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The active metabolite of vitamin D3, calcitriol (1,25D), is widely recognised for its direct anti-proliferative and pro-differentiation effects. However, 1,25D is calcaemic, which restricts its clinical use for cancer treatment. Non-calcaemic agonists of the vitamin D receptor (VDR) could be better candidates for cancer treatment. In this study, we examined the influence of the hydroxylated lithocholic acid derivative CAR-R on osteosarcoma (OS) cell (MG63) growth and differentiation. Treatment of MG63 cells with CAR-R inhibited growth under conventional and hypoxic conditions. Co-treating cells with CAR-R and a lysophosphatidic acid (LPA) analogue resulted in their differentiation, as supported by synergistic increases in alkaline phosphatase (ALP) activity. Under hypoxic conditions, however, this differentiation response was attenuated. The importance of observed increases in hypoxia inducible factors (HIFs) were investigated through targeted disruption using pharmacological and genetic approaches. Disruption elicited a reduction in ALP activity, suggesting an important role for HIFs in OS differentiation. Finally, we examined the expression of the VDR protein. Hypoxic MG63s expressed less VDR, with the levels increasing with CAR-R exposure. Whilst these findings are encouraging, future studies aimed at bolstering the pro-differentiating effect of CAR-R under hypoxic conditions are warranted if this agent is to gain traction in the treatment of OS.

Peptide-bile acid hybrids offer promising drug candidates due to enhanced pharmacological properties, such as improved protease resistance and oral bioavailability. However, it remains unknown whether bile acids can be incorporated into peptide chains by the ribosome to produce a peptide-bile acid hybrid macrocyclic peptide library for target-based de novo screening. In this study, we achieved the ribosomal incorporation of lithocholic acid (LCA)-d-tyrosine into peptide chains. This led to the construction of a peptide-LCA hybrid macrocyclic peptide library, which enabled the identification of peptides TP-2C-4L3 (targeting Trop2) and EP-2C-4L5 (targeting EphA2) with strong binding affinities. Notably, LCA was found to directly participate in binding to EphA2 and confer on the peptides improved stability and resistance to proteases. Cell staining experiments confirmed the high specificity of the peptides for targeting Trop2 and EphA2. This study highlights the benefits of LCA in peptides and paves the way for de novo discovery of stable peptide-LCA hybrid drugs.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Centenarians have a decreased susceptibility to ageing-associated illnesses, chronic inflammation and infectious diseases 1-3 . Here we show that centenarians have a distinct gut microbiome that is enriched in microorganisms that are capable of generating unique secondary bile acids, including various isoforms of lithocholic acid (LCA): iso-, 3-oxo-, allo-, 3-oxoallo- and isoallolithocholic acid. Among these bile acids, the biosynthetic pathway for isoalloLCA had not been described previously. By screening 68 bacterial isolates from the faecal microbiota of a centenarian, we identified Odoribacteraceae strains as effective producers of isoalloLCA both in vitro and in vivo. Furthermore, we found that the enzymes 5α-reductase (5AR) and 3β-hydroxysteroid dehydrogenase (3β-HSDH) were responsible for the production of isoalloLCA. IsoalloLCA exerted potent antimicrobial effects against Gram-positive (but not Gram-negative) multidrug-resistant pathogens, including Clostridioides difficile and Enterococcus faecium. These findings suggest that the metabolism of specific bile acids may be involved in reducing the risk of infection with pathobionts, thereby potentially contributing to the maintenance of intestinal homeostasis., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

7α-Hydroxysteroid dehydrogenase (7α-HSDH) catalyzes the dehydrogenation of a hydroxyl group at the 7α position in steroid substrates using NAD + or NADP + as a co-factor. Although studies have determined the binary and ternary complex structures, detailed structural changes induced by ligand and co-factor binding remain unclear, because ligand-free structures are not yet available. Here, we present the crystal structure of apo 7α-HSDH from Escherichia coli (Eco-7α-HSDH) at 2.7 Å resolution. We found that the apo form undergoes substantial conformational changes in the β4-α4 loop, α7-α8 helices, and C-terminus loop among the four subunits comprising the tetramer. Furthermore, a comparison of the apo structure with the binary (NAD + )-complex and ternary (NADH and 7-oxoglycochenodeoxycholic acid)-complex Eco-7α-HSDH structures revealed that only the ternary-complex structure has a fully closed conformation, whereas the binary-complex and apo structures have a semi-closed or open conformation. This open-to-closed transition forces several catalytically important residues (S146, Y159, and K163) into correct positions for catalysis. To confirm the catalytic activity, we used alcohol dehydrogenase for NAD + regeneration to allow efficient conversion of chenodeoxycholic acid to 7-ketolithocholic acid by Eco-7α-HSDH. These findings demonstrate that apo Eco-7α-HSDH exhibits intrinsically flexible characteristics with an open conformation. This structural information provides novel insight into the 7α-HSDH reaction mechanism., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Bile acids play crucial roles in host physiology by acting both as detergents that aid in digestion and as signaling molecules that bind to host receptors. Gut bacterial bile salt hydrolase (BSH) enzymes perform the gateway reaction leading to the conversion of host-produced primary bile acids into bacterially modified secondary bile acids. Small molecule probes that target BSHs will help elucidate the causal roles of these metabolites in host physiology. We previously reported the development of a covalent BSH inhibitor with low gut permeability. Here, we build on our previous findings and describe the development of a second-generation gut-restricted BSH inhibitor with enhanced potency, reduced off-target effects, and durable in vivo efficacy. Structure-activity relationship (SAR) studies focused on the bile acid core identified a compound, AAA-10, containing a C3-sulfonated lithocholic acid scaffold and an alpha-fluoromethyl ketone warhead as a potent pan-BSH inhibitor. This compound inhibits BSH activity in mouse and human fecal slurry, bacterial cultures, and purified BSH proteins and displays reduced toxicity against mammalian cells compared to first generation compounds. Oral administration of AAA-10 to wild-type mice for 5 days resulted in a decrease in the abundance of the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA) in the mouse GI tract with low systemic exposure of AAA-10, demonstrating that AAA-10 is an effective tool for inhibiting BSH activity and modulating bile acid pool composition in vivo .

In this study, we describe the engineering of sub-100 nm nanomicelles (DTX-PC NMs) derived from phosphocholine derivative of docetaxel (DTX)-conjugated lithocholic acid (DTX-PC) and poly(ethylene glycol)-tethered lithocholic acid. Administration of DTX-PC NMs decelerate tumor progression and increase the mice survivability compared to Taxotere (DTX-TS), the FDA-approved formulation of DTX. Unlike DTX-TS, DTX-PC NMs do not cause any systemic toxicity and slow the decay rate of plasma DTX concentration in rodents and non-rodent species including non-human primates. We further demonstrate that DTX-PC NMs target demethylation of CpG islands of Sparcl1 (a tumor suppressor gene) by suppressing DNA methyltransferase activity and increase the expression of Sparcl1 that leads to tumor regression. Therefore, this unique system has the potential to improve the quality of life in cancer patients and can be translated as a next-generation chemotherapeutic., (© 2020 Wiley-VCH GmbH.)

