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The nuclear receptor ssDAF-12 has been recognized as the key molecular player regulating the life cycle of the nematode parasite Strongyloides stercoralis. ssDAF-12 ligands permit the receptor to function as an on/off switch modulating infection, making it vulnerable to therapeutic intervention. In this study, we report the design and synthesis of a set of novel dafachronic acid derivatives, which were used to outline the first structure–activity relationship targeting the ssDAF-12 receptor and to unveil hidden properties shared by the molecular shape of steroidal ligands that are relevant to the receptor binding and modulation. Moreover, biological results led to the discovery of sulfonamide 3as a submicromolar ssDAF-12 agonist endowed with a high receptor selectivity, no toxicity, and improved properties, as well as to the identification of unprecedented ssDAF-12 antagonists that can be exploited in the search for novel chemical tools and alternative therapeutic approaches for treating parasitism such as Strongyloidiasis.

Retinol and its derivatives play an important role in epidermal growth and differentiation and represent chemopreventive agents in nonmelanoma skin cancer. Retinoic acid binding protein II (CRABP-II) is a cytoplasmic receptor that critically regulates all-trans-retinoic acid (ATRA) trafficking. We documented the marked reduced expression of CRABP-II and its promoter methylation in human poorly differentiated squamous cell carcinomas. To investigate the role of CRABP-II in skin carcinogenesis we used skin lesion induction by dimethylbenz[a]anthracene/12-O-tetradecanoyl-phorbol-13-acetate in CRABP-II–knockout C57BL/6 mice. We observed earlier and more diffuse epidermal dysplasia, greater incidence and severity of tumors, reduced expression of cytokeratin 1/cytokeratin 10 and involucrin, increased proliferation, and impaired ATRA inhibition of tumor promotion compared with wild-type animals. CRABP-II–transfected HaCaT, FaDu, and A431 cells showed expression of differentiation markers, retinoic acid receptor-β/-γ signaling, ATRA sensitivity, and suppression of EGFR/v-akt murine thymoma viral oncogene homolog 1 (AKT) pathways in a fatty acid binding protein 5/peroxisome proliferator–activated receptor-β/-δ–independent manner. The opposite was true in keratinocytes isolated from CRABP-II–knockout mice. Finally, CRABP-II accumulation induced ubiquitination-associated reduction of EGFR. Our results showed reduced CRABP-II expression in human poorly differentiated squamous cell carcinomas, and its gene deletion favored experimental skin carcinogenesis and impaired ATRA antitumor efficacy, likely modulating EGFR/AKT pathways and retinoic acid receptor-β/-γ signaling. Therapeutic interventions aimed at restoring CRABP-II–mediated signaling may amplify therapeutic retinoid efficacy in nonmelanoma skin cancer.

We report on the relationship between the structure-pharmacokinetics, metabolism, and therapeutic activity of semisynthetic bile acid analogs, including 6α-ethyl-3α,7α-dihydroxy-5β-cholan-24-oic acid (a selective farnesoid X receptor [FXR] receptor agonist), 6α-ethyl-23(S)-methyl-3α,7α,12α-trihydroxy-5β-cholan-24-oic acid (a specific Takeda G protein–coupled receptor 5 [TGR5] receptor agonist), and 6α-ethyl-3α,7α-dihydroxy-24-nor-5β-cholan-23-sulfate (a dual FXR/TGR5 agonist). We measured the main physicochemical properties of these molecules, including ionization constants, water solubility, lipophilicity, detergency, and protein binding. Biliary secretion and metabolism and plasma and hepatic concentrations were evaluated by high-pressure liquid chromatography- electrospray-mass spectrometry/mass spectrometry in bile fistula rat and compared with natural analogs chenodeoxycholic, cholic acid, and taurochenodexycholic acid and intestinal bacteria metabolism was evaluated in terms of 7α-dehydroxylase substrate-specificity in anaerobic human stool culture. The semisynthetic derivatives detergency, measured in terms of their critical micellar concentration, was quite similar to the natural analogs. They were slightly more lipophilic than the corresponding natural analogs, evaluated by their 1-octanol water partition coefficient (log P), because of the ethyl group in 6 position, which makes these molecules very stable toward bacterial 7-dehydroxylation. The hepatic metabolism and biliary secretion were different: 6α-ethyl-3α,7α-dihydroxy-5β-cholan-24-oic acid, as chenodeoxycholic acid, was efficiently conjugated with taurine in the liver and, only in this form, promptly and efficiently secreted in bile. 6α-Ethyl-23(S)-methyl-3α,7α,12α-trihydroxy-5β-cholan-24-oic acid was poorly conjugated with taurine because of the steric hindrance of the methyl at C23(S) position metabolized to the C23(R) isomer and partly conjugated with taurine. Conversely, 6α-ethyl-3α,7α-dihydroxy-24-nor-5β-cholan-23-sulfate was secreted in bile unmodified and as 3-glucuronide. Therefore, minor structural modifications profoundly influence the metabolism and biodistribution in the target organs where these analogs exert therapeutic effects by interacting with FXR and/or TGR5 receptors.

