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OleP is a bacterial cytochrome P450 involved in oleandomycin biosynthesis as it catalyzes regioselective epoxidation on macrolide intermediates. OleP has recently been reported to convert lithocholic acid (LCA) into murideoxycholic acid through a highly regioselective reaction and to unspecifically hydroxylate testosterone (TES). Since LCA and TES mainly differ by the substituent group at the C17, here we used X-ray crystallography, equilibrium binding assays, and molecular dynamics simulations to investigate the molecular basis of the diverse reactivity observed with the two steroids. We found that the differences in the structure of TES and LCA affect the capability of these molecules to directly form hydrogen bonds with N-terminal residues of OleP internal helix I. The establishment of these contacts, by promoting the bending of helix I, fosters an efficient trigger of the open-to-closed structural transition that occurs upon substrate binding to OleP and contributes to the selectivity of the subsequent monooxygenation reaction., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

In the study, we aimed to investigate the activity of nanoformulations containing 5-fluorouracil and polymer-magnetic hybrids bearing membrane-penetrating and ligand-receptor-recognizing agents against colorectal cancer cells. The formation and characterization of iron oxide particles covered with polymeric shells comprising lithocholic acid and folic acid moieties are presented. The efficiency of nanoformulations combined by the simple mixing of low doses of 5-fluorouracil with the obtained hybrids was demonstrated against DLD-1 and HT-29 colon cancer cells. The most pronounced cytotoxic potential against HT-29 cells was observed in the cases of particles based on block and randomly arranged copolymers functionalized by FA motifs with depletion of viable cells by approximately 50 % compared to control cells and cells treated by 5-FU applied in free form. In the case of the DLD-1 cell line, the percentage of viable DLD-1 cells decreased by about 30 to 40% after treatment with the block and randomly arranged copolymer decorated by FA-moiety, when compared to 5-FU at the free form and the untreated control. The induction of apoptosis associated with PS-translocation was determined to be the main mechanism of their cytotoxic effects. Moreover, the safety profiles of the nanoformulations were established through hemolysis assay and the analysis of the viability of human colorectal fibroblasts. It was indicated that all tested nanoparticles met the compatibility requirements at the in vitro level. It should be emphasized that in many cases, there was a significant improvement in the compatibility of hybrids with the FA motif compared to non-functionalized hybrids with the addition of 5-FU. These findings suggest that the presence of FA might modulate the toxicity of chemotherapeutic agents., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Agnieszka Zofia Wilczewska reports financial support was provided by National Science Centre Poland. If there are other authors, they 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. Published by Elsevier B.V.)

Lithocholic acid (LCA), a secondary bile acid, has emerged as a potential early diagnostic biomarker for various liver diseases. In this study, we introduce a novel near-infrared (NIR) polymethine dye-based biosensor, capable of sensitive and selective detection of LCA in phosphate buffer and artificial urine (AU) solutions. The detection mechanism relies on the formation of J-aggregates resulting from the interplay of 3,3-Diethylthiatricarbocyanine iodide (DiSC 2 (7)) dye molecules and LCA, which induces a distinctive red shift in both absorption and fluorescence spectra. The biosensor demonstrates a detection limit for LCA of 70 μM in PBS solution (pH 7.4), while in AU solution, it responds to an LCA concentration as low as ∼60 μM. Notably, the proposed biosensor exhibits outstanding selectivity for LCA, effectively distinguishing it from common interferents such as uric acid, ascorbic acid, and glucose. This rapid, straightforward, and cost-effective spectrometer-based method underscores its potential for early diagnosis of liver diseases by monitoring LCA concentrations., 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 B.V. All rights reserved.)

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.

Oxidative stress is pivotal in retinal disease progression, causing dysfunction in various retinal components. An effective antioxidant, such as probucol (PB), is vital to counteract oxidative stress and emerges as a potential candidate for treating retinal degeneration. However, the challenges associated with delivering lipophilic drugs such as PB to the posterior segment of the eye, specifically targeting photoreceptor cells, necessitate innovative solutions. This study uses formulation-based spray dry encapsulation technology to develop polymer-based PB-lithocholic acid (LCA) nanoparticles and assesses their efficacy in the 661W photoreceptor-like cell line. Incorporating LCA enhances nanoparticles' biological efficacy without compromising PB stability. In vitro studies demonstrate that PB-LCA nanoparticles prevent reactive oxygen species (ROS)-induced oxidative stress by improving cellular viability through the nuclear erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway. These findings propose PB-LCA nanoparticles as a promising therapeutic strategy for oxidative stress-induced retinopathies.

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.)

Cytochrome P450 enzyme with 7β-hydroxylation capacity has attracted widespread attentions due to the vital roles in the biosynthesis of ursodeoxycholic acid (UDCA), a naturally active molecule for the treatment of liver and gallbladder diseases. In this study, a novel P450 hydroxylase (P450 FE ) was screen out from Fusarium equiseti HG18 and identified by a combination of genome and transcriptome sequencing, as well as heterologous expression in Pichia pastoris. The biotransformation of lithocholic acid (LCA) by whole cells of recombinant Pichia pastoris further confirmed the C7β-hydroxylation with 5.2% UDCA yield. It was firstly identified a fungal P450 enzyme from Fusarium equiseti HG18 with the capacity to catalyze the LCA oxidation producing UDCA. The integration of homology modeling and molecular docking discovered the substrate binding to active pockets, and the key amino acids in active center were validated by site-directed mutagenesis, and revealed that Q112, V362 and L363 were the pivotal residues of P450 FE in regulating the activity and selectivity of 7β-hydroxylation. Specifically, V362I mutation exhibited 2.6-fold higher levels of UDCA and higher stereospecificity than wild-type P450 FE . This advance provided guidance for improving the catalytic efficiency and selectivity of P450 FE in LCA hydroxylation, indicative of the great potential in green synthesis of UDCA from biologically toxic LCA., 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.)