The family of ATP-gated purinergic P2X receptors comprises seven bunits (P2X1-7) that are unevenly distributed in the central and peripheral nervous systems as well as other organs. Endogenous modulators of P2X receptors are phospholipids, steroids and neurosteroids. Here, we analyzed whether bile acids, which are natural products derived from cholesterol, affect P2X receptor activity. We examined the effects of primary and secondary bile acids and newly synthesized derivatives of lithocholic acid on agonist-induced responses in HEK293T cells expressing rat P2X2, P2X4 and P2X7 receptors. Electrophysiology revealed that low micromolar concentrations of lithocholic acid and its structural analog 4-dafachronic acid strongly inhibit ATP-stimulated P2X2 but potentiate P2X4 responses, whereas primary bile acids and other secondary bile acids exhibit no or reduced effects only at higher concentrations. Agonist-stimulated P2X7 responses are significantly potentiated by lithocholic acid at moderate concentrations. Structural modifications of lithocholic acid at positions C-3, C-5 or C-17 abolish both inhibitory and potentiation effects to varying degrees, and the 3α-hydroxy group contributes to the ability of the molecule to switch between potentiation and inhibition. Lithocholic acid allosterically modulates P2X2 and P2X4 receptor sensitivity to ATP, reduces the rate of P2X4 receptor desensitization and antagonizes the effect of ivermectin on P2X4 receptor deactivation. Alanine-scanning mutagenesis of the upper halve of P2X4 transmembrane domain-1 revealed that residues Phe48, Val43 and Tyr42 are important for potentiating effect of lithocholic acid, indicating that modulatory sites for lithocholic acid and ivermectin partly overlap. Lithocholic acid also inhibits ATP-evoked currents in pituitary gonadotrophs expressing native P2X2, and potentiates ATP currents in nonidentified pituitary cells expressing P2X4 receptors. These results indicate that lithocholic acid is a bioactive steroid that may help to further unveil the importance of the P2X2, and P2X4 receptors in many physiological processes., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Bile salts tend to form micelles in aqueous media and can thereby contribute to drug solubilization; they also exhibit crystallization inhibition properties that can stabilize supersaturated drug solutions. Herein, we explore conjugation of bile salts with polysaccharides to create new, amphiphilic polysaccharide derivatives with intriguing properties, portending broad utility in various applications. We introduce efficient conjugation of cholesterol (as a model steroid), lithocholic acid, and deoxycholic acid by mild, modular olefin cross-metathesis reactions. These small molecules were first modified with an acrylate group from the A-ring hydroxyl, then reacted with cellulose derivatives bearing olefin-terminated metathesis "handles". Successful conjugation of bile acids has demonstrated chemoselective cross-metathesis with complex, polyfunctional structures, and large multi-ring systems. It also enabled an efficient, general pathway for polysaccharide-bile salt conjugates, which promise synergy for applications such as amorphous solid dispersion (ASD)., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The present study examined the significance of enterohepatic circulation and the effect of rifampicin [an inhibitor of organic anion-transporting polypeptide 1B (OATP1B)] on the plasma concentrations of bile acid-O-sulfates (glycochenodeoxycholate-O-sulfate, lithocholate-O-sulfate, glycolithocholate-O-sulfate, and taurolithocholate-O-sulfate) in monkeys and human liver-transplanted chimeric mice (PXB mouse). Rifampicin significantly increased the area under the curve of bile acid-O-sulfates in monkeys (13-69 times) and PXB mice (13-25 times) without bile flow diversion. Bile flow diversion reduced the concentration of plasma bile acid-O-sulfates under control conditions in monkeys and the concentration of plasma glycochenodeoxycholate-O-sulfate in PXB mice. It also diminished diurnal variation of plasma lithocholate-O-sulfate, glycolithocholate-O-sulfate, and taurolithocholate-O-sulfate in PXB mice under control conditions. Bile flow diversion did not affect the plasma concentration of bile acid-O-sulfates in monkeys and PXB mice treated with rifampicin. Plasma coproporphyrin I and III levels were constant in monkeys throughout the study, even with bile flow diversion. This study demonstrated that bile acid-O-sulfates are endogenous OATP1B biomarkers in monkeys and PXB mice. Enterohepatic circulation can affect the baseline levels of plasma bile acid-O-sulfates and modify the effect of OATP1B inhibition., (Copyright © 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Lithocholic acid (LCA) is a bile acid associated with adverse effects, including cholestasis, and it exists in vivo mainly as conjugates known as glyco-LCA (GLCA) and tauro-LCA (TLCA). Tamoxifen has been linked to the development of cholestasis, and it inhibits sulfotransferase 2A1 (SULT2A1)-catalyzed dehydroepiandrosterone (DHEA) sulfonation. The present study was done to characterize the sulfonation of LCA, GLCA, and TLCA and to investigate whether triphenylethylene (clomifene, tamoxifen, toremifene, ospemifene, droloxifene), benzothiophene (raloxifene, arzoxifene), tetrahydronaphthalene (lasofoxifene, nafoxidine), indole (bazedoxifene), and benzopyran (acolbifene) classes of selective estrogen receptor modulator (SERM) inhibit LCA, GLCA, and TLCA sulfonation. Human recombinant SULT2A1, but not SULT2B1b or SULT1E1, catalyzed LCA, GLCA, and TLCA sulfonation, whereas each of these enzymes catalyzed DHEA sulfonation. LCA, GLCA, and TLCA sulfonation is catalyzed by human liver cytosol, and SULT2A1 followed the substrate inhibition model with comparable apparent K m values (≤1 µ M). Each of the SERMs inhibited LCA, GLCA, and TLCA sulfonation with varying potency and mode of enzyme inhibition. The potency and extent of inhibition of LCA sulfonation were attenuated or increased by structural modifications to toremifene, bazedoxifene, and lasofoxifene. The inhibitory effect of raloxifene, bazedoxifene, and acolbifene on LCA sulfonation was also observed in HepG2 human hepatocellular carcinoma cells. Overall, among the SERMs investigated, bazedoxifene and raloxifene were the most effective inhibitors of LCA, GLCA, and TLCA sulfonation. These findings provide insight into the structural features of specific SERMs that contribute to their inhibition of SULT2A1-catalyzed LCA sulfonation. Inhibition of LCA, GLCA, and TLCA detoxification by a SERM may provide a biochemical basis for adverse effects associated with a SERM., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

7-Ketolithocholic acid (7-KLCA) is an important intermediate for the synthesis of ursodeoxycholic acid (UDCA). UDCA is the main effective component of bear bile powder that is used in traditional Chinese medicine for the treatment of human cholesterol gallstones. 7α-Hydroxysteroid dehydrogenase (7α-HSDH) is the key enzyme used in the industrial production of 7-KLCA. Unfortunately, the natural 7α-HSDHs reported have difficulty meeting the requirements of industrial application, due to their poor activities and strong substrate inhibition. In this study, a directed evolution strategy combined with high-throughput screening was applied to improve the catalytic efficiency and tolerance of high substrate concentrations of NADP+-dependent 7α-HSDH from Clostridium absonum. Compared with the wild type, the best mutant (7α-3) showed 5.5-fold higher specific activity and exhibited 10-fold higher and 14-fold higher catalytic efficiency toward chenodeoxycholic acid (CDCA) and NADP+, respectively. Moreover, 7α-3 also displayed significantly enhanced tolerance in the presence of high concentrations of substrate compared to the wild type. Owing to its improved catalytic efficiency and enhanced substrate tolerance, 7α-3 could efficiently biosynthesize 7-KLCA with a substrate loading of 100 mM, resulting in 99% yield of 7-KLCA at 2 h, in contrast to only 85% yield of 7-KLCA achieved for the wild type at 16 h.

