Development of a Novel, Potent, and Selective Sialyltransferase Inhibitor for Suppressing Cancer Metastasis.

Sialyltransferase-catalyzed membrane protein and lipid glycosylation plays a vital role as one of the most abundant post-translational modifications and diversification reactions in eukaryotes. However, aberrant sialylation has been associated with cancer malignancy and metastasis. Sialyltransferases thus represent emerging targets for the development of small molecule cancer drugs. Herein, we report the inhibitory effects of a recently discovered lithocholic acid derivative FCW393 on sialyltransferase catalytic activity, integrin sialyation, cancer-associated signal transduction, MDA-MB-231 and B16F10 cell migration and invasion, and in in vivo studies, on tumor growth, metastasis, and angiogenesis. FCW393 showed effective and selective inhibition of the sialyltransferases ST6GAL1 (IC50 = 7.8 µM) and ST3GAL3 (IC50 = 9.45 µM) relative to ST3GAL1 (IC50 > 400 µM) and ST8SIA4 (IC50 > 100 µM). FCW393 reduced integrin sialylation in breast cancer and melanoma cells dose-dependently and downregulated proteins associated with the integrin-regulated FAK/paxillin and GEF/Rho/ROCK pathways, and with the VEGF-regulated Akt/NFκB/HIF-1α pathway. FCW393 inhibited cell migration (IC50 = 2.6 µM) and invasion in in vitro experiments, and in in vivo studies of tumor-bearing mice, FCW393 reduced tumor size, angiogenesis, and metastatic potential. Based on its demonstrated selectivity, cell permeability, relatively low cytotoxicity (IC50 = 55 µM), and high efficacy, FCW393 shows promising potential as a small molecule experimental tool compound and a lead for further development of a novel cancer therapeutic.

Biological evaluation of sulfonate and sulfate analogues of lithocholic acid: A bioisosterism-guided approach towards the discovery of potential sialyltransferase inhibitors for antimetastatic study.

The naturally occurring bile acid lithocholic acid (LCA) has been a crucial core structure for many non-sugar-containing sialyltranferase (ST) inhibitors documented in literature. With the aim of elucidating the impact of the terminal carboxyl acid substituent of LCA on its ST inhibition, in this present study, we report the (bio)isosteric replacement-based design and synthesis of sulfonate and sulfate analogues of LCA. Among these compounds, the sulfate analogue SPP-002 was found to selectively inhibit N-glycan sialylation by at least an order of magnitude, indicating a substantial improvement in both potency and selectivity when compared to the unmodified parent bile acid. Molecular docking analysis supported the stronger binding of the synthetic analogue in the enzyme active site. Treatment with SPP-002 also hampered the migration, adhesion, and invasion of MDA-MB-231 cells in vitro by suppressing the expression of signaling proteins involved in the cancer metastasis-associated integrin/FAK/paxillin pathway. In totality, these findings offer not only a novel structural scaffold but also valuable insights for the future development of more potent and selective ST inhibitors with potential therapeutic effects against tumor cancer metastasis.

Bile acid metabolites enhance expression of cathelicidin antimicrobial peptide in airway epithelium through activation of the TGR5-ERK1/2 pathway.

Signals for the maintenance of epithelial homeostasis are provided in part by commensal bacteria metabolites, that promote tissue homeostasis in the gut and remote organs as microbiota metabolites enter the bloodstream. In our study, we investigated the effects of bile acid metabolites, 3-oxolithocholic acid (3-oxoLCA), alloisolithocholic acid (AILCA) and isolithocholic acid (ILCA) produced from lithocholic acid (LCA) by microbiota, on the regulation of innate immune responses connected to the expression of host defense peptide cathelicidin in lung epithelial cells. The bile acid metabolites enhanced expression of cathelicidin at low concentrations in human bronchial epithelial cell line BCi-NS1.1 and primary bronchial/tracheal cells (HBEpC), indicating physiological relevance for modulation of innate immunity in airway epithelium by bile acid metabolites. Our study concentrated on deciphering signaling pathways regulating expression of human cathelicidin, revealing that LCA and 3-oxoLCA activate the surface G protein-coupled bile acid receptor 1 (TGR5, Takeda-G-protein-receptor-5)-extracellular signal-regulated kinase (ERK1/2) cascade, rather than the nuclear receptors, aryl hydrocarbon receptor, farnesoid X receptor and vitamin D3 receptor in bronchial epithelium. Overall, our study provides new insights into the modulation of innate immune responses by microbiota bile acid metabolites in the gut-lung axis, highlighting the differences in epithelial responses between different tissues.