Tumour cells secrete various proangiogenic factors like VEGF, PDGF, and EGF that result in the formation of highly vascularized tumours with an immunosuppressive tumour microenvironment. As tumour growth and metastasis are highly dependent on angiogenesis, targeting tumour vasculature along with rapidly dividing tumour cells is a potential approach for cancer treatment. Here, we specifically engineered sub-100 sized nanomicelles (DTX-CA4 NMs) targeting proliferation and angiogenesis using an esterase-sensitive phosphocholine-tethered docetaxel conjugate of lithocholic acid (LCA) (PC-LCA-DTX) and a poly(ethylene glycol) (PEG) derivative of an LCA-combretastatin A4 conjugate (PEG-LCA-CA4). DTX-CA4 NMs effectively inhibit the tumour growth in syngeneic (CT26) and xenograft (HCT116) colorectal cancer models, inhibit tumour recurrence, and enhance the percentage survival in comparison with individual drug-loaded NMs. DTX-CA4 NMs enhance the T cell-mediated anti-tumour immune response and DTX-CA4 NMs in combination with an immune checkpoint inhibitor, anti-PDL1 antibody, enhance the anti-tumour response. We additionally showed that DTX-CA4 NMs effectively attenuate the production of ceramide-1-phosphate, a key metabolite of the sphingolipid pathway, by downregulating the expression of ceramide kinase at both transcriptional and translational levels. Therefore, this study presents the engineering of effective DTX-CA4 NMs for targeting the tumour microenvironment that can be explored further for clinical applications.

A high number of biologically active and low-calcemic secosteroidal ligands of the vitamin D receptor (VDR) have been developed, some of which are already used clinically although with limited success in the treatment of hyperproliferative diseases because the required pharmaceutical dosages induce toxicity. We describe here the in silico design, synthesis, structural analysis and biological evaluation of two novel active lithocholic acid derivatives hydroxylated at the side chain as highly potent inhibitors of atopic dermatitis-relevant keratinocyte inflammation of potential therapeutic interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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.)

The hypercalcemic effects of the hormone 1α,25-dihydroxyvitamin D 3 (calcitriol) and most of known vitamin D metabolites and analogs call for the development of non secosteroidal vitamin D receptor (VDR) ligands as new selective and noncalcemic agonists for treatment of hyperproliferative diseases. We report on the in silico design and stereoselective synthesis of six lithocholic acid derivatives as well as on the calcemic activity of a potent LCA derivative and its crystallographic structure in complex with zVDR LBD. The low calcemic activity of this compound in comparison with the native hormone makes it of potential therapeutic value. Structure-function relationships provide the basis for the development of even more potent and selective lithocholic acid-based VDR ligands., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Lithocholic acid ( 2 ) was identified as a second endogenous ligand of vitamin D receptor (VDR), though its activity is very weak. In this study, we designed novel lithocholic acid derivatives based on the crystal structure of VDR-ligand-binding domain (LBD) bound to 2 . Among the synthesized compounds, 6 bearing a 2-hydroxy-2-methylprop-1-yl group instead of the 3-hydroxy group at the 3α-position of 2 showed dramatically increased activity in HL-60 cell differentiation assay, being at least 10 000 times more potent than lithocholic acid ( 2 ) and 3 times more potent than 1α,25-dihydroxyvitamin D 3 ( 1 ). Although the binding affinities of 6 and its epimer 7 were less than that of 1 , their transactivation activities were greater than that of 1 . X-ray structure analyses of VDR LBD bound to 6 or 7 showed that the binding positions of these compounds in the ligand-binding pocket are similar to that of 1 .