Eph/ephrin system is an emerging target for cancer therapy but the lack of potent, stable and orally bioavailable compounds is impairing the development of the field. Since 2009 our research group has been devoted to the discovery and development of small molecules targeting Eph/ephrin system and our research culminated with the synthesis of UniPR129, a potent but problematic Eph/ephrin antagonist. Herein, we describe the in vitro pharmacological properties of two derivatives (UniPR139 and UniPR502) stemmed from structure of UniPR129. These two compounds acted as competitive and reversible antagonists of all Eph receptors reducing both ephrin-A1 and -B1 binding to EphAs and EphBs receptors in the low micromolar range. The compounds acted as antagonists inhibiting ephrin-A1-dependent EphA2 activation and UniPR139 exerted an anti-angiogenic effect, inhibiting HUVEC tube formation in vitro and VEGF-induced vessel formation in the chick chorioallantoic membrane assay. Finally, the oral bioavailability of UniPR139 represents a step forward in the search of molecules targeting the Eph/ephrin system and offers a new pharmacological tool useful for future in vivo studies., (Copyright © 2017 Elsevier Inc. All rights reserved.)

A novel series of 3-ketolithocholic acid derivatives as well as estrone derivatives bearing a small ring for the conformational fixation of the side chain were synthesized by using a catalytic [2+2] cycloaddition and a ring-contraction rearrangement. The steroidal derivatives were evaluated for transcriptional activation of vitamin D receptor by luciferase reporter assays. Among them, two estrone derivatives showed a higher efficacy of the transactivation of vitamin D receptor than 3-ketolithocholic acid, and the small ring moieties were found to be important for the efficacy., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

USP2a is a deubiquitinase responsible for stabilization of cyclin D1, a crucial regulator of cell-cycle progression and a proto-oncoprotein overexpressed in numerous cancer types. Here we report that lithocholic acid (LCA) derivatives are inhibitors of USP proteins, including USP2a. The most potent LCA derivative, LCA hydroxyamide (LCAHA), inhibits USP2a, leading to a significant Akt/GSK3β-independent destabilization of cyclin D1, but does not change the expression of p27. This leads to the defects in cell-cycle progression. As a result, LCAHA inhibits the growth of cyclin D1-expressing, but not cyclin D1-negative cells, independently of the p53 status. We show that LCA derivatives may be considered as future therapeutics for the treatment of cyclin D1-addicted p53-expressing and p53-defective cancer types., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Ursodeoxycholic acid (UDCA) is the main active ingredient of natural bear bile powder with multiple pharmacological functions. 7β-Hydroxysteroid dehydrogenase (HSDH) is a key biocatalyst for the synthesis of UDCA. However, all the 7β-HSDHs reported commonly suffer from poor activity and thermostability, resulting in limited productivity of UDCA. In this study, a multiobjective directed evolution (MODE) strategy was proposed and applied to improve the activity, thermostability, and pH optimum of a 7β-HSDH. The best variant (V 3-1 ) showed a specific activity 5.5-fold higher than and a half-life 3-fold longer than those of the wild type. In addition, the pH optimum of the variant was shifted to a weakly alkaline value. In the cascade reaction, the productivity of UDCA with V 3-1 increased to 942 g L -1 day -1 , in contrast to 141 g L -1 day -1 with the wild type. Therefore, this study provides a useful strategy for improving the catalytic efficiency of a key enzyme that significantly facilitated the bioproduction of UDCA.

Metadynamics (META-D) is emerging as a powerful method for the computation of the multidimensional free-energy surface (FES) describing the protein-ligand binding process. Herein, the FES of unbinding of the antagonist N-(3α-hydroxy-5β-cholan-24-oyl)-l-β-homotryptophan (UniPR129) from its EphA2 receptor was reconstructed by META-D simulations. The characterization of the free-energy minima identified on this FES proposes a binding mode fully consistent with previously reported and new structure-activity relationship data. To validate this binding mode, new N-(3α-hydroxy-5β-cholan-24-oyl)-l-β-homotryptophan derivatives were designed, synthesized, and tested for their ability to displace ephrin-A1 from the EphA2 receptor. Among them, two antagonists, namely compounds 21 and 22, displayed high affinity versus the EphA2 receptor and resulted endowed with better physicochemical and pharmacokinetic properties than the parent compound. These findings highlight the importance of free-energy calculations in drug design, confirming that META-D simulations can be used to successfully design novel bioactive compounds.

The chemical synthesis of the 9α-hydroxy derivatives of chenodeoxycholic and lithocholic acids is reported. For initiating the synthesis of the 9α-hydroxy derivative of chenodeoxycholic acid, cholic acid was used; for the synthesis of the 9α-hydroxy derivative of lithocholic acid, deoxycholic acid was used. The principal reactions involved were (1) decarbonylation of conjugated 12-oxo-Δ(9(11))-derivatives using in situ generated monochloroalane (AlH2Cl) prepared from LiAlH4 and AlCl3, (2) epoxidation of the deoxygenated Δ(9(11))-enes using m-chloroperbenzoic acid catalyzed by 4,4'-thiobis-(6-tert-butyl-3-methylphenol), (3) subsequent Markovnikov 9α-hydroxylation of the Δ(9(11))-enes with AlH2Cl, and (4) selective oxidation of the primary hydroxyl group at C-24 in the resulting 3α,9α,24-triol and 3α,7α,9α,24-tetrol to the corresponding C-24 carboxylic acids using sodium chlorite (NaClO2) in the presence of a catalytic amount of 2,2,6,6-tetramethylpiperidine 1-oxyl free radical (TEMPO) and sodium hypochlorite (NaOCl). The (1)H- and (13)C-NMR spectra are reported. The 3α,7α,9α-trihydroxy-5β-cholan-24-oic acid has been reported to be present in the bile of the Asian bear, and its 7-deoxy derivative is likely to be a bacterial metabolite. These bile acids are now available as authentic reference standards, permitting their identification in vertebrate bile acids.