Alcohol-induced gut microbiome dysbiosis enhances the colonization of Klebsiella pneumoniae on the mouse intestinal tract.

Chronic alcohol consumption, an important risk factor for diseases and deaths, can cause intestinal microbiota dysbiosis and increase the infection of some opportunistic pathogens. However, the current studies on the effects of alcohol-induced intestinal microbiota dysbiosis on gut colonization of Klebsiella pneumoniae are still scarce. In the present study, we established a binge-on-chronic alcohol model in mice to identify the characteristics of alcohol-induced intestinal microbiome and metabolite dysbiosis using multi-omics and explored the effects and potential mechanisms of these dysbioses on the intestinal colonization of K. pneumoniae. The results show that chronic alcohol consumption alters the diversity and composition of gut microbiota (including bacteria and fungi), decreases the complexity of the interaction between intestinal bacteria and fungi, disturbs the gut metabolites, and promotes the colonization of K. pneumoniae on the gut of mice. The relevance analyses find that alcohol-induced gut microbiome dysbiosis has a strong correlation with the alteration of secondary bile acids. In vitro results suggest that the high concentration of lithocholic acid, a secondary bile acid, could significantly inhibit the proliferation of K. pneumoniae, and the adhesion of K. pneumoniae to Caco-2 cells. Our results indicate that alcohol-induced microbiome dysbiosis contributes to decreased levels of secondary bile acids, which was one of the main reasons affecting the colonization of K. pneumoniae in mice's intestines. Some secondary bile acids (e.g., lithocholic acid) might be a potential drug to prevent the colonization and spread of K. pneumoniae.IMPORTANCEAlcohol is one of the most commonly misused substances in our lives. However, long-term heavy drinking will increase the colonization of some opportunistic pathogens (e.g., Klebsiella pneumoniae) in the body. Here, we revealed that binge-on-chronic alcohol consumption disrupted the balance between gut bacteria and fungi, induced the gut microbiome and metabolites dysbiosis, and promoted the colonization of K. pneumoniae in the intestine of mice. In particular, alcohol-taking disrupted intestinal bile acid metabolism and reduced the lithocholic acid concentration. However, a high concentration of lithocholic acid can protect against intestinal colonization of K. pneumoniae by inhabiting the bacterial growth and adhesion to the host cell. Hence, regulating the balance of gut microbiota and intestinal bile acid metabolism may be a potential strategy for reducing the risk of K. pneumoniae infection and spread.

Gut microbiota-bile acid-vitamin D axis plays an important role in determining oocyte quality and embryonic development.

OBJECTIVE: To reveal whether gut microbiota and their metabolites are correlated with oocyte quality decline caused by circadian rhythm disruption, and to search possible approaches for improving oocyte quality. DESIGN: A mouse model exposed to continuous light was established. The oocyte quality, embryonic development, microbial metabolites and gut microbiota were analyzed. Intragastric administration of microbial metabolites was conducted to confirm the relationship between gut microbiota and oocyte quality and embryonic development. RESULTS: Firstly, we found that oocyte quality and embryonic development decreased in mice exposed to continuous light. Through metabolomics profiling and 16S rDNA-seq, we found that the intestinal absorption capacity of vitamin D was decreased due to significant decrease of bile acids such as lithocholic acid (LCA), which was significantly associated with increased abundance of Turicibacter. Subsequently, the concentrations of anti-Mullerian hormone (AMH) hormone in blood and melatonin in follicular fluid were reduced, which is the main reason for the decline of oocyte quality and early embryonic development, and this was rescued by injection of vitamin D3 (VD3). Secondly, melatonin rescued oocyte quality and embryonic development by increasing the concentration of lithocholic acid and reducing the concentration of oxidative stress metabolites in the intestine. Thirdly, we found six metabolites that could rescue oocyte quality and early embryonic development, among which LCA of 30 mg/kg and NorDCA of 15 mg/kg had the best rescue effect. CONCLUSION: These findings confirm the link between ovarian function and gut microbiota regulation by microbial metabolites and have potential value for improving ovary function.

