Insights into estrogen receptor alpha modulation by cholestenoic acids.

Oxysterols are a family of over 25 cholesterol metabolites naturally produced by enzymatic or radical oxidation. They are involved in many physiological and pathological pathways. Although their activity has been mainly attributed to the modulation of the Liver X Receptors (LXR), it is currently accepted that oxysterols are quite promiscuous compounds, acting at several targets at the same time. The promiscuity of the oxysterols with the Estrogen Receptor α (ERα) is crucial in several pathologies such as ER+ breast cancer, inflammation and atherosclerosis. Regarding this matter, we have previously reported the synthesis, LXR activity and binding mode of a family of cholestenoic acid analogs with a modified side chain. Here we report the transcriptional activity on the ERα triggered by these compounds and details on the molecular determinants involved in their activities in order to establish structure-activity relationships to shed light over the molecular basis of the promiscuity of these compounds on ER/LXR responses. Our results show that 3ß-hydroxy-5-cholestenoic acid can interact with the ERα receptor in a way similar to 26-hydroxycholesterol and is an agonist of the receptor. Using molecular dynamics simulations, we were able to predict the ERα activity of a set of cholestenoic acid analogs with changes in the flexibility and/or steric requirements of the side chain, some of which exhibited selective activation of ERα or LXR.

Cholesterol metabolism pathways - are the intermediates more important than the products?

Every cell in vertebrates possesses the machinery to synthesise cholesterol and to metabolise it. The major route of cholesterol metabolism is conversion to bile acids. Bile acids themselves are interesting molecules being ligands to nuclear and G protein-coupled receptors, but perhaps the intermediates in the bile acid biosynthesis pathways are even more interesting and equally important. Here, we discuss the biological activity of the different intermediates generated in the various bile acid biosynthesis pathways. We put forward the hypothesis that the acidic pathway of bile acid biosynthesis has primary evolved to generate signalling molecules and its utilisation by hepatocytes provides an added bonus of producing bile acids to aid absorption of lipids in the intestine.

Cholestenoic acid analogues as inverse agonists of the liver X receptors.

Liver X Receptors (LXRs) are ligand dependent transcription factors activated by oxidized cholesterol metabolites (oxysterols) that play fundamental roles in the transcriptional control of lipid metabolism, cholesterol transport and modulation of inflammatory responses. In the last decade, LXRs have become attractive pharmacological targets for intervention in human metabolic diseases and thus, several efforts have concentrated on the development of synthetic analogues able to modulate LXR transcriptional response. In this sense, we have previously found that cholestenoic acid analogues with a modified side chain behave as LXR inverse agonists. To further investigate the structure-activity relationships and to explore how cholestenoic acid derivatives interact with the LXRs, we evaluated the LXR biological activity of new analogues containing a C24-C25 double bond. Furthermore, a microarray assay was performed to evaluate the recruitment of coregulators to recombinant LXR LBD upon ligand binding. Also, conventional and accelerated molecular dynamics simulations were applied to gain insight on the molecular determinants involved in the inverse agonism. As LXR inverse agonists emerge as very promising candidates to control LXR activity, the cholestenoic acid analogues here depicted constitute a new relevant steroidal scaffold to inhibit LXR action.

Secondary metabolites of petri-dish cultured Antrodia camphorata and their hepatoprotective activities against alcohol-induced liver injury in mice.

Antrodia camphorata, a well-known and highly valued edible medicinal mushroom with intriguing activities like liver protection, has been traditionally used for the treatment of alcoholic liver disease. A. camphorata shows highly medicinal and commercial values with the demand far exceeds the available supply. Thus, the petri-dish cultured A. camphorata (PDCA) is expected to develope as a substitute. In this paper, nineteen triterpenes were isolated from PDCA, and thirteen of them were the unique anthroic acids in A. camphorata, including the main content antcin K, which suggested that PDCA produced a large array of the same anthroic acids as the wild one. Furthermore, no obvious acute toxicity was found suggesting the edible safety of PDCA. In mice alcohol-induced liver injury model, triglyceride (TG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and malondialdehyde (MDA) had been reduced by the PDCA powder as well as the main content antcin K, which indicated that the PDCA could protect alcoholic liver injury in mice model and antcin K could be the effective component responsible for the hepatoprotective activities of PDCA against alcoholic liver diseases.

Antcins, triterpenoids from Antrodia cinnamomea, as new agonists for peroxisome proliferator-activated receptor α.