In order to identify structural features of lithocholic acid (LCA) critical for inhibition of the enzyme sialyltransferase (ST) novel analogues with modifications of the skeleton (7-9, 16-18 and 20) were designed and synthesized. Methyl 3α-acetoxy-7-oxo-cholanate (1), methyl 3α-acetoxy-12-oxo-cholanate (2) and methyl 3α,7α-diacetoxy-12-oxo-cholanate (3) were subjected to Baeyer-Villiger oxidation to provide homolactones (7-9) or to the Beckmann rearrangement of the corresponding oximes to give homolactams (16-18). Both reactions proceed regio- and stereoselectively. Ring B homolog of lithocholic acid (20) was efficiently synthesized. Among these compounds, 7, 9 and 16 were found to have the significant activity, with IC50 values ⩽3μM against α-2,6-(N)-ST selectively, which are 5-fold lower than that of Lith-O-Asp. Given the reality that LCA and its analogue, Lith-O-Asp, have been revealed to improve inhibitory efficacy of ST and to have a wide range of antimetastatic activities in different human cancer cells, the up-to-date findings have noteworthy pharmacological significance as they open a promising path to the improvement of a prospective molecular targeted application of modified LCA analogues as agents for the treatment of cancer metastasis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Lithocholic acid (LCA), a hepatotoxic and carcinogenic bile acid, is metabolized to 3-ketocholanoic acid (3-KCA) by cytochrome P450 3A (CYP3A). In the present study, the objectives were to develop and validate an ultra-high performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to quantify 3-KCA and apply it to the human liver microsomal CYP3A-dependent LCA 3-oxidation assay. Chromatographic separation was achieved on a Waters ACQUITY™ UPLC C18 column (50×2.1mm, 1.7μm) with a gradient system consisting of 0.1% v/v formic acid in water (solvent A) and 0.1% v/v formic acid in acetonitrile (solvent B). The retention time was 3.73min for 3-KCA and 2.73min for cortisol (internal standard). Positive electrospray ionization with multiple reaction monitoring (MRM) mode was used to quantify 3-KCA (m/z 375.4→135.2) and cortisol (m/z 363.5→121.0). The limit of detection of 3-KCA was 10μM, the lower limit of quantification was 33.3μM, and the calibration curve was linear from 0.05-10μM with r(2)>0.99. Intra-day and inter-day accuracy and precision were <13.7%. The quality control samples were stable when assessed after 4h at room temperature, 24h at 4°C, 14days at -20°C, and three freeze-thaw cycles. The liver microsomal matrix did not affect 3-KCA quantification. The amount of KCA formed in the human liver microsomal LCA 3-oxidation assay was linear with respect to the amount of microsomal protein (up to 40μg) and incubation time (5-30min). Enzyme kinetics experiment indicated that LCA 3-oxidation followed the Michaelis-Menten model with an apparent Km of 26±7μM and Vmax of 303±50pmol/min/mg protein. This novel UPLC-MS/MS method for quantifying 3-KCA offers a specific, sensitive, and fast approach to determine liver microsomal LCA 3-oxidation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Detoxification of lithocholic acid (LCA) to lithocholic acid sulfate (LCA-S) is catalyzed by sulfotransferases, mainly SULT2A1. We developed and validated an ultra-high performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to quantify human liver cytosolic-dependent LCA sulfation. Chromatographic separation was achieved on an UPLC C18 column (2.1×50mm, 1.7μm) and a gradient elution of 0.1% formic acid in water and acetonitrile. Negative electrospray ionization with multiple reaction monitoring (MRM) mode was used to quantify LCA-S (455.3→97.0) and cholic acid (407.2→343.3; internal standard). The retention time was 3.51min for LCA-S and 3.08min for cholic acid. The lower limit of quantification of LCA-S was 0.5nM (or 0.23ng/ml in 400μl total volume) and the assay was linear from 0.2 to 200pmol. Intra-day and inter-day accuracy and precision were <14%. The quality control samples were stable at room temperature for 4h, 4°C for 24h, -20°C for 14 days, and after three freeze-thaw cycles. The matrix (20-100μg cytosolic protein) did not affect LCA-S quantification. This is the first UPLC-MS/MS method applied to optimization of the human liver cytosolic LCA sulfation assay. The optimal levels of MgCl2 and 3'-phosphoadenosine 5'-phosphosulfate (PAPS) cofactor were 2.5mM and 20μM, respectively. Addition of reducing agents (2-mercaptoethanol and DL-dithiothreitol) did not affect LCA-S formation. Human liver cytosolic LCA sulfation was linear with 20-100μg of cytosolic protein and 5-30min incubation time. This UPLC-MS/MS approach offers a specific, sensitive, fast, and direct approach for quantifying human liver cytosolic LCA sulfation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Amino acid conjugates of lithocholic acid (LCA) have been recently described as effective disruptors of the EphA2-ephrin-A1 interaction able to inhibit EphA2 phosphorylation in intact cells and thus able to block prometastatic responses such as cellular retraction and angiogenesis. However, these LCA-based compounds were significantly more potent at disrupting the EphA2-ephrin-A1 interaction than at blocking phenotype responses in cells, which might reflect an unclear mechanism of action or a metabolic issue responsible for a reduction of the compound concentration at the cell's surface. Through the synthesis of new compounds and their examination by a combination of cell-based assays and real-time interaction analysis by surface plasmon resonance, we showed at molecular level that l-tryptophan conjugates of lithocholic acid disrupt EphA2-ephrin-A1 interaction by targeting the EphA 2 receptor and that the presence of a polar group in position 3 of steroid scaffold is a key factor to increase the effective concentration of the compounds in cancer cell lines., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In order to develop bioactive lithocholic acid derivatives, we prepared fifteen semi-synthetic compounds through modification at C-3 and/or C-24. The reactions showed yields ranging from 37% to 100%. The structures of all compounds obtained were identified on the basis of their spectral data (IR, MS, 1D- and 2D-NMR). The activity of lithocholic acid and derivatives was evaluated against the growth of Escherichia coli, Staphylococcus aureus, Bacillus cereus and Pseudomonas aeruginosa. The derivative 3α-formyloxy-5β-cholan-24-oic acid (LA-06) showed the best activity, with MIC values of 0.0790 mM against E. coli (Ec 27) and B. cereus in both cases, and 0.0395 mM against S. aureus (ATCC 12692). Lithocholic acid and the derivatives with MIC⩽1.2 mM were evaluated on the susceptibility of some bacterial pathogens to the aminoglycoside antibiotics neomycin, amikacin and gentamicin was evaluated. There are no previously reported studies about these compounds as modifiers of the action of antibiotics or any other drugs., (Copyright © 2015. Published by Elsevier Inc.)

Bile acids can regulate nutrient metabolism through the activation of the cell membrane receptor GPBAR1 and the nuclear receptor FXR. Developing an exogenous control over these receptors represents an attractive strategy for the treatment of enterohepatic and metabolic disorders. A number of dual GPBAR1/FXR agonists are known, however their therapeutic use is limited by multiple unwanted effects due to activation of the diverse downstream signals controlled by the two receptors. On the other hand, designing selective GPBAR1 and FXR agonists is challenging since the two proteins share similar structural requisites for ligand binding. Here, taking advantage of our knowledge of the two targets, we have identified through a rational drug design study a series of amine lithocholic acid derivatives as selective GPBAR1 agonists. The presence of the 3α-NH2 group on the steroidal scaffold is responsible for the selectivity over FXR unveiling unprecedented structural insights into bile acid receptors activity modulation.

Novel fucoxanthin derivatives that could change the size of mixed micelles were synthesized. The mixed micelles under consideration consist of a bile acid and some additives. To change the affinity against a bile acid, we designed the synthesis of a fucoxanthin-lithocholic acid complex. Lithocholic acid is one of the bile acids. The 3-OH on lithocholic acid was protected by a levulinyl group, and the protected lithocholic acid was selectively coupled via an ester linkage to the 3-OH on fucoxanthin to obtain levulinyl-protected lithocholyl fucoxanthin (LevLF). The levulinyl group was then selectively deprotected using hydrazine to obtain a lithocholyl fucoxanthin (LF). The average sizes of the micelles that contained these compounds (fucoxanthin, LevLF, and LF) with a bile acid (sodium taurocholate) were measured. The LevLF induced larger micelles than fucoxanthin or LF. Interestingly, the addition of 1-oleoyl-rac-glycerol induced a more efficient change in the micelle size. The large micelles grew larger, and the small micelles became smaller. Triple-mixed micelles with LevLF, sodium taurocholate, and 1-oleoyl-rac-glycerol formed the largest micelle with a diameter of 68 nm. On the other hand, triple-mixed micelles using LF, sodium taurocholate, and 1-oleoyl-rac-glycerol made the smallest micelles with diameters as low as 12 nm. We also investigated the hydrolysis of these compounds with enzymes (esterase from porcine liver, lipase from porcine pancreas, and cholesterol esterase from Pseudomonas sp.). The ester linkage between the lithocholic acid and fucoxanthin of LevLF was hydrolyzed with cholesterol esterase. In addition, the intestinal absorption was examined with Caco-2 cells, and no advantageous change in absorption efficiency was observed by chemically modifying the fucoxanthin unless different micelles sizes and increasing hydrophobicity are induced.