Design, Synthesis, Computational and Biological Evaluation of Novel Structure Fragments Based on Lithocholic Acid (LCA).

The regulation of bile acid pathways has become a particularly promising therapeutic strategy for a variety of metabolic disorders, cancers, and diseases. However, the hydrophobicity of bile acids has been an obstacle to clinical efficacy due to off-target effects from rapid drug absorption. In this report, we explored a novel strategy to design new structure fragments based on lithocholic acid (LCA) with improved hydrophilicity by introducing a polar "oxygen atom" into the side chain of LCA, then (i) either retaining the carboxylic acid group or replacing the carboxylic acid group with (ii) a diol group or (iii) a vinyl group. These novel fragments were evaluated using luciferase-based reporter assays and the MTS assay. Compared to LCA, the result revealed that the two lead compounds 1a-1b were well tolerated in vitro, maintaining similar potency and efficacy to LCA. The MTS assay results indicated that cell viability was not affected by dose dependence (under 25 µM). Additionally, computational model analysis demonstrated that compounds 1a-1b formed more extensive hydrogen bond networks with Takeda G protein-coupled receptor 5 (TGR5) than LCA. This strategy displayed a potential approach to explore the development of novel endogenous bile acids fragments. Further evaluation on the biological activities of the two lead compounds is ongoing.

An N-Cyanoamide Derivative of Lithocholic Acid Co-Operates with Lysophosphatidic Acid to Promote Human Osteoblast (MG63) Differentiation.

Less-calcaemic vitamin D receptor (VDR) agonists have the potential to promote osteoblast maturation in a bone regenerative setting. The emergence of lithocholic acid (LCA) as a bona fide VDR agonist holds promise as an adjunct for arthroplasty following reports that it was less calcaemic than calcitriol (1,25D). However, LCA and some earlier derivatives, e.g., LCA acetate, had to be used at much higher concentrations than 1,25D to elicit comparable effects on osteoblasts. However, recent developments have led to the generation of far more potent LCA derivatives that even outperform the efficacy of 1,25D. These new compounds include the cyanoamide derivative, Dcha-150 (also known as AY2-79). In light of this significant development, we sought to ascertain the ability of Dcha-150 to promote human osteoblast maturation by monitoring alkaline phosphatase (ALP) and osteocalcin (OC) expression. The treatment of MG63 cells with Dcha-150 led to the production of OC. When Dcha-150 was co-administered with lysophosphatidic acid (LPA) or an LPA analogue, a synergistic increase in ALP activity occurred, with Dcha-150 showing greater potency compared to 1,25D. We also provide evidence that this synergy is likely attributed to the actions of myocardin-related transcription factor (MRTF)-serum response factor (SRF) gene transcription following LPA-receptor-induced cytoskeletal reorganisation.

Lithocholic acid promotes skeletal muscle regeneration through the TGR5 receptor.

Lithocholic acid (LCA) is a classical secondary bile acid formed by the metabolism of gut microbiota. The TGR5 receptor (also known as G protein-coupled receptor 1, GPBAR1) is an important bile acid membrane receptor that mediates a variety of metabolic processes in vivo. In recent years, most studies have focused on the role of bile acid receptors in the intestine and liver. However, there are few reports on its effect on skeletal muscle regeneration, and the specific mechanism remains unclear. Therefore, it is necessary to investigate the mechanism of the TGR5 receptor in the regulation of skeletal muscle regeneration. The results demonstrate that muscle injection with LCA significantly reduces the necrosis rate of injured muscle and improves muscle injury. Moreover, treatment of C2C12 cells with LCA significantly increases AKT/mTOR/FoxO3 phosphorylation through the TGR5 receptor, enhances MyoG transcription and reduces FBXO32 transcription. These findings indicate that LCA can activate the TGR5/AKT signaling pathway, inhibit protein degradation and promote protein synthesis to enhance the myogenic process and promote skeletal muscle regeneration.