Two dedicated receptors for bile acids (BAs) have been identified, the nuclear hormone receptor farnesoid X receptor (FXR) and the G protein-coupled receptor TGR5, which represent attractive targets for the treatment of metabolic and chronic liver diseases. Previous work characterized 6α-ethyl-3α,7α-dihydroxy-5β-cholan-24-oic acid (INT-747), a potent and selective FXR agonist, as well as 6α-ethyl-23(S)-methyl-3α,7α,12α-trihydroxy-5β-cholan-24-oic acid (INT-777), a potent and selective TGR5 agonist. Here we characterize 6α-ethyl-3α,7α,23-trihydroxy-24-nor-5β-cholan-23-sulfate sodium salt (INT-767), a novel semisynthetic 23-sulfate derivative of INT-747. INT-767 is a potent agonist for both FXR (mean EC50, 30 nM by PerkinElmer AlphaScreen assay) and TGR5 (mean EC50, 630 nM by time resolved-fluorescence resonance energy transfer), the first compound described so far to potently and selectively activate both BA receptors. INT-767 does not show cytotoxic effects in HepG2 cells, does not inhibit cytochrome P450 enzymes, is highly stable to phase I and II enzymatic modifications, and does not inhibit the human ether-a-go-go-related gene potassium channel. In line with its dual activity, INT-767 induces FXR-dependent lipid uptake by adipocytes, with the beneficial effect of shuttling lipids from central hepatic to peripheral fat storage, and promotes TGR5-dependent glucagon-like peptide-1 secretion by enteroendocrine cells, a validated target in the treatment of type 2 diabetes. Moreover, INT-767 treatment markedly decreases cholesterol and triglyceride levels in diabetic db/db mice and in mice rendered diabetic by streptozotocin administration. Collectively, these preclinical results indicate that INT-767 is a safe and effective modulator of FXR and TGR5-dependent pathways, suggesting potential clinical applications in the treatment of liver and metabolic diseases.

Two dedicated receptors for bile acids (BAs) have been identified, the nuclear hormone receptor farnesoid X receptor (FXR) and the G protein-coupled receptor TGR5, which represent attractive targets for the treatment of metabolic and chronic liver diseases. Previous work characterized 6α-ethyl-3α,7α-dihydroxy-5β-cholan-24-oic acid (INT-747), a potent and selective FXR agonist, as well as 6α-ethyl-23(S)-methyl-3α,7α,12α-trihydroxy-5β-cholan-24-oic acid (INT-777), a potent and selective TGR5 agonist. Here we characterize 6α-ethyl-3α,7α,23-trihydroxy-24-nor-5β-cholan-23-sulfate sodium salt (INT-767), a novel semisynthetic 23-sulfate derivative of INT-747. INT-767 is a potent agonist for both FXR (mean EC50, 30 nM by PerkinElmer AlphaScreen assay) and TGR5 (mean EC50, 630 nM by time resolved-fluorescence resonance energy transfer), the first compound described so far to potently and selectively activate both BA receptors. INT-767 does not show cytotoxic effects in HepG2 cells, does not inhibit cytochrome P450 enzymes, is highly stable to phase I and II enzymatic modifications, and does not inhibit the human ether-a-go-go-related gene potassium channel. In line with its dual activity, INT-767 induces FXR-dependent lipid uptake by adipocytes, with the beneficial effect of shuttling lipids from central hepatic to peripheral fat storage, and promotes TGR5-dependent glucagon-like peptide-1 secretion by enteroendocrine cells, a validated target in the treatment of type 2 diabetes. Moreover, INT-767 treatment markedly decreases cholesterol and triglyceride levels in diabetic db/db mice and in mice rendered diabetic by streptozotocin administration. Collectively, these preclinical results indicate that INT-767 is a safe and effective modulator of FXR and TGR5-dependent pathways, suggesting potential clinical applications in the treatment of liver and metabolic diseases.