Background and Purpose: The Eph receptor tyrosine kinases and their ephrin ligands are key players in tumorigenesis and many reports have correlated changes in their expression with a poor clinical prognosis in many solid tumours. Agents targeting the Eph-ephrin system might emerge as new tools useful for the inhibition of different components of cancer progression. Even if different classes of small molecules targeting Eph-ephrin interactions have been reported, their use is hampered by poor chemical stability and low potency. Stable and potent ligands are crucial to achieve robust pharmacological performance., Experimental Approach: UniPR129 (the L-homo-Trp conjugate of lithocholic acid) was designed by means of computational methods, synthetized and tested for its ability to inhibit the interaction between the EphA2 receptor and the ephrin-A1 ligand in an elisa binding study. The ability of UniPR129 to disrupt EphA2-ephrin-A1 interaction was functionally evaluated in a prostate adenocarcinoma cell line and its anti-angiogenic effect was tested in vitro using cultures of HUVECs., Key Results: UniPR129 disrupted EphA2-ephrin-A1 interaction with Ki = 370 nM in an elisa binding assay and with low micromolar potency in cellular functional assays, including inhibition of EphA2 activation, inhibition of PC3 cell rounding and disruption of in vitro angiogenesis, without cytotoxic effects., Conclusions and Implications: The discovery of UniPR129 represents not only a major advance in potency compared with the existing Eph-ephrin antagonists but also an improvement in terms of cytotoxicity, making this molecule a useful pharmacological tool and a promising lead compound., (© 2014 The British Pharmacological Society.)

In this study we investigated resveratrol (trans-3,5,4'-trihydroxystilbene) solubilization with sodium 3α,12 α-dihydroxy-7-oxo-5 β-cholanoate (S7-OD) and sodium dodecyl sulfate (SDS). The investigation was aimed at determining whether large spherical micelles (SDS) or small longitudinal micelles (S7-OD) are more convenient for incorporation of resveratrol. Also, we studied resveratrol behavior in mixed micelles with mentioned surfactants using spectroflourimetric method as well as the effects of sodium chloride and urea on resveratrol solubilization capacity in the applied surfactants. Resveratrol solubilization curve was different in the investigated surfactants. Resveratrol solubilization curve for sodium 3α,12 α-dihydroxy-7-oxo-5 β-cholanoate at concentration 0.9 CMC reached saturation level of 60% dissolved resveratrol. The curve for sodium dodecyl sulfate was linear within the whole range of the investigated concentration; resveratrol solubilization rate reached 13% at 2 CMC. In S7-OD, NaCl increased capacity of resveratrol solubilization up to 1.4 CMC surfactant concentration, whilst maximum level of dissolved resveratrol (90%) was observed at 0.9 CMC. In SDS, NaCl decreased resveratrol solubilization capacity. Urea reduced resveratrol solubilization rate in sodium 3α ,12 α-dihydroxy-7-oxo-5 β-cholanoate, whereas it had inverse effect in sodium dodecyl sulfate. The obtained results strongly suggest that structure, that is, shape, of the surfactant micelles significantly affects their capacity of resveratrol solubilization. Also, presence of NaCl and urea influences solubilization capacities of investigated surfactants.

Bile acid composition in the colon is determined by bile acid flow in the intestines, the population of bile acid-converting bacteria, and the properties of the responsible bacterial enzymes. Ursodeoxycholic acid (UDCA) is regarded as a chemopreventive beneficial bile acid due to its low hydrophobicity. However, it is a minor constituent of human bile acids. Here, we characterized an UDCA-producing bacterium, N53, isolated from human feces. 16S rDNA sequence analysis identified this isolate as Ruminococcus gnavus, a novel UDCA-producer. The forward reaction that produces UDCA from 7-oxo-lithocholic acid was observed to have a growth-dependent conversion rate of 90-100% after culture in GAM broth containing 1 mM 7-oxo-lithocholic acid, while the reverse reaction was undetectable. The gene encoding 7β-hydroxysteroid dehydrogenase (7β-HSDH), which facilitates the UDCA-producing reaction, was cloned and overexpressed in Escherichia coli. Characterization of the purified 7β-HSDH revealed that the kcat/Km value was about 55-fold higher for the forward reaction than for the reverse reaction, indicating that the enzyme favors the UDCA-producing reaction. As R. gnavus is a common, core bacterium of the human gut microbiota, these results suggest that this bacterium plays a pivotal role in UDCA formation in the colon.

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) mediates glucocorticoid activation and is currently considered as therapeutic target to treat metabolic diseases; however, biomarkers to assess its activity in vivo are still lacking. Recent in vitro experiments suggested that human 11β-HSD1 metabolizes the secondary bile acid 7-oxolithocholic acid (7-oxoLCA) to chenodeoxycholic acid (CDCA) and minor amounts of ursodeoxycholic acid (UDCA). Here, we provide evidence from in vitro and in vivo studies for a major role of 11β-HSD1 in the oxidoreduction of 7-oxoLCA and compare its level and metabolism in several species. Hepatic microsomes from liver-specific 11β-HSD1-deficient mice were devoid of 7-oxoLCA oxidoreductase activity. Importantly, circulating and intrahepatic levels of 7-oxoLCA and its taurine conjugate were significantly elevated in mouse models of 11β-HSD1 deficiency. Moreover, comparative enzymology of 11β-HSD1-dependent oxidoreduction of 7-oxoLCA revealed that the guinea-pig enzyme is devoid of 7-oxoLCA oxidoreductase activity. Unlike in other species, 7-oxoLCA and its glycine conjugate are major bile acids in guinea-pigs. In conclusion, the oxidoreduction of 7-oxoLCA and its conjugated metabolites are catalyzed by 11β-HSD1, and the lack of this activity leads to the accumulation of these bile acids in guinea-pigs and 11β-HSD1-deficient mice. Thus, 7-oxoLCA and its conjugates may serve as biomarkers of impaired 11β-HSD1 activity.

The secondary bile acid lithocholic acid (LCA) and its derivatives act as selective modulators of the vitamin D receptor (VDR), although their structures fundamentally differ from that of the natural hormone 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3)]. Here, we have determined the crystal structures of the ligand-binding domain of rat VDR (VDR-LBD) in ternary complexes with a synthetic partial peptide of the coactivator MED1 (mediator of RNA polymerase II transcription subunit 1) and four ligands, LCA, 3-keto LCA, LCA acetate, and LCA propionate, with the goal of elucidating their agonistic mechanism. LCA and its derivatives bind to the same ligand-binding pocket (LBP) of VDR-LBD that 1,25(OH)2D3 binds to, but in the opposite orientation; their A-ring is positioned at the top of the LBP, whereas their acyclic tail is located at the bottom of the LBP. However, most of the hydrophobic and hydrophilic interactions observed in the complex with 1,25(OH)2D3 are reproduced in the complexes with LCA and its derivatives. Additional interactions between VDR-LBD and the C-3 substituents of the A-ring are also observed in the complexes with LCA and its derivatives. These may result in the observed difference in the potency among the LCA-type ligands.