Secondary bile acid lithocholic acid attenuates neurally evoked ion transport in the rat distal colon.

The inhibitory action of the secondary bile acid lithocholic acid (LCA) on neurally evoked Cl-/HCO3- secretion was investigated using the Ussing-chambered mucosal-submucosal preparation from the rat distal colon. Electrical field stimulation (EFS) evoked cholinergic and noncholinergic secretory responses in the rat distal colon. The responses were almost completely blocked by TTX (10-6 M) but not atropine (10-5 M) or hexamethonium (10-4 M). The selective antagonist for VIP receptor 1 (VPAC1) greatly reduced the EFS-evoked response. Thus, the rat distal colon may be predominantly innervated by noncholinergic VIP secretomotor neurons. Basolateral addition of 6 × 10-5 M LCA inhibited the EFS-evoked response. The inhibitory action of LCA was partly rescued by the Y2R antagonist BIIE0246. The bile acid receptor TGR5 agonist INT-777 mimicked the LCA-induced inhibitory action. Immunohistochemical staining showed the colocalization of TGR5 and PYY on L cells. TGR5 immunoreactivity was also found in VIP-immunoreactive submucosal neurons which also expressed the PYY receptor, Y2R. These results suggest that LCA inhibits neurally evoked Cl-/HCO3- secretion through the activation of TGR5 on L cells and cholinergic- and VIP-secretomotor neurons in the submucosal plexus. Furthermore, the inhibitory mechanism may involve TGR5-stimulated PYY release from L cells and Y2R activation in VIP-secretomotor neurons.

A Novel Lithocholic Acid Derivative Upregulates Detoxification-Related Genes in Human Induced Pluripotent Stem Cell-Derived Intestinal Organoids.

Vitamin D is a fat-soluble micronutrient that plays essential roles in a range of biological processes, including cell proliferation, inflammation, and metabolism. In this study, we investigated the effects of a novel synthetic lithocholic acid derivative with vitamin D activity (Dcha-20) on pharmacokinetic gene expression in human induced pluripotent stem cell-derived intestinal organoids. Compared with vitamin D3 treatment, Dcha-20 was found to upregulate the expression and enzyme activity of the drug-metabolizing enzyme CYP3A4, an indicator of intestinal functional maturation. In addition, Dcha-20 specifically increased expression levels of the xenobiotic detoxification enzyme UGT1A and excretion transporter MRP2. These results suggest that Dcha-20 promotes activity of the intrinsic defense system of the intestinal epithelium.

Lithocholic acid promotes rosacea-like skin inflammation via G protein-coupled bile acid receptor 1.

BACKGROUND: Rosacea is a chronic inflammatory skin disorder with unclear etiology. Evidence showed that immunoinflammatory dysregulation was involved in the pathogenesis. Bile acids, as important participants of hepatoenteric circulation, play a vital role in immunoinflammatory regulation through peripheral blood circulation. However, whether it has effects on rosacea remains unknown. METHODS: Here, we performed a bile acid analysis on the serum samples of rosacea patients and healthy controls. Then we gavage G protein-coupled bile acid receptor 1 (TGR5) knockout mice with lithocholic acid (LCA) based on a LL37-induced rosacea-like model. We further overexpress TGR5 in HaCaT keratinocytes to figure out the downstream pathway. RESULTS: We found varied bile acid profile in the peripheral blood circulation of patients, especially the most significant increase in LCA. LCA promoted skin inflammation in LL37-induced rosacea-like mouse model. Our in vivo and in vitro results further demonstrated that LCA induced inflammatory cytokines and chemokines, thus exacerbated rosacea-like skin inflammation, via TGR5 in keratinocytes and LL37-induced rosacea-like mouse model. CONCLUSIONS: Therefore, we conclude that LCA promotes skin inflammation of rosacea via TGR5, and LCA-TGR5 axis may be a novel therapeutic target for rosacea.

Lithocholic acid inhibits gallbladder cancer proliferation through interfering glutaminase-mediated glutamine metabolism.

