mediterranean countries and commonly used in traditional medicine for ear inflammation, ulcers and skin rashes as a refrigerant and astringent. OBJECTIVE: To demonstrate the therapeutic effects of the plant, various fractions were purified and characterised. The potential wound healing activity, proliferation rate and intracellular signalling cascades were investigated by using human epithelial colorectal carcinoma (HCT 116) cells. METHODOLOGY: An extraction method without organic solvents was applied for the first time. The purification was carried out by droplet counter current chromatography (DCCC) coupled with high-performance liquid chromatography (HPLC) and electrospray ionisation mass spectrometry (ESI-MS) data. By nuclear magnetic resonance (NMR) [1 H, 13 C and two-dimensional (2D) experiments] pure components were identified. Wound healing and cell proliferation assays were utilised to determine the role of the isolated S. tectorum (SVT) fraction on cellular migration and proliferation. The signalling pathways elicited from the SVT fractions, were analysed by Western blot analysis. RESULTS: In this study two rare natural components were identified, namely monosaccharide sedoheptulose and polyalcohol 2-C-methyl-D-erythritol, along with known organic acids and flavonoids. The fractions with high level of sedoheptulose enhance the proliferation and the cellular migration of epithelial HCT 116 cells. The intracellular signalling cascades elicited from the purified fractions induce the c-Src-mediated transactivation of EGFR and the activation of the STAT3 pathway which, in turn, are crucially involved in the cellular proliferation and migration. CONCLUSIONS: Our study demonstrates the efficacy of purified fractions of S. tectorum L. in enhancing cellular proliferation and migration, suggesting their potential role as topical therapeutic treatments for wound healing.

Phytochemical and Biological Studies of Nepeta asterotricha Rech. f. (Lamiaceae): Isolation of Nepetamoside.

The n-butanolic extract, from an Iranian specimen of Nepeta asterotricha Rech. f. (NABE), displayed anti-inflammatory effects on lipopolysaccharide (LPS)-stimulated J774A.1 macrophages, which reduced nitrites and cytokines production. Bioassay guided fractionation of the extract led to the isolation of four iridoid glycosides, including a new one known as nepetamoside (1), one hexenyl-diglycoside, and some polyphenol and flavonoid components. None of the isolated iridoid components displayed significant effects on nitrites formation in an in vitro LPS-induced model of inflammation, thus suggesting that the plant anti-inflammatory effect is probably due to a synergistic action among its constituents.

Novel Isoxazole Derivatives with Potent FXR Agonistic Activity Prevent Acetaminophen-Induced Liver Injury.

Acetaminophen misuse is a leading cause of acute liver failure and liver transplantation for which therapy is poorly effective. FXR ligands have shown effective in reducing liver injury in several experimental and clinical settings. In this Letter, we have elaborated on the structure of GW4064, the first nonsteroidal agonist for FXR, to identify novel isoxazoles endowed with FXR agonistic activity and improved ADME properties. The pharmacological characterization and molecular docking studies for the structure-activity rationalization allowed the identification of several FXR agonists with nanomolar potency in transactivation and SRC-1 recruitment assays. This characterization resulted in the identification of a potent FXR agonist, compound 20 that was orally active, and rescued mice from acute liver failure caused by acetaminophen overdose in a FXR-dependent manner.

Investigation around the Oxadiazole Core in the Discovery of a New Chemotype of Potent and Selective FXR Antagonists.

Recent findings have shown that Farnesoid X Receptor (FXR) antagonists might be useful in the treatment of cholestasis and related metabolic disorders. In this paper, we report the discovery of a new chemotype of FXR antagonists featured by a 3,5-disubstituted oxadiazole core. In total, 35 new derivatives were designed and synthesized, and notably, compounds 3f and 13, containing a piperidine ring, displayed the best antagonistic activity against FXR with promising cellular potency (IC50 = 0.58 ± 0.27 and 0.127 ± 0.02 µM, respectively). The excellent pharmacokinetic properties make compound 3f the most promising lead identified in this study.

Introduction of Nonacidic Side Chains on 6-Ethylcholane Scaffolds in the Identification of Potent Bile Acid Receptor Agonists with Improved Pharmacokinetic Properties.

As a cellular bile acid sensor, farnesoid X receptor (FXR) and the membrane G-coupled receptor (GPBAR1) participate in maintaining bile acid, lipid, and glucose homeostasis. To date, several selective and dual agonists have been developed as promising pharmacological approach to metabolic disorders, with most of them possessing an acidic conjugable function that might compromise their pharmacokinetic distribution. Here, guided by docking calculations, nonacidic 6-ethyl cholane derivatives have been prepared. In vitro pharmacological characterization resulted in the identification of bile acid receptor modulators with improved pharmacokinetic properties.

Discovery of ((1,2,4-oxadiazol-5-yl)pyrrolidin-3-yl)ureidyl derivatives as selective non-steroidal agonists of the G-protein coupled bile acid receptor-1.

The G-protein bile acid receptor 1 (GPBAR1) has emerged in the last decade as prominent target for the treatment of metabolic and inflammatory diseases including type 2 diabetes, obesity, and non-alcoholic steatohepatitis. To date numerous bile acid derivatives have been identified as GPBAR1 agonists, however their clinical application is hampered by the lack of selectivity toward the other bile acid receptors. Therefore, non-steroidal GPBAR1 ligands able to selectively activate the receptor are urgently needed. With this aim, we here designed, synthesized and biologically evaluated ((1,2,4-oxadiazol-5-yl)pyrrolidin-3-yl) urea derivatives as novel potent GPBAR1 agonists. Particularly, compounds 9 and 10 induce the mRNA expression of the GPBAR1 target gene pro-glucagon and show high selectivity over the other bile acid receptors FXR, LXRα, LXRß and PXR, and the related receptors PPARα and PPARγ. Computational studies elucidated the binding mode of 10 to GPBAR1, providing important structural insights for the design of non-steroidal GPBAR1 agonists. The pharmacokinetic properties of 9 and 10 suggest that the ((1,2,4-oxadiazol-5-yl)pyrrolidin-3-yl)ureydil scaffold might be exploited to achieve effective drug candidates to treat GPBAR1 related disorders.