Protein tyrosine phosphatase 1B (PTP1B) is a major negative regulator of both insulin and leptin signals. For years, inhibiting of PTP1B has been considered to be a potential therapeutics for treating Type 2 diabetes and obesity. Recently, we recognized lithocholic acid (LCA) as a natural inhibitor against PTP1B (IC(50)=12.74 μM) by a vertical screen for the first time. Further SAR research was carried out by synthesizing and evaluating a series of compounds bearing two methyls at C-4 position and a fused heterocycle to ring A. Among them, compound 14b achieved a PTP1B inhibitory activity about eightfold than LCA and a 14-fold selectivity over the homogenous enzyme TCPTP., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The oxidized bile acid 7-oxoLCA (7-oxolithocholic acid), formed primarily by gut micro-organisms, is reduced in human liver to CDCA (chenodeoxycholic acid) and, to a lesser extent, UDCA (ursodeoxycholic acid). The enzyme(s) responsible remained unknown. Using human liver microsomes, we observed enhanced 7-oxoLCA reduction in the presence of detergent. The reaction was dependent on NADPH and stimulated by glucose 6-phosphate, suggesting localization of the enzyme in the ER (endoplasmic reticulum) and dependence on NADPH-generating H6PDH (hexose-6-phosphate dehydrogenase). Using recombinant human 11β-HSD1 (11β-hydroxysteroid dehydrogenase 1), we demonstrate efficient conversion of 7-oxoLCA into CDCA and, to a lesser extent, UDCA. Unlike the reversible metabolism of glucocorticoids, 11β-HSD1 mediated solely 7-oxo reduction of 7-oxoLCA and its taurine and glycine conjugates. Furthermore, we investigated the interference of bile acids with 11β-HSD1-dependent interconversion of glucocorticoids. 7-OxoLCA and its conjugates preferentially inhibited cortisone reduction, and CDCA and its conjugates inhibited cortisol oxidation. Three-dimensional modelling provided an explanation for the binding mode and selectivity of the bile acids studied. The results reveal that 11β-HSD1 is responsible for 7-oxoLCA reduction in humans, providing a further link between hepatic glucocorticoid activation and bile acid metabolism. These findings also suggest the need for animal and clinical studies to explore whether inhibition of 11β-HSD1 to reduce cortisol levels would also lead to an accumulation of 7-oxoLCA, thereby potentially affecting bile acid-mediated functions.

Increased sialyltransferase (ST) activity promotes cancer cell metastasis, and overexpression of cell surface sialic acid correlates with poor prognosis in cancer patients. To seek therapies targeting metastasis for cancer treatment, we developed a novel ST inhibitor, Lith-O-Asp, and investigated its antimetastatic and antiangiogenic effects and mechanisms. We found that cells treated with Lith-O-Asp showed a reduction of activity on various ST enzymes by in vitro and cell-based activity analyses. Lith-O-Asp inhibited migration and invasion abilities in various cancer cell lines and showed inhibitory effect on the angiogenic activity of human umbilical vein endothelial cells. Indeed, Lith-O-Asp treatment consequently delayed cancer cell metastasis in experimental and spontaneous metastasis assays in animal models. Importantly, Lith-O-Asp decreased the sialic acid modification of integrin-β1 and inhibited the expression of phospho-FAK, phospho-paxillin, and the matrix metalloprotease (MMP) 2 and MMP9. Lith-O-Asp attenuated the Rho GTPase activity leading to actin dynamic impairment. In addition, 2DE-MS/MS and immunoblotting analyses showed that Lith-O-Asp altered the protein expression level and phosphorylation status of various proteins involved in crucial metastasis and angiogenesis pathways such as vimentin and ribonuclease/angiogenin inhibitor RNH1. Furthermore, Lith-O-Asp treatment significantly inhibited the invasive ability exerted by ectopic overexpression of various ST enzymes catalyzing α-2,6- or α-2,3-sialylation. Our results provide compelling evidence that the potential pan-ST inhibitor, Lith-O-Asp, suppressed cancer cell metastasis likely by inhibiting FAK/paxillin signaling and expressing antiangiogenesis factors. Lith-O-Asp is worthy for further testing as a novel antimetastasis drug for cancer treatment., (© 2011 AACR.)

Aberrant sialylation catalyzed by sialyltransferases (STs) is frequently found in cancer cells and is associated with increased cancer metastasis. However, ST inhibitors developed till now are not applicable for clinical use because of their poor cell permeability. In this study, a novel ST inhibitor AL10 derived from the lead compound lithocholic acid identified in our previous study is synthesized and the anti-cancer effect of this compound is studied. AL10 is cell-permeable and effectively attenuates total sialylation on cell surface. This inhibitor shows no cytotoxicity but inhibits adhesion, migration, actin polymerization and invasion of alpha-2,3-ST-overexpressing A549 and CL1.5 human lung cells. Inhibition of adhesion and migration by AL10 is associated with reduced sialylation of various integrin molecules and attenuated activation of the integrin downstream signaling mediator focal adhesion kinase. More importantly, AL10 significantly suppresses experimental lung metastasis in vivo without affecting liver and kidney function of experimental animals as determined by serum biochemical assays. Taken together, AL10 is the first ST inhibitor, which exhibits potent anti-metastatic activity in vivo and may be useful for clinical cancer treatment.

To develop an effective long-acting antidiabetic, the GLP-1 analogue of exendin-4 was modified with three different bile acids (BAs; cholic, deoxycholic, or lithocholic acid), at its two lysine residues. The biological, pharmaceutical, and physicochemical characteristics of these exendin-4 analogues were carefully investigated. Biological activity tests demonstrated that the monobile acid substitutions of exendin-4 showed well preserved receptor binding efficacy without noticeable insulinotropic or antidiabetic activity loss. However, physicochemical and pharmacokinetic studies revealed that the albumin-binding properties and in vivo elimination half-lives of BAM1-Ex4s (Lys(27)-BA-Ex4s) were significantly enhanced by increasing the hydrophobicities of the conjugated BAs. Furthermore, the protracted antidiabetic effects of the BAM1-Ex4s were also verified by the prolonged restoration of normoglycemia in type 2 diabetic mice. Accordingly, the present study suggests that the derivatization of exendin-4 with BAs offers a means of producing long-acting GLP-1 receptor agonists for type 2 diabetic therapy.