We engineered the cytochrome P450 monooxygenase CYP107D1 (OleP) from Streptomyces antibioticus for the stereo- and regioselective 7β-hydroxylation of lithocholic acid (LCA) to yield ursodeoxycholic acid (UDCA). OleP was previously shown to hydroxylate testosterone at the 7β-position but LCA is exclusively hydroxylated at the 6β-position, forming murideoxycholic acid (MDCA). Structural and 3DM analysis, and molecular docking were used to identify amino acid residues F84, S240, and V291 as specificity-determining residues. Alanine scanning identified S240A as a UDCA-producing variant. A synthetic "small but smart" library based on these positions was screened using a colorimetric assay for UDCA. We identified a nearly perfectly regio- and stereoselective triple mutant (F84Q/S240A/V291G) that produces 10-fold higher levels of UDCA than the S240A variant. This biocatalyst opens up new possibilities for the environmentally friendly synthesis of UDCA from the biological waste product LCA., (© 2020 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The present study was intended to develop a papain grafted S-protected hyaluronic acid-lithocholic acid co-block (PAP-HA-ss-LCA) polymeric excipient as an amphiphilic muco permeating stabilizer for targeting breast cancer epithelial cells overexpressed with CD44 receptors. The mucopermeating, stabilizing and targeting capability of the PAP-HA-ss-LCA polymeric excipient was investigated by manufacturing tamoxifen (TMX) loaded self-nanoemulsifying drug delivery system (SNEDDS). TMX loaded PAP-HA-ss-LCA incorporated SNEDDS (TMX-PAP-HA-ss-LCA SNEDDS) were characterized for their surface chemistry, drug release, permeation enhancement, biocompatibility and antitumor activity. FTIR spectroscopic analysis showed successful synthesis of PAP-HA-ss-LCA polymer. X-ray diffraction (XRD) showed the amorphous form of TMX inside SNEDDS. The observed hydrodynamic diameter of TMX-PAP-HA-ss-LCA SNEDDS was 367.5 nm. Furthermore, Hyaluronic Acid-based Mucoadhesive Self Nanoemulsifying Drug Delivery System (SNEDDS) of TMX showed homogeneity in synthesis with low polydispersity and negative zeta potential due to stabilization with PAP-HA-ss-LCA polymer. The distinct spherical shape of the nanodroplets was evident by transmission electron microscopy (TEM). In vitro release kinetics indicated approximately >80% release within 48 h under sink conditions. Ex-vivo permeation study displayed 7.11-folds higher permeation of TMX by TMX-PAP-HA-ss-LCA in contrast to pure TMX. The biocompatibility study proved that SNEDDS formulation was safe and compatible against macrophages. In vitro cytotoxicity studies demonstrated that TMX-PAP-HA-ss-LCA SNEDDS could efficiently kill MCF-7 breast cancer cells as compared to the native TMX drug. Systemic toxicity studies proved the non-toxic nature of TMX-PAP-HA-ss-LCA in contrast to pure TMX. Based on these evidences, TMX-PAP-HA-ss-LCA SNEDDS formulation seems to be promising mucopermeating, augmented intracellular uptake with strong targeting potential for anti-proliferative activity., (Copyright © 2020. Published by Elsevier B.V.)

The gut microbiota synthesize hundreds of molecules, many of which influence host physiology. Among the most abundant metabolites are the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA), which accumulate at concentrations of around 500 μM and are known to block the growth of Clostridium difficile 1 , promote hepatocellular carcinoma 2 and modulate host metabolism via the G-protein-coupled receptor TGR5 (ref. 3 ). More broadly, DCA, LCA and their derivatives are major components of the recirculating pool of bile acids 4 ; the size and composition of this pool are a target of therapies for primary biliary cholangitis and nonalcoholic steatohepatitis. Nonetheless, despite the clear impact of DCA and LCA on host physiology, an incomplete knowledge of their biosynthetic genes and a lack of genetic tools to enable modification of their native microbial producers limit our ability to modulate secondary bile acid levels in the host. Here we complete the pathway to DCA and LCA by assigning and characterizing enzymes for each of the steps in its reductive arm, revealing a strategy in which the A-B rings of the steroid core are transiently converted into an electron acceptor for two reductive steps carried out by Fe-S flavoenzymes. Using anaerobic in vitro reconstitution, we establish that a set of six enzymes is necessary and sufficient for the eight-step conversion of cholic acid to DCA. We then engineer the pathway into Clostridium sporogenes, conferring production of DCA and LCA on a nonproducing commensal and demonstrating that a microbiome-derived pathway can be expressed and controlled heterologously. These data establish a complete pathway to two central components of the bile acid pool.

Probucol (PB) is a drug that exhibits significant hydrophobicity and substantial intra and inter individual variability in oral absorption, with a miniature bioavailability and complex three compartmental pharmacokinetic modelling due to its high lipid affinity, low stability and high octanol to water partition coefficient. Multiple attempts to formulate PB have not produced satisfactory stable matrices, drug-release profile or rheological flow properties for optimum manufacturing conditions, and with positive and none toxic biological effects. Lithocholic acid (LCA) has recently shown to optimise formulation and cell uptake of drugs. Hence, the aim of this study was to design new PB delivery system, using LCA, and examine its morphology, rheology, stability, and cellular effects. PB was formulated with LCA and sodium alginate (PB-LCA-SA) using various microencapsulation methodologies, and best formulation was investigated in vitro and ex vivo. Using our Ionic Gelation Vibrational Jet flow technology, PB-LCA-SA microcapsules showed good stability and significantly enhanced cell viability, cellular respiration, and reduced inflammation suggesting potential LCA applications in PB delivery and biological effects.

Objective: Regio- and stereoselective hydroxylation of lithocholic acid (LCA) using CYP107D1 (OleP), a cytochrome P450 monooxygenase from the oleandomycin synthesis pathway of Streptomyces antibioticus., Results: Co-expression of CYP107D1 from S. antibioticus and the reductase/ferredoxin system PdR/PdX from Pseudomonas putida was performed in Escherichia coli whole cells. In vivo hydroxylation of LCA exclusively yielded the 6β-OH product murideoxycholic acid (MDCA). In resting cells, 19.5% of LCA was converted to MDCA within 24 h, resulting in a space time yield of 0.04 mmol L -1 h -1 . NMR spectroscopy confirmed the identity of MDCA as the sole product., Conclusions: The multifunctional P450 monooxygenase CYP107D1 (OleP) can hydroxylate LCA, forming MDCA as the only product.

