Elucidation of Distinct Modular Assemblies of Smoothened Receptor by Bitopic Ligand Measurement.

Class F G protein-coupled receptors are characterized by a large extracellular domain (ECD) in addition to the common transmembrane domain (TMD) with seven α-helixes. For smoothened receptor (SMO), structural studies revealed dissected ECD and TMD, and their integrated assemblies. However, distinct assemblies were reported under different circumstances. Using an unbiased approach based on four series of cross-conjugated bitopic ligands, we explore the relationship between the active status and receptor assembly. Different activity dependency on the linker length for these bitopic ligands corroborates the various occurrences of SMO assembly. These results reveal a rigid "near" assembly for active SMO, which is in contrast to previous results. Conversely, inactive SMO adopts a free ECD, which would be remotely captured at "far" assembly by cholesterol. Altogether, we propose a mechanism of cholesterol flow-caused SMO activation involving an erection of ECD from far to near assembly.

New 20-hydroxycholesterol-like compounds with fluorescent NBD or alkyne labels: Synthesis, in silico interactions with proteins and uptake by yeast cells.

20-hydroxycholesterol is a signaling oxysterol with immunomodulating functions and, thus, structural analogues with reporter capabilities could be useful for studying and modulating the cellular processes concerned. We have synthesized three new 20-hydroxycholesterol-like pregn-5-en-3ß-ol derivatives with fluorescent 7-nitrobenzofurazan (NBD) or Raman-sensitive alkyne labels in their side-chains. In silico computations demonstrated the compounds possess good membrane permeability and can bind within active sites of known 20-hydroxycholesterol targets (e.g. Smoothened and yeast Osh4) and some other sterol-binding proteins (human LXRß and STARD1; yeast START-kins Lam4S2 and Lam2S2). Having found good predicted membrane permeability and binding to some yeast proteins, we tested the compounds on microorganisms. Fluorescent microscopy indicated the uptake of the steroids by both Saccharomyces cerevisiae and Yarrowia lipolytica, whereas only S. cerevisiae demonstrated conversion of the compounds into 3-O-acetates, likely because 3-O-acetyltransferase Atf2p is present only in its genome. The new compounds provide new options to study the uptake, intracellular distribution and metabolism of sterols in yeast cells as well as might be used as ligands for sterol-binding proteins.

The synthetic neuroactive steroid SGE-516 reduces seizure burden and improves survival in a Dravet syndrome mouse model.

Dravet syndrome is an infant-onset epileptic encephalopathy with multiple seizure types that are often refractory to conventional therapies. Treatment with standard benzodiazepines like clobazam, in combination with valproate and stiripentol, provides only modest seizure control. While benzodiazepines are a first-line therapy for Dravet syndrome, they are limited by their ability to only modulate synaptic receptors. Unlike benzodiazepines, neuroactive steroids potentiate a wider-range of GABAA receptors. The synthetic neuroactive steroid SGE-516 is a potent positive allosteric modulator of both synaptic and extrasynaptic GABAA receptors. Prior work demonstrated anticonvulsant activity of SGE-516 in acute seizure assays in rodents. In this study, we evaluated activity of SGE-516 on epilepsy phenotypes in the Scn1a +/- mouse model that recapitulates many features of Dravet syndrome, including spontaneous seizures, premature death and seizures triggered by hyperthermia. To evaluate SGE-516 in Scn1a +/- mice, we determined the effect of treatment on hyperthermia-induced seizures, spontaneous seizure frequency and survival. SGE-516 treatment protected against hyperthermia-induced seizures, reduced spontaneous seizure frequency and prolonged survival in the Scn1a +/- mice. This provides the first evidence of SGE-516 activity in a mouse model of Dravet syndrome, and supports further investigation of neuroactive steroids as potential anticonvulsant compounds for refractory epilepsies.

Synthesis of 24(S)-hydroxycholesterol esters responsible for the induction of neuronal cell death.

We synthesized several candidates of 24(S)-hydroxycholesterol (24S-OHC) esters, which are involved in neuronal cell death, through catalysis with acyl-CoA:cholesterol acyltransferase-1 (ACAT-1). We studied the regioselectivity of the acylation of the secondary alcohol at the 3- or 24-position of 24S-OHC. The appropriate saturated and unsaturated long-chain fatty acids were esterified with the protected 24S-OHC and then de-protected to afford the desired esters at a satisfactory yield. We then confirmed by HPLC monitoring that the retention times of four esters of 24S-OHC, namely 3-oleate, 3-linoleate, 3-arachidonoate and 3-docosahexaenoate, were consistent with those of 24S-OHC esters observed in 24S-OHC-treated SH-SY5Y cells.