Peroxisome proliferator-activated receptor α (PPARα) is a nuclear hormone receptor that transcriptionally regulates lipid metabolism and inflammation; therefore, PPARα agonists are promising agents to treat dyslipidemia and metabolic disorders. PPARα full agonists, such as fibrates, are effective anti-hypertriglyceride agents, but their use is limited by adverse side effects. Hence, the aim of this study was to identify small molecules that can activate PPARα while minimizing the adverse effects. Antrodia cinnamomea, a rare medical mushroom, has been used widely in Asian countries for the treatment of various diseases, including liver diseases. Antcin B, H and K (antcins) and ergostatrien-3ß-ol (EK100) are bioactive compounds isolated from A. cinnamomea with anti-inflammatory actions. Antcins, ergostane-type triterpenoids, contain the polar head with carboxylate group and the sterol-based body. Here, we showed at the first time that sterol-based compounds, antcins, but not EK100, activate PPARα in a cell-based transactivation study. The in silico docking studies presented several significant molecular interactions of antcins, including Tyr314, and His440 in the ligand-binding domain of PPARα, and these interactions are required for helix 12 (H12) stabilization. We propose that PPARα activation activity of antcins is related to their binding mode which requires conventional H12 stabilization, and that antcins can be developed as safe selective PPARα modulators.

Identification of 7α,24-dihydroxy-3-oxocholest-4-en-26-oic and 7α,25-dihydroxy-3-oxocholest-4-en-26-oic acids in human cerebrospinal fluid and plasma.

Dihydroxyoxocholestenoic acids are intermediates in bile acid biosynthesis. Here, using liquid chromatography - mass spectrometry, we confirm the identification of 7α,24-dihydroxy-3-oxocholest-4-en-26-oic and 7α,25-dihydroxy-3-oxocholest-4-en-26-oic acids in cerebrospinal fluid (CSF) based on comparisons to authentic standards and of 7α,12α-dihydroxy-3-oxocholest-4-en-26-oic and 7α,x-dihydroxy-3-oxocholest-4-en-26-oic (where hydroxylation is likely on C-22 or C-23) based on exact mass measurement and multistage fragmentation. Surprisingly, patients suffering from the inborn error of metabolism cerebrotendinous xanthomatosis, where the enzyme CYP27A1, which normally introduces the (25 R)26-carboxylic acid group to the sterol side-chain, is defective still synthesise 7α,24-dihydroxy-3-oxocholest-4-en-26-oic acid and also both 25 R- and 25 S-epimers of 7α,12α-dihydroxy-3-oxocholest-4-en-26-oic acid. We speculate that the enzymes CYP46A1 and CYP3A4 may have C-26 carboxylase activity to generate these acids. In patients suffering from hereditary spastic paraplegia type 5 the CSF concentrations of the 7α,24- and 7α,25-dihydroxy acids are reduced, suggesting an involvement of CYP7B1 in their biosynthesis in brain.

Improved chromatographic diastereoresolution of cyclopropyl dafachronic acid derivatives using chiral anion exchangers.

In the present paper we describe the optimization and the application of a chromatographic method suitable to get all four diastereoisomers of C24-C25 cyclopropyl dafachronic acid derivatives in sufficient amount for their biological appraisal towards the nuclear hormone receptor transcription factor DAF-12. A preliminary column screening of six anion exchange type Cinchona alkaloid-based chiral stationary phases (CSPs) allowed to identify the one with a quinine scaffold and carrying a O-9-(3,5-bis(trifluoromethyl)phenyl) moiety at C9 position as the best CSP. Few modifications of the experimental conditions revealed that a content of 18mM acetic acid used as counterion and displacer in acetonitrile and a column temperature fixed at 35°C were optimal for the simultaneous discrimination of all four diastereoisomers with a 1.0mL/min flow rate. With such conditions, transoid (S,S) and (R,R) diastereoisomers were resolved with RS>1.4. With non chiral reversed-phase columns, neither the cisoid nor the transoid diastereoisomers could be resolved. This way, ca. 1.0mg of each stereoisomer was isolated with a diastereomeric purity >98%, suitable for the following biological tests. The indirect stereochemical assignments of the four diastereoisomers, and hence the corresponding chromatographic elution order (24R,25R)<(24S,25S)<(24R,25S)<(24S,25R) were made in an analogy manner on the basis of the resolution of fully assigned and structurally very similar ursodeoxycholic acid derivatives. As support of this indirect way of assigning the absolute configuration of the C24 and C25 chiral centre a molecular modeling procedure based on dynamic simulation was successfully applied.