Metal-organic frameworks (MOFs) for enzyme encapsulation-induced biomimetic mineralization under mild reaction conditions are commonly microporous and hydrophobic, which result in a rather high mass transfer resistance of the reactants and restrain the enzyme catalytic activity. Herein, we prepared a type of hierarchical porous and hydrophilic MOF through the biomimetic mineralization of enzymes, zinc ions, 2-methylimidazole, and lithocholic acid. The hierarchical porous structure accelerated the diffusion process of the reactants and the increased hydrophilicity conferred interfacial activity and increased the enzyme catalytic activity. The immobilized enzyme retained higher catalytic activity than the free enzyme and exhibited enhanced resistance to alkaline, organic, and high-temperature conditions. The nanobiocatalyst was reusable and showed long-term storage stability.

The EphA2 receptor has been validated in animal models as new target for treating tumors depending on angiogenesis and vasculogenic mimicry. In the present work, we extended our current knowledge on structure-activity relationship (SAR) data of two related classes of antagonists of the EphA2 receptor, namely 5β-cholan-24-oic acids and 5β-cholan-24-oyl l-β-homotryptophan conjugates, with the aim to develop new antiangiogenic compounds able to efficiently prevent the formation of blood vessels. As a result of our exploration, we identified UniPR505, N-[3α-(Ethylcarbamoyl)oxy-5β-cholan-24-oyl]-l-β-homo-tryptophan (compound 14), as a submicromolar antagonist of the EphA2 receptor capable to block EphA2 phosphorylation and to inhibit neovascularization in a chorioallantoic membrane (CAM) assay., 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 © 2020 Elsevier Masson SAS. All rights reserved.)

Bile acids are abundant in the mammalian gut, where they undergo bacteria-mediated transformation to generate a large pool of bioactive molecules. Although bile acids are known to affect host metabolism, cancer progression and innate immunity, it is unknown whether they affect adaptive immune cells such as T helper cells that express IL-17a (T H 17 cells) or regulatory T cells (T reg cells). Here we screen a library of bile acid metabolites and identify two distinct derivatives of lithocholic acid (LCA), 3-oxoLCA and isoalloLCA, as T cell regulators in mice. 3-OxoLCA inhibited the differentiation of T H 17 cells by directly binding to the key transcription factor retinoid-related orphan receptor-γt (RORγt) and isoalloLCA increased the differentiation of T reg cells through the production of mitochondrial reactive oxygen species (mitoROS), which led to increased expression of FOXP3. The isoalloLCA-mediated enhancement of T reg cell differentiation required an intronic Foxp3 enhancer, the conserved noncoding sequence (CNS) 3; this represents a mode of action distinct from that of previously identified metabolites that increase T reg cell differentiation, which require CNS1. The administration of 3-oxoLCA and isoalloLCA to mice reduced T H 17 cell differentiation and increased T reg cell differentiation, respectively, in the intestinal lamina propria. Our data suggest mechanisms through which bile acid metabolites control host immune responses, by directly modulating the balance of T H 17 and T reg cells.

Polyethyleneimine (PEI) is widely used for the delivery of nucleic acids, but its clinical application is limited due to high cytotoxicity and instability in biological fluids. To overcome these challenges, linear PEI (2.5 kDa) was modified with lithocholic acid (LCA) to produce a LCA-PEI conjugate (lp), and its complex with plasmid DNA (pDNA) was covered with hyaluronic acid (HA). Ternary complexes of pDNA, lp, and HA ("DlpH") were prepared in different ratios and tested in cells and tumor-bearing mice for gene transfection efficiency. DlpH with a relatively high lp/pDNA ratio (Hi-DlpH) was more resistant to DNase and heparin treatment and showed more efficient gene transfection than DlpH with a lower lp/pDNA ratio (Lo-DlpH) in vitro. In contrast, Hi- and Lo-DlpH showed distinct transfection efficiency in vivo in a tumor-size dependent manner, where Hi-DlpH showed relatively high gene transfection in tumors of <300 mm 3 but performed poorly in tumors of >500 mm 3 and Lo-DlpH did the opposite. Tumor-associated macrophages, which increase with tumor growth and preferentially intercept Hi-DlpH, may account for the poor performance of Hi-DlpH in relatively large tumors. Accordingly, suggestions are made for future in vitro screening of new gene formulations to better predict their in vivo performances., (Copyright © 2019 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.)