Lithocholic acid (LCA), one of the most common metabolic products of bile acids (BAs), is originally synthesized in the liver, stored in the gallbladder, and released to the intestine, where it assists absorption of lipid-soluble nutrients. LCA has recently emerged as a powerful reagent to inhibit tumorigenesis; however, the anti-tumor activity and molecular mechanisms of LCA in gallbladder cancer (GBC) remain poorly acknowledged. Here, we analyzed serum levels of LCA in human GBC and found that LCA was significantly downregulated in these patients, and reduced LCA levels were associated with poor clinical outcomes. Treatment of xenografts with LCA impeded tumor growth. Furthermore, LCA treatment in GBC cell lines decreased glutaminase (GLS) expression, glutamine (Gln) consumption, and GSH/GSSG and NADPH/NADP+ ratios, leading to cellular ferroptosis. In contrast, GLS overexpression in tumor cells fully restored GBC proliferation and decreased ROS imbalance, thus suppressing ferroptosis. Our findings reveal that LCA functions as a tumor-suppressive factor in GBC by downregulating GLS-mediated glutamine metabolism and subsequently inducing ferroptosis. This study may offer a new therapeutic strategy tailored to improve the treatment of GBC.

Establishment of In Vitro and In Vivo Anticolorectal Cancer Efficacy of Lithocholic Acid-Based Imidazolium Salts.

Imidazolium salts (IMSs) are the subject of many studies showing their anticancer activities. In this research, a series of novel imidazolium salts substituted with lithocholic acid (LCA) and alkyl chains of various lengths (S1-S10) were evaluated against colon cancer cells. A significant reduction in the viability and metabolic activity was obtained in vitro for DLD-1 and HT-29 cell lines when treated with tested salts. The results showed that the activities of tested agents are directly related to the alkyl chain length, where S6-S8 compounds were the most cytotoxic against the DLD-1 line and S4-S10 against HT-29. The research performed on the xenograft model of mice demonstrated a lower tendency of tumor growth in the group receiving compound S6, compared with the group receiving 5-fluorouracil (5-FU). Obtained results indicate the activity of S6 in the induction of apoptosis and necrosis in induced colorectal cancer. LCA-based imidazolium salts may be candidates for chemotherapeutic agents against colorectal cancer.

Lithocholic acid inhibits dendritic cell activation by reducing intracellular glutathione via TGR5 signaling.

Dendritic cells (DCs) are the major antigen-presenting cells and play an important role in autoimmune uveitis. Emerging evidence suggests that bile acids (BAs) regulate DCs maturation. However, the underlying mechanisms by which BAs regulate the function of DCs still need to be clarified. Here, we demonstrate that lithocholic acid (LCA) inhibits the production of pro-inflammatory cytokines and the expression of surface molecules in bone marrow-derived dendritic cells (BMDCs). LCA attenuates the severity of EAU by modulating the maturation of splenic CD11C+MHCIIhigh DCs. Notably, Takeda G-protein coupled receptor 5 (TGR5) deficiency partially reverses the inhibitory effect of LCA on DCs in vitro and in vivo. TGR5 activation also downregulates the NF-κB and MAPK pathways by inhibiting glutathione production and inducing oxidative stress in DCs, which leads to apoptosis and autophagy in DCs. In addition, LCA or INT-777 treatment increases the TGR5 expression in monocyte-derived dendritic cells (MD-DCs) of patients with active BD, whereas both LCA and TGR5 agonists inhibit the activation of MD-DCs. These results suggest that LCA and TGR5 agonists might be potential therapeutic drugs for the treatment of autoimmune uveitis.

In vitro protoscolicidal effects of lithocholic acid on protoscoleces of Echinococcus granulosus and its mechanism.