The estrogen receptor as a mediator of the pathological actions of cholesterol in breast cancer.

Despite increased survivorship among patients, breast cancer remains the most common cancer among women and is the second leading cause of cancer death in women. The magnitude of this problem provides a strong impetus for new chemopreventative strategies and/or lifestyle changes that reduce cancer incidence. It is of significance, therefore, that several studies positively correlate obesity to the development of breast cancer. Importantly, obesity is also highly associated with elevated cholesterol, and cholesterol itself is a risk factor for breast cancer. Furthermore, patients taking statins demonstrate a lower breast cancer incidence and decreased recurrence. The recent observation that 27-hydroxycholesterol (27HC) is produced in a stoichiometric manner from cholesterol, together with our recent demonstration that it exerts partial agonist activity on both the estrogen and liver X receptors, suggested a potential mechanistic link between hyper-cholesterolemia and breast cancer incidence. Using genetic and pharmacological approaches, we have recently shown that elevation of circulating 27HC significantly increases tumor growth and metastasis in murine models of breast cancer. Further, we have demonstrated in appropriate animal models that the impact of high-fat diet on tumor pathogenesis can be mitigated by statins or by small molecule inhibitors of CYP27A1. These findings suggest that pharmacological or dietary modifications that lower total cholesterol, and by inference 27HC, are likely to reduce the impact of obesity/metabolic syndrome on breast cancer incidence.

Identification of novel regulatory cholesterol metabolite, 5-cholesten, 3ß,25-diol, disulfate.

Oxysterol sulfation plays an important role in regulation of lipid metabolism and inflammatory responses. In the present study, we report the discovery of a novel regulatory sulfated oxysterol in nuclei of primary rat hepatocytes after overexpression of the gene encoding mitochondrial cholesterol delivery protein (StarD1). Forty-eight hours after infection of the hepatocytes with recombinant StarD1 adenovirus, a water-soluble oxysterol product was isolated and purified by chemical extraction and reverse-phase HPLC. Tandem mass spectrometry analysis identified the oxysterol as 5-cholesten-3ß, 25-diol, disulfate (25HCDS), and confirmed the structure by comparing with a chemically synthesized compound. Administration of 25HCDS to human THP-1-derived macrophages or HepG2 cells significantly inhibited cholesterol synthesis and markedly decreased lipid levels in vivo in NAFLD mouse models. RT-PCR showed that 25HCDS significantly decreased SREBP-1/2 activities by suppressing expression of their responding genes, including ACC, FAS, and HMG-CoA reductase. Analysis of lipid profiles in the liver tissues showed that administration of 25HCDS significantly decreased cholesterol, free fatty acids, and triglycerides by 30, 25, and 20%, respectively. The results suggest that 25HCDS inhibits lipid biosynthesis via blocking SREBP signaling. We conclude that 25HCDS is a potent regulator of lipid metabolism and propose its biosynthetic pathway.

Tracking the subcellular fate of 20(s)-hydroxycholesterol with click chemistry reveals a transport pathway to the Golgi.

Oxysterols, oxidized metabolites of cholesterol, are endogenous small molecules that regulate lipid metabolism, immune function, and developmental signaling. Although the cell biology of cholesterol has been intensively studied, fundamental questions about oxysterols, such as their subcellular distribution and trafficking pathways, remain unanswered. We have therefore developed a useful method to image intracellular 20(S)-hydroxycholesterol with both high sensitivity and spatial resolution using click chemistry and fluorescence microscopy. The metabolic labeling of cells with an alkynyl derivative of 20(S)-hydroxycholesterol has allowed us to directly visualize this oxysterol by attaching an azide fluorophore through cyclo-addition. Unexpectedly, we found that this oxysterol selectively accumulates in the Golgi membrane using a pathway that is sensitive to ATP levels, temperature, and lysosome function. Although previous models have proposed nonvesicular pathways for the rapid equilibration of oxysterols between membranes, direct imaging of oxysterols suggests that a vesicular pathway is responsible for differential accumulation of oxysterols in organelle membranes. More broadly, clickable alkynyl sterols may represent useful tools for sterol cell biology, both to investigate the functions of these important lipids and to decipher the pathways that determine their cellular itineraries.

Investigation on the synthesis of 25-hydroxycholesterol.

A very efficient and environmentally benign method has been developed for the synthesis of 25-hydroxycholesterol. The reaction was performed in THF-water (4:1, v/v) using NBS as the brominating agent, followed by the easy reduction of C-Br with lithium aluminum hydride in THF, to yield the final product corresponding to a Markovnikov's rule. Excellent yields and regioselectivity have been obtained.

Kinetics of 25-hydroperoxycholesterol formation during photo-oxidation of crystalline cholesterol.