Despite some efforts have been made in the research of supramolecular hyperbranched polymers (SHPs) self-assemblies, the study which has not been consideration to date is the influence of incoming stimuli-responsive polymer chain on their self-assembly property undergo outer stimuli. The introduction of stimuli-responsive segments which could maintain their hydrophilic property are expected to affect the self-assembly behaviour of SHPs and expand their further biomedical application. In this paper, AB 2 -type macromolecular monomer, LA-(CD-PDMA) 2 , which consisted one lithocholic acid (LA) and two β-cyclodextrin terminated poly(2-(dimethylamino)ethyl methacrylate) segments (CD-PDMA) was synthesized. LA-(CD-PDMA) 2 based SHP were obtained based on the host-guest inclusion interactions of CD/LA moietes and with PDMA as pH-responsive hydrophilic chains. As a control to study the influence of incoming PDMA chains, both LA-(CD-PDMA) 2 based SHPs-1 and LA-CD 2 based SHPs-2 self-assemblies were comparatively investiged through 2D 1 H NMR ROESY, transmission electron microscopy (TEM) and dynamic light scattering (DLS). The results suggested that except for the higher drug loading efficiency LA-(CD-PDMA) 2 based SHPs-1 pocessing, the release rates of SHPs-1 increased notably at pH 5.0 than that of pH 7.4 due to the repulsion and stretch of protonated PDMA chains while the release rates of SHPs-2 showed no obvious difference. Finally, basic cell experiments demonstrated that the SHPs based self-assemblies can be internalized into cancer cells, indicating their potential application in the drug delivery field., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Lipid modification of proteins plays a significant role in the activation of cellular signals such as proliferation. Thus, the demand for lipidated proteins is rising. However, getting a high yield and purity of lipidated proteins has been challenging. We developed a strategy for modifying proteins with a wide variety of synthetic lipids using microbial transglutaminase (MTG), which catalyzes the cross-linking reaction between a specific glutamine (Q) in a protein and lysine (K) in the lipid-fused peptide. The synthesized lipid-G 3 S-MRHKGS lipid (lipid: fatty acids, tocopherol, lithocholic acid, cholesterol) was successfully conjugated to a protein fused with LLQG (Q-tagged protein) by an MTG reaction, yielding >90 % conversion of the Q-tagged protein in a lipidated form. The purified lipid-protein conjugates were used for labeling the cell membrane in vitro, resulting in best-anchoring ability of cholesterol modification. Furthermore, in situ cell-surface decoration with the protein was established in a simple manner: subjection of cells to a mixture of cholesterol-fused peptides, Q-tagged proteins and MTG., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We have reported synthesis of a novel 1,2,3-triazole conjugate of lithocholic acid by 1,3-dipolar cycloaddition reaction. The molecular properties such as geometry, conformations, bond lengths and dihedral angles were investigated theoretically. The bond order analysis was performed using Wiberg bond order (WBO), Fuzzy bond order (FBO) and Laplacian bond order (MBO) method. Electronic properties of molecule such as electrostatic surface potential analysis, frontier molecular orbital analysis, reduced density gradient, total density of states, and global chemical reactivity indices have been investigated. The nonlinear optical properties were also investigated. Total dipole moment, mean polarizability and hyperpolarizability were found to be much higher than standard urea molecule which suggests that it could act as potential NLO material. The molecular docking calculations are also performed to investigate its potential as PTP 1B enzyme inhibitor., (Copyright © 2018. Published by Elsevier Ltd.)

The human gut hosts trillions of microorganisms that exert a profound influence on human biology. Gut bacteria communicate with their host by secreting small molecules that can signal to distant organs in the body. Bile acids are one class of these signaling molecules, synthesized by the host and chemically transformed by the gut microbiota. Among bile acid metabolizers, bile acid 7-dehydroxylating bacteria are commensals of particular importance as they carry out the 7-dehydroxylation of liver-derived primary bile acids to 7-dehydroxylated bile acids. The latter represents a major fraction of the secondary bile acid pool. The microbiology of this group of gut microorganisms is understudied and warrants more attention. Here, we detail the bile acid transformations carried out by the 7-dehydroxylating bacterium Clostridium scindens in vitro and in vivo. In vitro, C. scindens exhibits not only 7α-dehydroxylating capabilities but also, the ability to oxidize other hydroxyl groups and reduce ketone groups in primary and secondary bile acids. This study revealed 12-oxolithocholic acid as a major transient product in the 7α-dehydroxylation of cholic acid. Furthermore, the in vivo study included complementing a gnotobiotic mouse line (devoid of the ability to 7-dehydroxylate bile acids) with C. scindens and investigating its colonization dynamics and bile acid transformations. Using NanoSIMS (Nanoscale Secondary Ion Mass Spectrometry), we demonstrate that the large intestine constitutes a niche for C. scindens , where it efficiently 7-dehydroxylates cholic acid to deoxycholic acid. Overall, this work reveals a novel transient species during 7-dehydroxylation as well as provides direct evidence for the colonization and growth of 7-dehydroxylating bacteria in the large intestine.

GLP-1 analogs suffer from the main disadvantage of a short in vivo half-life. Lithocholic acid (LCA), one of the four main bile acids in the human body, possesses a high albumin binding rate. We therefore envisioned that a LCA-based peptide delivery system could extend the half-life of GLP-1 analogs by facilitating the noncovalent binding of peptides to human serum albumin. On the basis of our previously identified Xenopus GLP-1 analogs (1-3), a series of LCA-modified Xenopus GLP-1 conjugates were designed (4a-4r), and the bioactivity studies of these conjugates were performed to identify compounds with balanced in vitro receptor activation potency and plasma stability. 4c, 4i, and 4r were selected, and their LCA side chains were optimized to further increase their stability, affording 5a-5c. Compound 5b showed a more increased albumin affinity and prolonged in vitro stability than that of 4i and liraglutide. In db/ db mice, 5b exhibited comparable hypoglycemic and insulinotropic activity to liraglutide and semaglutide. Importantly, the enhanced albumin affinity of 5b resulted in a prolonged in vivo antidiabetic duration. Finally, chronic treatment investigations of 5b demonstrated the therapeutic effects of 5b on HbA1c, body weight, blood glucose, and pancreatic endocrine deficiencies on db/ db mice. Our studies revealed 5b as a promising antidiabetic candidate. Furthermore, our study suggests the derivatization of Xenopus GLP-1 analogs with LCA represents an effective strategy to develop potent long-acting GLP-1 receptor agonists for the treatment of type 2 diabetes.