A novel function for glucocerebrosidase as a regulator of sterylglucoside metabolism.

BACKGROUND: Sterols are major cell membrane lipids, and in many organisms they are modified with glucose to generate sterylglucosides. Glucosylation dramatically changes the functional properties of sterols. The formation of sterylglucosides from sterols in plants, fungi, and bacteria uses UDP-glucose as a glucose donor. By contrast, sterylglucoside biosynthesis in mammals is catalyzed by the transglucosylation activity of glucocerebrosidases, with glucosylceramide acting as the glucose donor. Recent success in isolation and structural determination of sterylglucosides in the vertebrate central nervous system shows that transglucosylation also occurs in vivo. These analyses also revealed that sterylglucoside aglycons are composed of several cholesterol-related metabolites, including a plant-type sitosteryl. SCOPE OF REVIEW: In this review, we discuss the biological functions and metabolism of sterylglucosides. We also summarize new findings from studies on the metabolism of vertebrate sterylglucosides and review the circumstances underlying the recent discovery of sterylglucosides in vertebrate brain. Finally, we discuss the role of sterylglucosides in a variety of neurodegenerative disorders such as Gaucher disease and Parkinson's disease. MAJOR CONCLUSIONS: The biological significance of UDP-glucose-independent sterol glucosylation is still unknown, but it is plausible that glucosylation may provide sterols with novel biological functions. Even though sterol glucosylation is a simple reaction, it can dramatically change the physical properties of sterols. GENERAL SIGNIFICANCE: Sterylglucosides may play roles in various physiological processes and in the pathogenesis of different diseases. Arriving at a better understanding of them at the organ and cellular level may open up new approaches to developing therapeutics for a variety of diseases. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa.

Charge-tagging liquid chromatography-mass spectrometry methodology targeting oxysterol diastereoisomers.

The introduction of a hydroxy group to the cholesterol skeleton introduces not only the possibility for positional isomers but also diastereoisomers, where two or more isomers have different configurations at one or more of the stereocentres but are not mirror images. The differentiation of diastereoisomers is important as differing isomers can have differing biochemical properties and are formed via different biochemical pathways. Separation of diasterioisomers is not always easy by chromatographic methods Here we demonstrate, by application of charge-tagging and derivatisation with the Girard P reagent, the separation and detection of biologically relevant diastereoisomers using liquid chromatography - mass spectrometry with multistage fragmentation.

Synthesis of new C-25 and C-26 steroidal acids as potential ligands of the nuclear receptors DAF-12, LXR and GR.

A new methodology to obtain C-25 and C-26 steroidal acids starting from pregnenolone is described. Construction of the side chain was achieved by applying the Mukaiyama aldol reaction with a non-hydrolytic work-up to isolate the trapped silyl enol ether with higher yields. Using this methodology we synthesized three new steroidal acids as potential ligands of DAF-12, Liver X and Glucocorticoid nuclear receptors and studied their activity in reporter gene assays. Our results show that replacement of the 21-CH3 by a 20-keto group in the side chains of the cholestane scaffold of DAF-12 or Liver X receptors ligands causes the loss of the activity.

Synthesis and Live-Cell Imaging of Fluorescent Sterols for Analysis of Intracellular Cholesterol Transport.

Cellular cholesterol homeostasis relies on precise control of the sterol content of organelle membranes. Obtaining insight into cholesterol trafficking pathways and kinetics by live-cell imaging relies on two conditions. First, one needs to develop suitable analogs that resemble cholesterol as closely as possible with respect to their biophysical and biochemical properties. Second, the cholesterol analogs should have good fluorescence properties. This interferes, however, often with the first requirement, such that the imaging instrumentation must be optimized to collect photons from suboptimal fluorophores, but good cholesterol mimics, such as the intrinsically fluorescent sterols, cholestatrienol (CTL) or dehydroergosterol (DHE). CTL differs from cholesterol only in having two additional double bonds in the ring system, which is why it is slightly fluorescent in the ultraviolet (UV). In the first part of this protocol, we describe how to synthesize and image CTL in living cells relative to caveolin, a structural component of caveolae. In the second part, we explain in detail how to perform time-lapse experiments of commercially available BODIPY-tagged cholesterol (TopFluor-cholesterol®; TF-Chol) in comparison to DHE. Finally, using two-photon time-lapse imaging data of TF-Chol, we demonstrate how to use our imaging toolbox SpatTrack for tracking sterol rich vesicles in living cells over time.

Fluorinated oxysterol analogues: Synthesis, molecular modelling and LXRß activity.