BACKGROUND: 25-Hydroxycholesterol (25-OH), a side-chain product of cholesterol oxidation, has emerged as one of the important issues in food chemistry and biochemistry, because of its involvement in several human pathologies. This oxysterol is derived from both enzymatic and non-enzymatic pathways. However, the latter mechanism has been scarcely studied in either food or model systems. In this work, a kinetic model was developed to evaluate the formation of 25-OH and its precursor 25-hydroperoxycholesterol (25-OOH) during photo-oxidation of cholesterol for 28 days under fluorescent light. 25-OOH was estimated by an indirect method, using thin-layer chromatography coupled with gas chromatography-mass spectrometry. RESULTS: Peroxide value (POV) and cholesterol oxidation products (COPs) were determined. POV showed a hyperbolic behavior, typical of a crystalline system in which the availability of cholesterol is the limiting factor. Further reactions of hydroperoxides were followed; in particular, after photo-oxidation, 25-OOH (0.55 mg g(-1) ) and 25-OH (0.08 mg g(-1) ) were found in cholesterol, as well as seven other oxysterols, including 7-hydroxy and 5,6-epoxy derivatives. The application of kinetic models to the data showed good correlation with theoretical values, allowing derivation of the kinetic parameters for each oxidation route. CONCLUSIONS: The results of this work confirm that cholesterol in the crystalline state involves different oxidation patterns as compared to cholesterol in solution. Moreover, the numerical fit proved that hydroperoxidation is the rate-limiting step in 25-OH formation.

Synthesis and initial biological evaluation of new mimics of the LXR-modulator 22(S)-hydroxycholesterol.

The generic, synthetic oxysterol 22(S)-hydroxycholesterol (22SHC) has shown antagonistic effects towards liver X receptor (LXR) in vitro and promising effects on plasma triacylglycerol level and body weight-gain in animal studies. On the contrary, the endogenic LXR agonist 22(R)-hydroxycholesterol (22RHC) and synthetic LXR agonists convincingly have shown agonistic effects on genes involved in lipogenesis, and inhibitory effects on cell proliferation in vitro and in vivo. We hypothesized that the carbon side chain containing the hydroxyl group at the 22-position was a pharmacophore affecting these opposite effects on LXR. This prompted us to initiate a rational drug design incorporating the 22-hydroxylated 20-27 cholesterol moiety into cholesterol-mimicking building blocks. The two enantiomers of the 22-hydroxylated 20-27 cholesterol moiety were synthesized with an excellent enantiomeric excess and the stereochemistry are supported by X-ray crystallography. Molecular modelling of the new compounds showed promising LXR selectivity (LXRß over LXRα) and initial in vitro biological evaluation in human myotubes showed that compound 16b had agonistic effects on the gene expression of SCD1 and increased lipogenesis.

Oxysterols are allosteric activators of the oncoprotein Smoothened.

Oxysterols are a class of endogenous signaling molecules that can activate the Hedgehog pathway, which has critical roles in development, regeneration and cancer. However, it has been unclear how oxysterols influence Hedgehog signaling, including whether their effects are mediated through a protein target or indirectly through effects on membrane properties. To answer this question, we synthesized the enantiomer and an epimer of the most potent oxysterol, 20(S)-hydroxycholesterol. Using these molecules, we show that the effects of oxysterols on Hedgehog signaling are exquisitely stereoselective, consistent with the hypothesis that they function through a specific protein target. We present several lines of evidence that this protein target is the seven-pass transmembrane protein Smoothened, a major drug target in oncology. Our work suggests that these enigmatic sterols, which have multiple effects on cell physiology, may act as ligands for signaling receptors and provides a generally applicable framework for probing sterol signaling mechanisms.

A novel synthetic approach to (20R)- and (20S)-21-hydroxy steroids. Synthesis of a marine sterol 21-hydroxycholesterol.

A short and efficient synthesis of steroid synthons, di(tert-butyldimethylsilyl) ethers of 3,21-dihydroxy-24-nor-chol-5-en-23-al (8 and 10) and of ethyl 3,21-dihydroxy-25-homo-chola-5,23-dien-25-oate (9 and 11), having natural (20R) and unnatural (20S) configuration from 3ß-(tert-butyldimethylsilyloxy)-14α,20ξ-card-5-enolide (2) is reported. Further elongation of the side chain of these synthons provides a new method for the synthesis of (20R) and (20S)-21-hydroxy steroids. The utility of the method was exemplified by the synthesis of a natural marine sterol - 21-hydroxycholesterol (18).

[Synthesis of secasterol and 24-episecasterol and their toxicity in MCF-7 cells].