A method for the preparation of 11α-hydroxy derivatives of lithocholic and chenodeoxycholic acids, recently discovered to be natural bile acids, is described. The principal reactions involved were (1) elimination of the 12α-mesyloxy group of the methyl esters of 3α-acetate-12α-mesylate and 3α,7α-diacetate-12α-mesylate derivatives of deoxycholic acid and cholic acid with potassium acetate/hexamethylphosphoramide; (2) simultaneous reduction/hydrolysis of the resulting △ 11 -3α-acetoxy and △ 11 -3α,7α-diacetoxy methyl esters with lithium aluminum hydride; (3) stereoselective 11α-hydroxylation of the △ 11 -3α,24-diol and △ 11 -3α,7α,24-triol intermediates with B 2 H 6 /tetrahydrofuran (THF); and (4) selective oxidation at C-24 of the resulting 3α,11α,24-triol and 3α,7α,11α,24-tetrol to the corresponding C-24 carboxylic acids with NaClO 2 catalyzed by 2,2,6,6-tetramethylpiperidine 1-oxyl free radical (TEMPO) and NaClO. In summary, 3α,11α-dihydroxy-5β-cholan-24-oic acid and 3α,7α,11α-trihydroxy-5β-cholan-24-oic acid have been synthesized and their nuclear magnetic resonance (NMR) spectra characterized. These compounds are now available as reference standards to be used in biliary bile acid analysis., (© 2018 AOCS.)

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.)

Weakly basic drugs display poor solubility and tend to precipitate in the stomach's acidic environment causing reduced oral bioavailability. Tracing of these orally delivered therapeutic agents using molecular probes is challenged due to their poor absorption in the gastrointestinal tract (GIT). Therefore, we designed a gastric pH stable bile acid derived amphiphile where Tamoxifen (as a model anticancer drug) is conjugated to lithocholic acid derived phospholipid (LCA-Tam-PC). In vitro studies suggested the selective nature of LCA-Tam-PC for cancer cells over normal cells as compared to the parent drug. Fluorescent labeled version of the conjugate (LCA-Tam-NBD-PC) displayed an increased intracellular uptake compared to Tamoxifen. We then investigated the antitumor potential, toxicity, and median survival in 4T1 tumor bearing BALB/c mice upon LCA-Tam-PC treatment. Our studies confirmed a significant reduction in the tumor volume, tumor weight, and reduced hepatotoxicity with a significant increase in median survival on LCA-Tam-PC treatment as compared to the parent drug. Pharmacokinetic and biodistribution studies using LCA-Tam-NBD-PC witnessed the enhanced gut absorption, blood circulation, and tumor site accumulation of phospholipid-drug conjugate leading to improved antitumor activity. Therefore, our studies revealed that conjugation of chemotherapeutic/imaging agents to bile acid phospholipid can provide a new platform for oral delivery and tracing of chemotherapeutic drugs.

In this paper, a new and concise synthetic route of lithocholic acid (LCA) using commercially available steroid source deoxycholic acid is reported. A series of amide derivatives of LCA were also synthesized and investigated for their activity against the growth of MCF-7 and MCF-7/ADR cells using the sulforhodamine B assay. For MCF-7, the most potent compound 20 showed a 20-fold higher antitumor activity than LCA. For MCF-7/ADR, the most potent compound 24 showed a 22-fold higher antitumor activity than LCA. The transwell migration assay of 20 was evaluated on MDA-MB-231 cells. The colony formation and apoptosis assays of 20 were performed on MCF-7 and MCF-7/ADR cell lines., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Lipid composition in general determines the drug encapsulation efficacy and release kinetics from liposomes that impact the clinical outcomes of cancer therapy. We synthesized three bile acid phospholipids by conjugating the phosphocholine headgroup to the 3'-hydroxyl group of benzylated lithocholic acid (LCA), deoxycholic acid (DCA), and cholic acid (CA); and investigated the impact of membrane rigidity on drug encapsulation efficacy, drug release kinetics, anticancer effects, and mice survival. Liposomes with a hydrodynamic diameter of 100-110 nm were subsequently developed using these phospholipids. Fluorescence-probe based quantification revealed a more fluidic nature of DCA-PC- and CA-PC-derived liposomes, whereas the LCA-PC-derived ones are rigid in nature. Doxorubicin encapsulation studies showed ∼75% encapsulation and ∼38% entrapment efficacy of doxorubicin using more fluidic DCA-PC and CA-PC derived liposomes as compared to ∼58% encapsulation and ∼18% entrapment efficacy in the case of LCA-PC derived liposomes. In vivo anticancer studies in the murine model confirmed that doxorubicin entrapped CA-PC liposomes compromise mice survival, whereas rigid drug entrapped LCA-PC-derived-liposomes increased mice survival with ∼2-fold decrease in tumor volume. Pharmacokinetic and biodistribution studies revealed an ∼1.5-fold increase in plasma drug concentration and an ∼4.0-fold rise in tumor accumulation of doxorubicin on treatment with drug entrapped LCA-PC liposomes as compared to doxorubicin alone. In summary, this study presents the impact of bile acid derived liposomes with different rigidities on drug delivery and mice survivability.