Liver X receptors (LXRs) are nuclear receptors that play central roles in the transcriptional control of lipid metabolism. The ability of LXRs to integrate metabolic and inflammation signalling makes them attractive targets for intervention in human metabolic diseases. Several oxidized metabolites of cholesterol (oxysterols) are endogenous LXR ligands, that modulate their transcriptional responses. While 25R-cholestenoic acid is an agonist of the LXRs, the synthetic analogue 27-norcholestenoic acid that lacks the 25-methyl is an inverse agonist. This change in the activity profile is triggered by a disruption of a key interaction between residues His435 and Trp457 that destabilizes the H11-H12 region of the receptor and favors the binding of corepressors. The introduction of fluorine atoms on the oxysterol side chain can favor both hydrophobic interactions as well as hydrogen bonds with the fluorine atoms and may thus induce changes in the receptor that may lead to changes in the activity profile. To evaluate these effects we have synthesized two fluorinated 27-nor-steroids, analogues of 27-norcholestenoic acid, the 25,25-difluoroacid and the corresponding 26-alcohol. The key step was a Reformatsky reaction on the C-24 cholenaldehyde, with ethyl bromodifluoroacetate under high intensity ultrasound (HIU) irradiation, followed by a Barton-McCombie type deoxygenation. Activity was evaluated in a luciferase reporter assay in the human HEK293T cells co-transfected with full length human LXRß expression vector. The 25,25-difluoro-27-norcholestenoic acid was an inverse agonist and antagonist similar to its non-fluorinated analogue while its reduced derivative 25,25-difluoro-27-norcholest-5-ene-3ß,26-diol was an agonist. Molecular dynamics simulation of the ligand-receptor complexes showed that the difluoroacid disrupted the His435-Trp457 interaction although the resulting conformational changes were different from those induced by the non-fluorinated analogue. In the case of the difluoroalcohol, the fluorine atoms actively participated in the interaction with several residues in the ligand binding pocket leading to a stabilization of the active receptor conformation.

C(16)-C(22) oxygen-bridged analogues of ceDAF-12 and LXR ligands.

The DAF-12 receptor in nematodes and the Liver X Receptor (LXR) in mammals are structurally related transcription factors that play key roles in determining the life span of the organism. Both types of receptors are activated by oxysterols, cholesterol metabolites with oxidized side chains. Restricting the movement of the oxysterol side chain to certain orientations may have profound effects in the activity profile, however this has not been explored so far. In a first attempt to obtain analogues of natural ligands of DAF-12 and LXR with restricted side chain mobility we introduced a 16,22-oxygen bridge in 26-hydroxycholesterol, a cholestenoic acid and a dafachronic acid (5-7). Diosgenin was used as starting material, the key step to obtain the 16,22 epoxy functionality was the one pot formation and reduction of a cyclic hemiketal via the oxocarbenium ion using sodium cyanoborohydride. All new compounds were characterized by NMR and mass spectrometry and assayed as ceDAF-12 or LXR ligands in transactivation cell-based assays. The dafachronic acid analogue 7 behaved as a ceDAF-12 agonist.

The oxysterol and cholestenoic acid profile of mouse cerebrospinal fluid.

Oxysterols and cholestenoic acids are oxidised forms of cholesterol with a host of biological functions. The possible roles of oxysterols in various neurological diseases makes the analysis of these metabolites in the central nervous system of particular interest. Here, we report the identification and quantification of a panel of twelve sterols in mouse cerebrospinal fluid (CSF) using liquid chromatography-mass spectrometry exploiting enzyme assisted derivatisation for sterol analysis technology. We found low levels of oxysterols and cholestenoic acids in CSF in the range of 5pg/mL-2.6ng/mL. As found in man, these concentrations are one to two orders of magnitude lower than in plasma.

Site-selective and metal-free aliphatic C-H oxidation enabled synthesis of [5,24,25-D3]-(25S)-Δ(7)-dafachronic acid.

Steroid hormones play significant roles in both worms and mammalians. (25S)-Δ(7)-Dafachronic acid (Δ(7)-DA, 1) is a member of the dafachronic acid hormonal series that regulates both development and lifespan of C. elegans. Despite its importance, effective tools for the illumination of its mode of action are lacking. Herein, we report an efficient synthesis of trideuterated Δ(7)-DA, [5,24,25-D3]-(25S)-Δ(7)-dafachronic acid ([D3]-Δ(7)-DA, 2), as a useful chemical tool for subsequent biological studies. Key steps for this bioinspired synthesis approach include site-selective aliphatic C-H oxidation mediated by methyl(trifluoromethyl)dioxirane (TFDO), and the iridium/phosphine-oxazoline-catalyzed late-stage asymmetric deuterium reduction.