A convergent synthesis of biosynthetic precursors of brassinosteroids - secasterol and 24-episecasterol with Δ²-bond in cycle A is described. The key stages in the construction of the side chain of these compounds were Julia olefination of steroid 22-aldehyde followed by asymmetric Sharpless dihydroxylation of the intermediate Δ²²-olefin. Toxicity of synthesized compounds against breast carcinoma MCF-7 cells was studied.

A facile synthesis of C-24 and C-25 oxysterols by in situ generated ethyl(trifluoromethyl)dioxirane.

Experiments were performed to compare the regioselective hydroxylation of the isopropyl C-H bond at C-25 in 5alpha-cholestan-3beta-yl acetate by in situ generated dimethyldioxirane, methyl(trifluoromethyl)dioxirane, hexafluoro(dimethyl)dioxirane or ethyl(trifluoromethyl)dioxirane (ETDO). The dioxiranes were generated from the corresponding ketones and potassium peroxymonosulfate in aq. NaHCO(3), pH 7.5-8.0. Of the four dioxiranes examined, partially fluorinated, sterically bulky ETDO displayed the highest reactivity and regioselectivity. Using in situ generated ETDO, a facile, synthesis was developed for two naturally occurring oxysterols, i.e., 25-hydroxycholesterol, as well as its 3-sulfate (overall yield of the sulfate, 24%) and 24-oxocholesterol (16%), starting from cholesterol.

[Synthesis of sterols oxygenated in the terminal fragment of their side chains].

Methods of stereoselective synthesis of oxysterols are considered by the examples of (25R)-26-hydroxycholesterol, (24S)-24,25-epoxycholesterol, and (24S)-24-hydroxycholesterol containing functional groups in the terminal fragments of their side chains. Special attention is paid to the problems of construction of chiral centers C24 and C25.

[A new synthesis of cerebrosterol and its 24-epimer from lithocholic acid].

A new method for the synthesis of both isomers of 24-hydroxycholesterol starting from lithocholic acid is proposed.

Clemmensen reduction of diosgenin and kryptogenin: synthesis of [16,16,22,22,23,23-(2)H(6)]-(25R)-26-hydroxycholesterol.

A new experimental protocol has been established for the Clemmensen reduction of diosgenin and kryptogenin with the aim to prepare deuterated isotopomers of (25R)-26-hydroxycholesterol. Uncontrolled deuteration has been achieved from diosgenin, whereas [16,16,22,22,23,23-(2)H(6)]-(25R)-26-hydroxycholesterol (1) can be synthesized from kryptogenin.

Synthesis of (25R)-26-hydroxycholesterol.

We describe the synthesis of (25R)-cholest-5-en-3beta,26-diol ((25R)-26-hydroxycholesterol) from diosgenin in four steps in 58% overall, yield via a modified Clemmensen reduction followed by a Barton deoxygenation reaction.

Pharmacophore analysis of the nuclear oxysterol receptor LXRalpha.

A cell-free assay was developed for the orphan nuclear receptor LXRalpha that measures the ligand-dependent recruitment of a peptide from the steroid receptor coactivator 1 (SRC1) to the nuclear receptor. Using this ligand-sensing assay (LiSA), the structural requirements for activation of the receptor by oxysterols and related compounds were studied. The minimal pharmacophore for receptor activation was shown to be a sterol with a hydrogen bond acceptor at C24. 24(S),25-Epoxycholesterol (1), which meets this criterion, is among the most efficacious of the oxysterols and is an attractive candidate as the LXRalpha natural hormone. Cholenic acid dimethylamide (14) showed increased efficacy compared to 1, whereas the unnatural oxysterol 22(S)-hydroxycholesterol (4) was shown to be an antagonist of 1 in the LiSA. The structural requirements for SRC1 recruitment in the LiSA correlated with the transcriptional activity of compounds in a cell-based reporter assay employing LXRalpha-GAL4 chimeric receptors. Site-directed mutagenesis identified Trp(443) as an amino acid critical for activation of LXRalpha by oxysterol ligands. This information was combined with the structure-activity relationship developed from the LiSA to develop a 3D homology model of LXRalpha. This model may aid the design of synthetic drugs targeted at this transcriptional regulator of cholesterol homeostasis.

A new chemical synthesis of 5alpha-cholest-8(14)-en-3beta,5alpha-diol.

Reduction of 3beta-benzoyloxy-14alpha,15alpha-epoxy-5alpha-cholest-7-ene with lithium in ethylenediamine gave 5alpha-cholest-8(14)-en-3beta, 5alpha-diol in high yield. This procedure offers an alternate synthesis through the reductive rearrangement of an alpha,beta-unsaturated steroidal epoxide.