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.)

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.

By imitating the scaffold of lithocholic acid (LCA), a natural steroidal compound displaying Protein Tyrosine Phosphatase 1B (PTP1B) inhibitory activity, a series of stilbene derivatives containing phenyl-substituted isoxazoles were designed and synthesized. The structures of the title compounds were confirmed by ¹H-NMR, 13 C-NMR and HRMS. Activities of the title compounds were evaluated on PTP1B and the homologous enzyme TCPTP by using a colorimetric assay. Most of the target compounds had good activities against PTP1B. Among them, compound 29 (IC 50 = 0.91 ± 0.33 μM), characterized by a 5-(2,3-dichlorophenyl) isoxazole moiety, exhibited an activity about 14-fold higher than the lead compound LCA and a 4.2-fold selectivity over TCPTP. Compound 29 was identified as a competitive inhibitor of PTP1B with a K i value of 0.78 μM in enzyme kinetic studies.

Control of higher-order polymer structures attracts a great deal of interest for many researchers when they lead to the development of materials having various advanced functions. Among them, conjugated polymers that are useful as starting materials in the design of molecular wires are particularly attractive. However, an equilibrium existing between isolated chains and bundled aggregates is inevitable and has made their physical properties very complicated. As an attempt to simplify this situation, we previously reported that a polymer chain of a water-soluble polythiophene could be isolated through complexation with a helix-forming polysaccharide. More recently, a covalently self-threading polythiophene was reported, the main chain of which was physically protected from self-folding and chain-chain π-stacking. In this report, we wish to report a new strategy to isolate a water-soluble polythiophene and to control its higher-order structure by a supramolecular approach: that is, among a few bile acids, lithocholate can form stoichiometric complexes with cationic polythiophene to isolate the polymer chain, and the higher-order structure is changeable by the molar ratio. The optical and morphological studies have been thoroughly performed, and the resultant complex has been applied to the selective recognition of two AMP structural isomers.

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.

The crystal structure of a Li(+) salt of a glucosyl derivative of lithocholic acid (lithium 3α-(α-d-glucopyranosyl)-5β-cholan-24-oate) has been solved. The crystal belongs to the orthorhombic system, P212121 spatial group, and includes acetone and water in the structure with a 1:1:2 stoichiometry. Monolayers, having a hydrophobic interior and hydrophilic edges, are recognized in the crystal structure. Li(+) is coordinated to three hydroxyl groups of three different glucose residues, with two of them belonging to the same monolayer. A fourth molecule, located in this monolayer, is involved in the coordination of the cation through the carboxylate ion by an electrostatic interaction, thus completing a distorted tetrahedron. All Li(+)-oxygen distances values are very close to the sum of the ionic radius of Li(+) and van der Waals radius of oxygen. Each steroid molecule is linked to other five steroid molecules through hydrogen bonds. Water and acetone are also involved in the hydrogen bond network. A hierarchical organization can be recognized in the crystal, the helical assembly along 21 screw axes being left-handed., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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.)

Four novel bile acid ethyl amides were synthetized using a well-known method. All the four compounds were characterized by IR, SEM, and X-ray crystal analyses. In addition, the cytotoxicity of the compounds was tested. Two of the prepared compounds formed organogels. Lithocholic acid derivative 1 formed hydrogels as 1% and 2% (w/v) in four different aqueous solutions. This is very intriguing regarding possible uses in biomedicine., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A systematic study with phase 1 and phase 2 metabolites of cholesterol and vitamin D was conducted to determine whether their biological activity is mediated by the vitamin D receptor (VDR). The investigation necessitated the development of novel synthetic routes for lithocholic acid (LCA) glucuronides (Gluc). Biochemical and cell-based assays were used to demonstrate that hydroxylated LCA analogs were not able to bind VDR. This excludes VDR from mediating their biological and pharmacological activities. Among the synthesized LCA conjugates a novel VDR agonist was identified. LCA Gluc II increased the expression of CYP24A1 in DU145 cancer cells especially in the presence of the endogenous VDR ligand 1,25(OH)2D3. Furthermore, the methyl ester of LCA was identified as novel VDR antagonist. For the first time, we showed that calcitroic acid, the assumed inactive final metabolite of vitamin D, was able to activate VDR-mediated transcription to a higher magnitude than bile acid LCA. Due to a higher metabolic stability in comparison to vitamin D, a very low toxicity, and high concentration in bile and intestine, calcitroic acid is likely to be an important mediator of the protective vitamin D properties against colon cancer., (Copyright © 2016 Elsevier Masson SAS. 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.)

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). The complexes of the ligand-binding domain of rat VDR (VDR-LBD) with LCA and its derivatives revealed that the ligands bound 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 was positioned at the top of the LBP, whereas their acyclic tail was located at the bottom of the LBP. However, most of the hydrophobic and hydrophilic interactions observed in the complex with 1,25(OH)2D3 were reproduced in the complexes with LCA and its derivatives. Additional interactions between VDR-LBD and the C-3 substituents of the A-ring were also observed in the complexes, probably related to the observed difference in the potency among the LCA-type ligands. Recently, zebrafish VDR has been reported to have the second LBP on the outside of the canonical LBP, although its physiological function is unclear., (© 2016 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.)