Synthetic DAF-12 modulators with potential use in controlling the nematode life cycle.

Dafachronic acids (DAs) are 3-keto cholestenoic acids bearing a carboxylic acid moiety at the end of the steroid side chain. These compounds interact with the DAF-12 receptor, a ligand-dependent transcription factor that acts as a molecular switch mediating the choice between arrest at diapause or progression to reproductive development and adult lifespan in different nematodes. Recently, we reported that the 27-nor-Δ4-DA was able to directly activate DAF-12 in a transactivation cell-based luciferase assay and rescued the Mig phenotype of daf-9(rh50) Caenorhabditis elegans mutants. In the present paper, to investigate further the relationship between the structure of the steroid side chain and DAF-12 activity, we evaluated the in vitro and in vivo activity of Δ4-DA analogues with modified side chains using transactivation cell-based assays and daf-9(dh6) C. elegans mutants. Our results revealed that introduction of a 24,25-double bond on the cholestenoic acid side chain did not affect DAF-12 activity, whereas shortening the side chain lowered the activity. Most interestingly, the C24 alcohol 24-hydroxy-4-cholen-3-one (6) was an antagonist of the DAF-12 receptor both in vitro and in vivo.

Fast separation and quantification of steroid hormones Δ4- and Δ7-dafachronic acid in Caenorhabditis elegans.

Separation of isomeric molecular species, e.g. double bond position isomers, is a challenging task for liquid chromatography. The two steroid hormones Δ4- and Δ7-dafachronic acid (DA) represent such an isomeric pair. DAs are 3-ketosteroids found in the nematode Caenorhabditis elegans and generated from cholesterol. Δ4- and Δ7-DA have important biological activities and are produced by two different biological pathways in C. elegans. Here we have described a fast separation method for these two isomers using a 1.3 µm core-shell particle in less than 10 min together with a simple MeOH extraction. Using this method we were able to independently quantify Δ4- and Δ7-DA in C. elegans independently from each other and limits of detection of about 5 ng/ml for each isomer were achieved with a good day-to-day reproducibility. As proof-of-principle the method has been applied to the quantification of DAs in worms fed ad libitum or under bacterial deprivation.

Studies on chemical constituents of Ophiopogon japonicus.

Two new and six known steroidal glucosides were isolated from the tuber of Ophiopogon japonicus. The new steroidal glucosides were established as (20R,25R)-26-O-ß-d-glucopyranosyl-3ß,26-dihydroxycholest-5-en-16,22-dioxo-3-O-α-l-rhamnopyranosyl-(1 â†’ 2)-ß-d-glucopyranoside (1) and 26-O-ß-d-glucopyranosyl-(25R)-furost-5-en-3ß,14α,17α,22α,26-pentaol-3-O-α-l-rhamnopyranosyl-(1 â†’ 2)-ß-d-glucopyranoside (3) on the basis of spectroscopic data as well as chemical evidence.

A new alkaloid from the fruit of Nandina domestica Thunb.

A new steroidal alkaloid, (20S,22R,24R)-24-ethyl-3-oxocholest-4-en-22-amino, named as nandsterine (1), together with 10 known alkaloids, palmatine (2), O-methylbulbocapnine (3), nantenine (4), dehydronantenine (5), glaucine (6), didehydroglaucine (7), dehydrocorydaline (8), jatrorrhizine (9), magnoflorine (10) and berberine (11), was isolated from the fruit of Nandina domestica Thunb. Their structures were elucidated by using spectroscopic methods as well as by comparing with the published data. Compound 1 was a new class of steroidal alkaloid isolated from the family Berberidaceae, meanwhile compounds 2, 3, 6-8 and 10 were obtained from N. domestica for the first time. Compound 1 exhibited cytotoxicity against HL-60 cells (human leukaemia) with IC50 values of 52.1 µM.

Mixed bioengineering-chemical synthesis approach for the efficient preparation of Δ7-dafachronic acid.

Combining bioengineering with chemical synthesis has enabled an efficient method for producing Δ7-dafachronic acid, a steroidal hormone associated with nematode germline longevity. Saccharomyces cerevisiae was engineered to produce 7,24-cholestadienol, a convenient starting material for a four-step synthesis of Δ7-dafachronic acid.