3alpha-Hydroxy-5 beta-cholan-24-oic (lithocholic) acid is a relatively minor component of hepatic bile acids in humans but is highly cytotoxic. Hepatic microsomal oxidation offers a potential mechanism for effective detoxification and elimination of bile acids. The aim of the present study was to investigate the biotransformation of lithocholic acid by human hepatic microsomes and to assess the contribution of cytochrome P450 (P450) enzymes in human hepatic microsomes using human recombinant P450 enzymes and chemical inhibitors. Metabolites were identified, and metabolite formation was quantified using a liquid chromatography/mass spectrometry-based assay. Incubation of lithocholic acid with human liver microsomes resulted in the formation of five metabolites, which are listed in order of their rates of formation: 3-oxo-5 beta-cholan-24-oic (3-ketocholanoic) acid, 3 alpha,6 alpha-dihydroxy-5 beta-cholan-24-oic (hyodeoxycholic) acid, 3 alpha,7 beta-dihydroxy-5 beta-cholan-24-oic (ursodeoxycholic) acid, 3 alpha,6 beta-dihydroxy-5 beta-cholan-24-oic (murideoxycholic) acid, and 3 alpha-hydroxy-6-oxo-5 beta-cholan-24-oic (6-ketolithocholic) acid. 3-Ketocholanoic acid was the major metabolite, exhibiting apparent K(m) and V(max) values of 22 muM and 336 pmol/min/mg protein, respectively. Incubation of lithocholic acid with a of human recombinant P450 enzymes revealed that all five metabolites were formed by recombinant CYP3A4. Chemical inhibition studies with human liver microsomes and recombinant P450 enzymes confirmed that CYP3A4 was the predominant enzyme involved in hepatic microsomal biotransformation of lithocholic acid. In summary, the results indicate that oxidation of the third carbon of the cholestane ring is the preferred position of oxidation by P450 enzymes for lithocholic acid biotransformation in humans and suggest that formation of lithocholic acid metabolites leads to enhanced hepatic detoxification and elimination.

The use of a bis-urea lithocholamide linker within a guanosine-sterol dimer resulted in formation of large and stable ion channels. The channels were longer-lived than those formed by the corresponding bis-carbamate.

Secondary bile acid-producing bacteria were isolated from human feces to improve our appreciation of the functional diversity and redundancy of the intestinal microbiota. In total, 619 bacterial colonies were isolated using a nutrient-poor agar medium and the level of secondary bile acid formation was examined in each by a liquid culture, followed by thin-layer chromatography. Of five strains analyzed by 16S rRNA gene sequencing and biochemical testing, one was identified as Bacteroides intestinalis AM-1, which was not previously recognized as a secondary bile-acid producer. GC-MS revealed that B. intestinalis AM-1 converts cholic acid (CA) and chenodeoxycholic acid into their 7-oxo derivatives, 7-oxo-deoxycholic acid (7-oxo-DCA) and 7-oxo-lithocholic acid, respectively. Thus, B. intestinalis AM-1 possesses 7alpha-hydroxysteroid dehydrogenase (7alpha-HSDH) activity. In liquid culture, B. intestinalis AM-1 showed a relatively higher productivity of 7-oxo-DCA than Escherichia coli HB101 and Bacteroides fragilis JCM11019(T), which are known to possess 7alpha-HSDH activity. The level of 7alpha-HSDH activity was higher in B. intestinalis AM-1 than in the other two strains under the conditions tested. The 7alpha-HSDH activity in each of the three strains is not induced by CA; instead, it is regulated in a growth phase-dependent manner.

Six new synthetic bile acid derivatives were synthesized and tested in vitro against various human cancer cells (glioblastoma multiforme (GBM), multiple myeloma (KMS-11), and colonic carcinoma (HCT-116) cell lines. The best activity was obtained with compound IIIb on multiple myeloma cells (LD(50): 8.5+/-0.5 microM). This activity was associated with Mcl-1 and PARP-1 cleavage, inhibition of NFkappaB signaling, and DNA fragmentation, demonstrating an apoptotic cell death signaling pathway.

Lithocholic acid (LCA) acetate induced the differentiation of human leukemia cells. Treatment with a combination of LCA acetate and cotylenin A, an inducer of the differentiation of leukemia cells, was more effective than that with LCA acetate or cotylenin A alone at inducing monocytic differentiation. LCA acetate activated mitogen-activated protein kinase (MAPK) before inducing differentiation. Cotylenin A did not activate MAPK, suggesting that cotylenin A has a different mode of action. The cooperative effects of LCA acetate and cotylenin A on inducing differentiation were, at least partly, due to the enhancement of LCA acetate-induced MAPK activation by cotylenin A.

1alpha,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], a vitamin D receptor (VDR) ligand, regulates calcium homeostasis and also exhibits noncalcemic actions on immunity and cell differentiation. In addition to disorders of bone and calcium metabolism, VDR ligands are potential therapeutic agents in the treatment of immune disorders, microbial infections, and malignancies. Hypercalcemia, the major adverse effect of vitamin D(3) derivatives, limits their clinical application. The secondary bile acid lithocholic acid (LCA) is an additional physiological ligand for VDR, and its synthetic derivative, LCA acetate, is a potent VDR agonist. In this study, we found that an additional derivative, LCA propionate, is a more selective VDR activator than LCA acetate. LCA acetate and LCA propionate induced the expression of the calcium channel transient receptor potential vanilloid type 6 (TRPV6) as effectively as that of 1alpha,25-dihydroxyvitamin D(3) 24-hydroxylase (CYP24A1), whereas 1,25(OH)(2)D(3) was more effective on TRPV6 than on CYP24A1 in intestinal cells. In vivo experiments showed that LCA acetate and LCA propionate effectively induced tissue VDR activation without causing hypercalcemia. These bile acid derivatives have the ability to function as selective VDR modulators.

The major bile acids present in the gallbladder bile of the common Australian wombat (Vombatus ursinus) were isolated by preparative HPLC and identified by NMR as the taurine N-acylamidates of chenodeoxycholic acid (CDCA) and 15alpha-hydroxylithocholic acid (3alpha,15alpha-dihydroxy-5beta-cholan-24-oic acid). Taurine-conjugated CDCA constituted 78% of biliary bile acids, and (taurine-conjugated) 15alpha-hydroxylithocholic acid constituted 11%. Proof of structure of the latter compound was obtained by its synthesis from CDCA via a Delta14 intermediate. The synthesis of its C-15 epimer, 15beta-hydroxylithocholic acid (3alpha,15beta-dihydroxy-5beta-cholan-24-oic acid), is also reported. The taurine conjugate of 15alpha-hydroxylithocholic acid was synthesized and shown to have chromatographic and spectroscopic properties identical to those of the compound isolated from bile. It is likely that 15alpha-hydroxylithocholic acid is synthesized in the wombat hepatocyte by 15alpha-hydroxylation of lithocholic acid that was formed by bacterial 7alpha-dehydroxylation of CDCA in the distal intestine. Thus, the wombat appears to use 15alpha-hydroxylation as a novel detoxification mechanism for lithocholic acid.

The aim of this study was to evaluate the usefulness of human intestinal LS180 cells for studying the induction of CYP3A4 mRNA expression via vitamin D receptor (VDR). CYP3A4 mRNA expression in LS180 cells treated with 100 nM 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) for 6 and 24 h was approximately 80- and 500-fold higher than the control, respectively. A protein kinase (PK) inhibitor (staurosporine), c-jun N-terminal kinase (JNK) pathway inhibitor (curcumin), and JNK inhibitor (SP600125) attenuated 1alpha,25(OH)(2)D(3)-induced CYP3A4 mRNA expression, suggesting that the PK-JNK pathway contributed to the rapid and drastic induction of CYP3A4 expression via VDR in LS180 cells. The ability of CYP3A4 mRNA induction in LS180 cells was highly dependent on the site and number of vitamin D(3) and D(2) hydroxylation. In addition, short-time (6 h) treatment of LS180 cells with cytotoxic secondary bile acids, lithocholic acid (LCA) and 3-keto-LCA also significantly induced the mRNA expression of CYP3A4. LS180 cells may be useful to quickly investigate the CYP3A4-inducing effect of drugs, xenobiotics, and/or endogenous substrates in the intestinal epithelia.

