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

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

A Gut-Restricted Lithocholic Acid Analog as an Inhibitor of Gut Bacterial Bile Salt Hydrolases.

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.

Novel bile acid biosynthetic pathways are enriched in the microbiome of centenarians.

Centenarians have a decreased susceptibility to ageing-associated illnesses, chronic inflammation and infectious diseases1-3. 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 3ß-hydroxysteroid dehydrogenase (3ß-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.

Crystal structure of an apo 7α-hydroxysteroid dehydrogenase reveals key structural changes induced by substrate and co-factor binding.

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 ß4-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.

Bile Acid Tethered Docetaxel-Based Nanomicelles Mitigate Tumor Progression through Epigenetic Changes.

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.

Lithocholic acid inhibits P2X2 and potentiates P2X4 receptor channel gating.

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.

Conjugation of bile esters to cellulose by olefin cross-metathesis: A strategy for accessing complex polysaccharide structures.

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).

Inhibition of Human Sulfotransferase 2A1-Catalyzed Sulfonation of Lithocholic Acid, Glycolithocholic Acid, and Taurolithocholic Acid by Selective Estrogen Receptor Modulators and Various Analogs and Metabolites.

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 µM). 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.

Effect of Rifampicin on the Plasma Concentrations of Bile Acid-O-Sulfates in Monkeys and Human Liver-Transplanted Chimeric Mice With or Without Bile Flow Diversion.

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.

Enhanced activity and substrate tolerance of 7α-hydroxysteroid dehydrogenase by directed evolution for 7-ketolithocholic acid production.

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.

Pharmacological evaluation of new bioavailable small molecules targeting Eph/ephrin interaction.

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.

Synthesis and biological evaluation of steroidal derivatives bearing a small ring as vitamin D receptor agonists.

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.

Lithocholic Acid Hydroxyamide Destabilizes Cyclin D1 and Induces G0/G1 Arrest by Inhibiting Deubiquitinase USP2a.

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/GSK3ß-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.

Engineering 7ß-Hydroxysteroid Dehydrogenase for Enhanced Ursodeoxycholic Acid Production by Multiobjective Directed Evolution.

Ursodeoxycholic acid (UDCA) is the main active ingredient of natural bear bile powder with multiple pharmacological functions. 7ß-Hydroxysteroid dehydrogenase (HSDH) is a key biocatalyst for the synthesis of UDCA. However, all the 7ß-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 7ß-HSDH. The best variant (V3-1) 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 V3-1 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 for Perspective Drug Design: Computationally Driven Synthesis of New Protein-Protein Interaction Inhibitors Targeting the EphA2 Receptor.

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-5ß-cholan-24-oyl)-l-ß-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-5ß-cholan-24-oyl)-l-ß-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.

Chemical Synthesis of Uncommon Natural Bile Acids: The 9α-Hydroxy Derivatives of Chenodeoxycholic and Lithocholic Acids.

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-5ß-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.

An ultra-high performance liquid chromatography-tandem mass spectrometric assay for quantifying 3-ketocholanoic acid: Application to the human liver microsomal CYP3A-dependent lithocholic acid 3-oxidation assay.

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.

Synthesis of B- and C-ring-modified lithocholic acid analogues as potential sialyltransferase inhibitors.

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.

Fast and sensitive quantification of human liver cytosolic lithocholic acid sulfation using ultra-high performance liquid chromatography-tandem mass spectrometry.

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.

Biochemical characterization of EphA2 antagonists with improved physico-chemical properties by cell-based assays and surface plasmon resonance analysis.

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.