Surgery has been found to be the best choice of treatment for hydatidosis. However, leakage of cyst contents during surgery is the foremost reason for recurrence of hydatidosis. In this study, we investigated the in vitro efficacy of lithocholic acid (LCA) against Echinococcus granulosus protoscoleces. The protoscoleces were divided into a control group, an albendazole (ABZ) positive control group and LCA intervention groups at concentrations of 0.5, 1, 2, and 3 mmol/L and stained with 0.1% eosin for observation using an inverted microscope; the protoscolecal ultrastructure was examined with SEM and TEM; the activities of ROS, SOD, and caspase-3 were investigated using an ROS kit, SOD kit, and caspase-3 kit, respectively; the contents of HO-1 and NQO-1 were analyzed by enzyme-linked immunosorbent assay; and the expression level of cytochrome c (Ctyc) was analyzed by western blotting. Results: As the concentration of LCA increased, the survival rate of protoscoleces gradually decreased. The microstructure shows that the external shape and internal structure were gradually deformed and collapse. SOD, GSH, HO-1 and NQO-1 decreased more significantly in the 3 mmol/L LCA group. However, ROS levels gradually increased. LCA treatment for 3 days at all concentrations significantly increased caspase-3 activity and expression in a dose-dependent manner. LCA decreased the level of Ctyc protein in vitro. LCA demonstrated a parasiticidal effect on the protoscoleces of Echinococcus granulosus in vitro. LCA may induce apoptosis of E. granulosus protoscoleces by oxidative stress and mitochondrial pathways.

The inhibition of enterocyte proliferation by lithocholic acid exacerbates necrotizing enterocolitis through downregulating the Wnt/ß-catenin signalling pathway.

OBJECTIVES: Necrotizing enterocolitis (NEC) is a catastrophic gastrointestinal emergency in preterm infants, whose exact aetiology remains unknown. The role of lithocholic acid (LCA), a key component of secondary bile acids (BAs), in NEC is unclear. METHODS: Clinical data were collected to analyse the changes of BAs in NEC patients. In vitro studies, the cell proliferation and cell death were assessed. In vivo experiments, the newborn rats were administered with low or high dose of LCA and further induced NEC. RESULTS: Clinically, compared with control group, total BAs in the NEC patients were significantly higher when NEC occurred. In vitro, LCA treatment significantly inhibited the cell proliferation through arresting cell cycle at G1/S phase without inducing apoptosis or necroptosis. Mechanistically, the Wnt/ß-catenin pathway was involved. In vivo, LCA inhibited intestinal cell proliferation leading to disruption of intestinal barrier, and thereby increased the severity of NEC. Specifically, LCA supplementation caused higher levels of FITC-labelled dextran in serum, reduced PCNA expression and inhibited the activity of Wnt/ß-catenin pathway in enterocytes. The LC-MS/MS test found that LCA was significantly higher in intestinal tissue of NEC group, and more obviously in the NEC-L and NEC-H group compared with the DM group. CONCLUSION: LCA exacerbates NEC by inhibiting intestinal cell proliferation through downregulating the Wnt/ß-catenin pathway.

Synergistic Effect of Lithocholic Acid with Gentamicin against Gram-Positive Bacteria but Not against Gram-Negative Bacteria.

Listeria monocytogenes (L. monocytogenes) is an important Gram-positive food-borne pathogen that severely threatens public health. A checkerboard microdilution method was performed to evaluate the synergistic effect of lithocholic acid (LCA) with Gentamicin (Genta) against L. monocytogenes. BacLight LIVE/DEAD staining, scanning electron microscopy and biofilm inhibition assays were further used to explore the bactericidal effect and antibiofilm effect of this combination on L. monocytogenes. Additionally, the synergistic effects of LCA derivatives with Genta were also evaluated against L. monocytogenes, S.aureus and S. suis. The results indicated that a synergistic bactericidal effect was observed for the combined therapy of LCA at the concentration without affecting bacteria viability, with Genta. Additionally, LCA in combination with Genta had a synergistic effect against Gram-positive bacteria (L. monocytogenes, S. aureus and S. suis) but not against Gram-negative bacteria (E. coli, A. baumannii and Salmonella). BacLight LIVE/DEAD staining and scanning electron microscopy analysis revealed that the combination of LCA with Genta caused L. monocytogenes membrane injury, leading to bacteria death. We found that 8 µg/mL LCA treatment effectively improved the ability of Genta to eradicate L. monocytogenes biofilms. In addition, we found that chenodeoxycholic acid, as a cholic acid derivative, also improved the bactericidal effect of Genta against Gram-positive bacteria. Our results indicate that LCA represents a broad-spectrum adjuvant with Genta for infection caused by L. monocytogenes and other Gram-positive pathogens.