Hyodeoxycholic acid derivatives as liver X receptor α and G-protein-coupled bile acid receptor agonists.

Bile acids are extensively investigated for their potential in the treatment of human disorders. The liver X receptors (LXRs), activated by oxysterols and by a secondary bile acid named hyodeoxycholic acid (HDCA), have been found essential in the regulation of lipid homeostasis in mammals. Unfortunately, LXRα activates lipogenic enzymes causing accumulation of lipid in the liver. In addition to LXRs, HDCA has been also shown to function as ligand for GPBAR1, a G protein coupled receptor for secondary bile acids whose activation represents a promising approach to liver steatosis. In the present study, we report a library of HDCA derivatives endowed with modulatory activity on the two receptors. The lead optimization of HDCA moiety was rationally driven by the structural information on the binding site of the two targets and results from pharmacological characterization allowed the identification of hyodeoxycholane derivatives with selective agonistic activity toward LXRα and GPBAR1 and notably to the identification of the first example of potent dual LXRα/GPBAR1 agonists. The new chemical entities might hold utility in the treatment of dyslipidemic disorders.

Navigation in bile acid chemical space: discovery of novel FXR and GPBAR1 ligands.

Bile acids are signaling molecules interacting with nuclear receptors and membrane G-protein-coupled receptors. Among these receptors, the farnesoid X receptor (FXR) and the membrane G-coupled receptor (GPBAR1) have gained increasing consideration as druggable receptors and their exogenous dual regulation represents an attractive strategy in the treatment of enterohepatic and metabolic disorders. However, the therapeutic use of dual modulators could be associated to severe side effects and therefore the discovery of selective GPBAR1 and FXR agonists is an essential step in the medicinal chemistry optimization of bile acid scaffold. In this study, a new series of 6-ethylcholane derivatives modified on the tetracyclic core and on the side chain has been designed and synthesized and their in vitro activities on FXR and GPBAR1 were assayed. This speculation resulted in the identification of compound 7 as a potent and selective GPBAR1 agonist and of several derivatives showing potent dual agonistic activity.

Insights on FXR selective modulation. Speculation on bile acid chemical space in the discovery of potent and selective agonists.

Bile acids are the endogenous modulators of the nuclear receptor FXR and the membrane receptor GPBAR1. FXR represents a promising pharmacological target for the treatment of cholestatic liver disorders. Currently available semisynthetic bile acid derivatives cover the same chemical space of bile acids and therefore they are poorly selective toward BA receptors, increasing patient risk for adverse side effects. In this report, we have investigated around the structure of CDCA describing the synthesis and the in vitro and in vivo pharmacological characterization of a novel family of compounds modified on the steroidal tetracyclic core and on the side chain. Pharmacological characterization resulted in the identification of several potent and selective FXR agonists. These novel agents might add utility in the treatment of cholestatic disorders by potentially mitigating side effects linked to unwanted activation of GPBAR1.

Investigation on bile acid receptor regulators. Discovery of cholanoic acid derivatives with dual G-protein coupled bile acid receptor 1 (GPBAR1) antagonistic and farnesoid X receptor (FXR) modulatory activity.

Bile acids, the end products of cholesterol metabolism, activate multiple mechanisms through the interaction with membrane G-protein coupled receptors including the bile acid receptor GPBAR1 and nuclear receptors such as the bile acid sensor, farnesoid X receptor (FXR). Even if dual FXR/GPBAR1 agonists are largely considered a novel opportunity in the treatment of several liver and metabolic diseases, selective targeting of one of these receptors represents an attractive therapeutic approach for a wide range of disorders in which dual modulation is associated to severe side effects. In the present study we have investigated around the structure of LCA generating a small library of cholane derivatives, endowed with dual FXR agonism/GPBAR1 antagonism. To the best of our knowledge, this is the first report of bile acid derivatives able to antagonize GPBAR1.

Exploitation of cholane scaffold for the discovery of potent and selective farnesoid X receptor (FXR) and G-protein coupled bile acid receptor 1 (GP-BAR1) ligands.

Nuclear and G-protein coupled receptors are considered major targets for drug discovery. FXR and GP-BAR1, two bile acid-activated receptors, have gained increasing consideration as druggable receptors. Because endogenous bile acids often target both receptor families, the development of selective ligands has been proven difficult, exposing patients to side effects linked to an unwanted activation of one of the two receptors. In the present study, we describe a novel library of semisynthetic bile acid derivatives obtained by modifications on the cholane scaffold. The pharmacological characterization of this library led to the discovery of 7α-hydroxy-5ß-cholan-24-sulfate (7), 6ß-ethyl-3α,7ß-dihydroxy-5ß-cholan-24-ol (EUDCOH, 26), and 6α-ethyl-3α, 7α-dihydroxy-24-nor-5ß-cholan-23-ol (NorECDCOH, 30) as novel ligands for FXR and GP-BAR1 that might hold utility in the treatment of FXR and GP-BAR1 mediated disorders.