Purpose: Anitangiogenic and apoptotic properties of a novel chemically modified heparin derivative with low anticoagulant activity were evaluated on the experimental in vitro and in vivo model., Materials and Methods: Heparin-lithocholate conjugate (HL) was initially synthesized by covalently bonding lithocholate to heparin. Folate-HL conjugate (FHL) was further synthesized by conjugating folate to HL. Antiangiogenic and apoptotic abilities of HL and FHL were characterized in vitro and in vivo experimentations., Results: Compared to unmodified heparin, both HL and FHL represented a low anticoagulant activity (38 and 28%, respectively). HL and FHL maintained antiangiogenic activity even further modification from the results of Matrigel plugs assay. FHL specifically induced apoptosis on KB cells having highly expressed folate receptor after cellular internalization. Both administered HL and FHL had similar antiangiogenic activity and inhibitory effect on tumor growth in vivo although FHL induced higher apoptosis on tumor tissues., Conclusions: In vivo tumor growth inhibition was possibly due to the decrease of vessel density and apoptotic cell death, although antiangiogenic effect of FHL seemed more actively affected on growth inhibition than apoptotic potential in vivo system. Thus, Low anticoagulant FHL having antiangiogenic and apoptotic properties would provide benefits for the development of a new class of anticancer agent.

A new type of noncompetitive alpha-2,3-sialyltransferase inhibitor has been synthesized; we report the discovery, preparation and inhibitory activity of sixteen lithocholic acid analogues.

Emerging evidence indicates that some secondary bile acids interact functionally with muscarinic cholinergic receptors. Using thoracic aortic rings prepared from rats and mice, we examined the mechanism of deoxycholyltaurine-induced vasorelaxation. Increasing concentrations of both acetylcholine (1 nM to 0.1 mM) and deoxycholyltaurine (0.1 microM to 1 mM) stimulated relaxation of phenylephrine-constricted rings prepared from rat thoracic aortae. These effects were reduced by endothelial denudation and by treatment with an inhibitor of nitric oxide formation and with a synthetic acetylcholine:bile acid hybrid that acts as a muscarinic receptor antagonist. Likewise, both acetylcholine (1 nM to 0.1 mM) and deoxycholyltaurine (0.1 microM to 0.1 mM) stimulated relaxation of phenylephrine-constricted rings prepared from mouse thoracic aortae. These effects were reduced by endothelial denudation, addition of an inhibitor of nitric oxide formation, and by muscarinic M(3) receptor knockout. We conclude that the systemic vasodilatory actions of deoxycholyltaurine are mediated in part by a nitric oxide-, muscarinic M(3) receptor-dependent mechanism. In advanced liver disease, interaction of serum bile acids with endothelial muscarinic receptors may explain nitric oxide overproduction in the systemic circulation and resulting peripheral arterial vasodilation.

The vitamin D receptor (VDR), a member of the nuclear receptor superfamily, mediates the biological actions of the active form of vitamin D, 1alpha,25-dihydroxyvitamin D(3). It regulates calcium homeostasis, immunity, cellular differentiation, and other physiological processes. Recently, VDR was found to respond to bile acids as well as other nuclear receptors, farnesoid X receptor (FXR) and pregnane X receptor (PXR). The toxic bile acid lithocholic acid (LCA) induces its metabolism through VDR interaction. To elucidate the structure-function relationship between VDR and bile acids, we examined the effect of several LCA derivatives on VDR activation and identified compounds with more potent activity than LCA. LCA acetate is the most potent of these VDR agonists. It binds directly to VDR and activates the receptor with 30 times the potency of LCA and has no or minimal activity on FXR and PXR. LCA acetate effectively induced the expression of VDR target genes in intestinal cells. Unlike LCA, LCA acetate inhibited the proliferation of human monoblastic leukemia cells and induced their monocytic differentiation. We propose a docking model for LCA acetate binding to VDR. The development of VDR agonists derived from bile acids should be useful to elucidate ligand-selective VDR functions.

Four lithocholic acid dimers were synthesised via esterification. The ester-linked dimer, 3-oxo-5beta-cholan-24-oic acid (cholan-24-oic acid methyl ester)-3-yl ester, (3alpha,5beta), was obtained by condensation of methyl lithocholate with 3-oxo-5beta-cholan-24-oic acid. Borohydride reduction of this ester-linked dimer gave 3alpha-hydroxy-5beta-cholan-24-oic acid (cholan-24-oic acid methyl ester)-3-yl ester, (3alpha,5beta), which was acetylated to 3alpha-acetoxy-5beta-cholan-24-oic acid (cholan-24-oic acid methyl ester)-3-yl ester, (3alpha,5beta). Reaction of methyl lithocholate with oxalyl chloride yielded the oxalate dimer, bis(5beta-cholan-24-oic acid methyl ester)-3alpha-yl oxalate.

Besides their known physiological actions, bile acids are signaling molecules that alter cell function by interacting with muscarinic and nuclear receptors. Bile acid interaction with nuclear receptors modulates bile acid and cholesterol metabolism, whereas the potential consequences of muscarinic receptor activation are much broader. This review examines recent discoveries regarding bile acid interaction with muscarinic receptors. Selective and functional bile acid interaction has been reported with M3 receptors expressed in guinea pig gastric chief cells, human colon cancer cells, and transfected Chinese hamster ovary cells. Interaction of bile acids with chief cells may contribute to mucosal damage and other pathophysiological consequences of bile reflux. Bile acid-induced stimulation of muscarinic receptors on colon cancer cells may contribute to cellular proliferation and neoplasia. Potential consequences of bile acid interaction with muscarinic receptors on gastrointestinal myocytes, biliary epithelium, vascular endothelium and dermal neurons are discussed. Elucidation of molecular mechanisms underlying interaction of bile acids with muscarinic receptors may suggest new treatments for conditions that result from such interactions.

Bile acids alter regulatory pathways in several cell types. The molecular basis for these actions is not fully elucidated, but lithocholyltaurine interacts functionally with muscarinic receptors on gastric chief cells. In the present report, we demonstrate selective interaction of bile acids with Chinese hamster ovary (CHO) cells expressing each of the five muscarinic receptors. Lithocholyltaurine decreases binding of a radioligand to muscarinic M3 receptors, but not to other muscarinic receptors. Sulfated lithocholyltaurine, the major human metabolite, inhibits radioligand binding to muscarinic M1, but not to M2 or M3 receptors. Post-receptor actions of lithocholyltaurine include modulation of acetylcholine-induced increases in inositol phosphate formation and mitogen-activated protein (MAP) kinase phosphorylation. Molecular modeling suggests that the specific and functional interaction of lithocholyltaurine with muscarinic receptors is most likely due to similar shape and surface charge distribution of portions of acetylcholine and the bile acid. We propose that bile acids are signaling molecules whose effects may be mediated by interaction with muscarinic receptors.