Human gut bacteria produce ΤΗ17-modulating bile acid metabolites.

The microbiota modulates gut immune homeostasis. Bacteria influence the development and function of host immune cells, including T helper cells expressing interleukin-17A (TH17 cells). We previously reported that the bile acid metabolite 3-oxolithocholic acid (3-oxoLCA) inhibits TH17 cell differentiation1. Although it was suggested that gut-residing bacteria produce 3-oxoLCA, the identity of such bacteria was unknown, and it was unclear whether 3-oxoLCA and other immunomodulatory bile acids are associated with inflammatory pathologies in humans. Here we identify human gut bacteria and corresponding enzymes that convert the secondary bile acid lithocholic acid into 3-oxoLCA as well as the abundant gut metabolite isolithocholic acid (isoLCA). Similar to 3-oxoLCA, isoLCA suppressed TH17 cell differentiation by inhibiting retinoic acid receptor-related orphan nuclear receptor-γt, a key TH17-cell-promoting transcription factor. The levels of both 3-oxoLCA and isoLCA and the 3α-hydroxysteroid dehydrogenase genes that are required for their biosynthesis were significantly reduced in patients with inflammatory bowel disease. Moreover, the levels of these bile acids were inversely correlated with the expression of TH17-cell-associated genes. Overall, our data suggest that bacterially produced bile acids inhibit TH17 cell function, an activity that may be relevant to the pathophysiology of inflammatory disorders such as inflammatory bowel disease.

Synthesis and identification of lithocholic acid 3-sulfate as RORγt ligand to inhibit Th17 cell differentiation.

Primary bile acids (BAs), products of cholesterol metabolism and clearance, are synthesized in the liver and released into the intestine to facilitate the digestion and absorption of lipids. BAs are further converted by gut commensal bacteria into secondary colonic BAs and the metabolism disorder is closely linked to cholestatic liver diseases via regulating immune response. However, the effect and underlying mechanism of these host-microorganism biliary metabolites on T lymphocyte remain unclear. In the current study, we synthesized a sulfated product of lithocholic acid (LCA), lithocholic acid 3-sulfate (LCA-3-S), and investigated the binding affinity of the BAs metabolites on RORγt, the transcription factor of IL-17A. Our results demonstrated that the sulfate of LCA, LCA-3-S, exhibited better effect than its oxidated metabolite, 3-oxo-LCA, binding to RORγt. The results further demonstrated that LCA-3-S selectively suppressed Th17 cell differentiation without influence on Th1, Th2, and Treg cells. Collectively, we synthesized the sulfated biliary metabolite LCA-3-S and demonstrated that LCA-3-S selectively inhibited Th17 cell differentiation by targeting RORγt, indicating that metabolite disorder of BAs resulting in the decrease of LCA-3-S probably contributes to the pathogenesis of cholestatic liver diseases.

Lithocholic Acid Amides as Potent Vitamin D Receptor Agonists.

1α,25-Dihydroxyvitamin D3 [1α,25(OH)2D3, 1] is an active form of vitamin D3 and regulates various biological phenomena, including calcium and phosphate homeostasis, bone metabolism, and immune response via binding to and activation of vitamin D receptor (VDR). Lithocholic acid (LCA, 2) was identified as a second endogenous agonist of VDR, though its potency is very low. However, the lithocholic acid derivative 3 (Dcha-20) is a more potent agonist than 1α,25(OH)2D3, (1), and its carboxyl group has similar interactions to the 1,3-dihydroxyl groups of 1 with amino acid residues in the VDR ligand-binding pocket. Here, we designed and synthesized amide derivatives of 3 in order to clarify the role of the carboxyl group. The synthesized amide derivatives showed HL-60 cell differentiation-inducing activity with potency that depended upon the substituent on the amide nitrogen atom. Among them, the N-cyanoamide 6 is more active than either 1 or 3.