Hydrophobically modified glycol chitosan (HGC) nanoparticles loaded with mono-lithocholic acid-conjugated exendin-4 at the Lys(27) residue (LAM1-Ex4) were prepared and characterized by particle size measurement, proteolytic stability, in vitro drug-release profile, and in vivo antidiabetic effects in a db/db diabetic mouse model. Compared with Ex-4-loaded HGC nanoparticles (Ex4/HGC NPs) prepared as a control, LAM1-Ex4-loaded HGC nanoparticles (LAM1-Ex4/HGC NPs) showed improved drug-loading efficiency, small particle size, enhanced resistance against proteolytic digestion, and an extended in vitro drug release profile. These findings may be attributable to the strong hydrophobic interaction between LAM1-Ex4 and the inner core of HGC. Furthermore, LAM1-Ex4/HGC NPs showed prolonged hypoglycemic efficacy in db/db mice, lasting 1 week after a single subcutaneous administration. The present study demonstrated that LAM1-Ex4/HGC NPs have considerable potential as a long-acting sustained-release antidiabetic system for type 2 diabetes., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Organic nanotubes form in aqueous solution near physiological pH by self-assembly of lithocholic acid (LCA) with inner diameters of 20-40nm. The encapsulation of enhanced green fluorescent protein (eGFP) and resultant confinement effect for eGFP within these nanotubes is studied via confocal microscopy. Timed release rate studies of eGFP encapsulated in LCA nanotubes and fluorescence recovery after photobleaching (FRAP) indicate that the diffusive transport of eGFP out of and/or within the nanotubes is very slow, in contrast to the rapid introduction of eGFP into the nanotubes. By encapsulating two fluorescent proteins in LCA nanotubes, eGFP and mCherry, as a fluorescence resonance energy transfer (FRET) pair, the FRET efficiencies are determined using FRET imaging microscopy at three different protein concentrations with a fixed donor-to-acceptor ratio of 1:1. Förster theory reveals that the proteins are spatially separated by 4.8-7.2nm in distance inside these nanotubes. The biomimetic nanochannels of LCA nanotubes not only afford a confining effect on eGFP that results in enhanced chemical and thermal stability under conditions of high denaturant concentration and temperature, but also function as protein concentrators for enriching protein in the nanochannels from a diluted protein solution by up to two orders of magnitude., (Published by Elsevier Inc.)

The gelation behavior of lithocholate (LC(-) ) with different metal ions in water was investigated. The microstructures of hydrogels were determined to be three-dimensional (3D) networks of fibrous aggregates. The formation of fibrils was speculated to be mainly driven by the coordination between carboxylate of LC(-) and metal ions, accompanied by the assistance of noncovalent interactions such as electrostatic and hydrophobic interactions. The hydrogels, which can maintain the mechanical strength at higher temperature, exhibit thermal stability. Their gelation capability was enhanced with the increase in acidity. The hydrogels of LC(-) and Cu(2+) mixtures served as the precursors for producing network nanostructures of CuS nanoparticles. These new CuS networks exhibit high fluorescence quenching ability and can act as an effective fluorescent sensing platform for ssDNA detection., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Sulfotransferases catalyze transfer of the sulfuryl-group (-SO3) from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to a large number of substrates. They play an important role in phase II metabolic process. The impact of the cofactor (PAPS) on the structure and flexibility of the enzyme has been studied extensively, and the response of the active-cap region to cofactor binding was proposed as the molecular basis for substrate selectivity. In this study, individual and cooperative effects of the cofactor and substrate on the structure and flexibility of the enzyme were investigated. Molecular dynamics simulations were performed for four systems, including free enzyme, binary complexes (cofactor or substrate bound enzyme) and ternary complex (both cofactor and substrate bound enzyme). The influence of ligands (the cofactor and the substrate) on the structure and flexibility of the enzyme, especially that of the active-site cap region, was analyzed. Moreover, mutual structural impact of the ligands was examined as well. The results show that the impact of both the cofactor and the substrate was significant. Our study indicated that the substrate, such as lithocholic acid (LCA), participated in regulating the structure and flexibility of the enzyme actively rather than merely being selected passively. Additionally, the observed synergistic effects of the cofactor and the substrate demonstrated the importance of examining both ligands in understanding enzymes.

Delivering drugs selectively to cancer cells but not to nearby normal cells is a major obstacle in drug therapy. In this study, lithocholic acid (LCA), a potent anti-cancer drug, is converted to two forms of poly(ethyleneglycol) (PEG) conjugates, viz., PEG-LCA (PL) and lactobionic acid (LBA) conjugated PEG-LCA (LPL). The latter form contains a galactose ligand in LBA to target the hepatocytes. Both forms are self-assembled to form nanoparticle formulation, and they have high potency than LCA to kill HepG2 cancer cells, sparing normal LO2 cells. Besides, LPL has high specificity to mouse liver cells in vivo. Western blot results confirm that the cell death is occurred through apoptosis induced by LPL nanoparticles. In conclusion, the induction of apoptosis and cell death is much more efficient with LPL nanoparticles than LCA molecules., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)