The farnesoid X receptor (FXR) is a nuclear receptor that functions as an endogenous sensor for bile acids (BAs). FXR is bound to and activated by bile acid, and chenodeoxycholic acid (CDCA) is the natural most active ligand. Upon activation, FXR heterodimerizes with the 9-cisretinoic X receptor (RXR) and regulates genes involved in cholesterol and BA homeostasis. 6-Ethyl CDCA (6-ECDCA) is a synthetic BA that binds FXR and induces gene transcription by recruiting coactivators, such as steroid receptor coactivator-1, with histone acetyltransferase activity. In addition to acetylation, histone methylation is critically involved in regulating eukaryotic gene expression. In the present study, we demonstrated that 6-ECDCA activates FXR to interacts with Protein Arginine Methyl-Transferase type I (PRMT1), which induces up-regulation of bile salt export pump (BSEP) and the small heterodimer partner (SHP) mRNA expression and causes a down-regulation of P450 cholesterol 7α-hydroxylase and Na+taurocholate cotransport peptide genes. Chromatin immunoprecipitation assay suggests that 6-ECDCA induces both the recruitment of PRMT1 and the H4 methylation to the promoter of BSEP and SHP genes. We also provide evidence that a methyltransferase inhibitor blocks the activation of FXR-responsive genes. Our results indicate that histone methylation, similar to acetylation, regulates transcriptional activation of genes involved in cholesterol and BAs homeostasis.

The farnesoid X receptor (FXR) is a nuclear receptor that functions as an endogenous sensor for bile acids (BAs). FXR is bound to and activated by bile acid, and chenodeoxycholic acid (CDCA) is the natural most active ligand. Upon activation, FXR heterodimerizes with the 9-cis retinoic X receptor (RXR) and regulates genes involved in cholesterol and BA homeostasis. 6-Ethyl CDCA (6-ECDCA) is a synthetic BA that binds FXR and induces gene transcription by recruiting coactivators, such as steroid receptor coactivator-1, with histone acetyltransferase activity. In addition to acetylation, histone methylation is critically involved in regulating eukaryotic gene expression. In the present study, we demonstrated that 6-ECDCA activates FXR to interacts with Protein Arginine Methyl-Transferase type I (PRMT1), which induces up-regulation of bile salt export pump (BSEP) and the small heterodimer partner (SHP) mRNA expression and causes a down-regulation of P450 cholesterol 7α-hydroxylase and Na+ taurocholate cotransport peptide genes. Chromatin immunoprecipitation assay suggests that 6-ECDCA induces both the recruitment of PRMT1 and the H4 methylation to the promoter of BSEP and SHP genes. We also provide evidence that a methyltransferase inhibitor blocks the activation of FXR-responsive genes. Our results indicate that histone methylation, similar to acetylation, regulates transcriptional activation of genes involved in cholesterol and BAs homeostasis.

MINA53 is a JmjC domain 2-oxoglutarate-dependent oxygenase that catalyzes ribosomal hydroxylation and is a target of the oncogenic transcription factor c-MYC. Despite its anticancer target potential, no small-molecule MINA53 inhibitors are reported. Using ribosomal substrate fragments, we developed mass spectrometry assays for MINA53 and the related oxygenase NO66. These assays enabled the identification of 2-(aryl)alkylthio-3,4-dihydro-4-oxoypyrimidine-5-carboxylic acids as potent MINA53 inhibitors, with selectivity over NO66 and other JmjC oxygenases. Crystallographic studies with the JmjC demethylase KDM5B revealed active site binding but without direct metal chelation; however, molecular modeling investigations indicated that the inhibitors bind to MINA53 by directly interacting with the iron cofactor. The MINA53 inhibitors manifest evidence for target engagement and selectivity for MINA53 over KDM4–6. The MINA53 inhibitors show antiproliferative activity with solid cancer lines and sensitize cancer cells to conventional chemotherapy, suggesting that further work investigating their potential in combination therapies is warranted.