From Marine Metabolites to the Drugs of the Future: Squalamine, Trodusquemine, Their Steroid and Triterpene Analogues.

This review comprehensively describes the recent advances in the synthesis and pharmacological evaluation of steroid polyamines squalamine, trodusquemine, ceragenins, claramine, and their diverse analogs and derivatives, with a special focus on their complete synthesis from cholic acids, as well as an antibacterial and antiviral, neuroprotective, antiangiogenic, antitumor, antiobesity and weight-loss activity, antiatherogenic, regenerative, and anxiolytic properties. Trodusquemine is the most-studied small-molecule allosteric PTP1B inhibitor. The discovery of squalamine as the first representative of a previously unknown class of natural antibiotics of animal origin stimulated extensive research of terpenoids (especially triterpenoids) comprising polyamine fragments. During the last decade, this new class of biologically active semisynthetic natural product derivatives demonstrated the possibility to form supramolecular networks, which opens up many possibilities for the use of such structures for drug delivery systems in serum or other body fluids.

Cholestane-3ß, 5α, 6ß-triol: Further insights into the performance of this oxysterol in diagnosis of Niemann-Pick disease type C.

In recent years the oxysterol species cholestane-3ß, 5α, 6ß-triol (C-triol) has found application as a diagnostic biomarker for Niemann-Pick disease type C. Other studies have described increased C-triol in patients with Niemann-Pick disease type A/B and milder increases in lysosomal acid lipase deficiency (LALD), whereas they note normal C-triol levels in Smith-Lemli-Opitz syndrome (SLOS) and familial hypercholesterolaemia (FH) patients. Herein, we review data collected in our laboratory during method evaluation along with 5 years of routine analysis and present findings which differ from those reported by other groups with respect to LALD, SLOS and FH in particular, whilst providing further evidence regarding the clinical sensitivity and specificity of this biomarker, which are difficult to accurately assess. All of our Wolman disease (severe LALD) patients have demonstrated gross elevations of C-triol at diagnosis, with reduction to normal levels after induction of enzyme replacement therapy. In diagnostic specimens from SLOS patients we observed very low or undetectable C-triol levels whereas in post-therapeutic SLOS patients demonstrated normalised levels; we also describe a homozygous FH patient in which C-triol is significantly elevated. Upon investigation, we found that C-triol was formed artefactually from cholesterol during our sample preparation, i.e. this is a false positive of analytical origin; at present it is unclear whether similar effects occur during sample preparation in other laboratories. Our data demonstrates clinical sensitivity of 100% during routine application to diagnostic specimens; this is in keeping with other estimates, yet in a small proportion of patients diagnosed prior to C-triol measurement, either by Filipin staining of fibroblasts or molecular genetics, we have observed normal C-triol concentrations. Clinical specificity of C-triol alone is 93.4% and 95.3% when performed in conjunction with lysosomal enzymology. These performance statistics are very similar to those achieved with Filipin staining of cultured fibroblasts in the 5 years preceding introduction of C-triol to routine use in our laboratory. It is increasingly apparent to us that although this analyte is a very useful addition to the diagnostic tools available for NPC, with considerable advantages over more invasive and time-consuming methods, the interpretation of results is complex and should be undertaken only in light of clinical details and results of other analyses including enzymology for lysosomal acid lipase and acid sphingomyelinase.

A natural product inhibits the initiation of α-synuclein aggregation and suppresses its toxicity.

The self-assembly of α-synuclein is closely associated with Parkinson's disease and related syndromes. We show that squalamine, a natural product with known anticancer and antiviral activity, dramatically affects α-synuclein aggregation in vitro and in vivo. We elucidate the mechanism of action of squalamine by investigating its interaction with lipid vesicles, which are known to stimulate nucleation, and find that this compound displaces α-synuclein from the surfaces of such vesicles, thereby blocking the first steps in its aggregation process. We also show that squalamine almost completely suppresses the toxicity of α-synuclein oligomers in human neuroblastoma cells by inhibiting their interactions with lipid membranes. We further examine the effects of squalamine in a Caenorhabditis elegans strain overexpressing α-synuclein, observing a dramatic reduction of α-synuclein aggregation and an almost complete elimination of muscle paralysis. These findings suggest that squalamine could be a means of therapeutic intervention in Parkinson's disease and related conditions.

Developing an Enzyme-Assisted Derivatization Method for Analysis of C27 Bile Alcohols and Acids by Electrospray Ionization-Mass Spectrometry.

Enzyme-assisted derivatization for sterol analysis (EADSA) is a technology designed to enhance sensitivity and specificity for sterol analysis using electrospray ionization⁻mass spectrometry. To date it has only been exploited on sterols with a 3ß-hydroxy-5-ene or 3ß-hydroxy-5α-hydrogen structure, using bacterial cholesterol oxidase enzyme to convert the 3ß-hydroxy group to a 3-oxo group for subsequent derivatization with the positively charged Girard hydrazine reagents, or on substrates with a native oxo group. Here we describe an extension of the technology by substituting 3α-hydroxysteroid dehydrogenase (3α-HSD) for cholesterol oxidase, making the method applicable to sterols with a 3α-hydroxy-5ß-hydrogen structure. The 3α-HSD enzyme works efficiently on bile alcohols and bile acids with this stereochemistry. However, as found by others, derivatization of the resultant 3-oxo group with a hydrazine reagent does not go to completion in the absence of a conjugating double bond in the sterol structure. Nevertheless, Girard P derivatives of bile alcohols and C27 acids give an intense molecular ion ([M]⁺) upon electrospray ionization and informative fragmentation spectra. The method shows promise for analysis of bile alcohols and 3α-hydroxy-5ß-C27-acids, enhancing the range of sterols that can be analyzed at high sensitivity in sterolomic studies.

Potent Antitrypanosomal Activities of 3-Aminosteroids against African Trypanosomes: Investigation of Cellular Effects and of Cross-Resistance with Existing Drugs.

Treatment of animal African trypanosomiasis (AAT) requires urgent need for safe, potent and affordable drugs and this has necessitated this study. We investigated the trypanocidal activities and mode of action of selected 3-aminosteroids against Trypanosoma brucei brucei. The in vitro activity of selected compounds of this series against T. congolense (Savannah-type, IL3000), T. b. brucei (bloodstream trypomastigote, Lister strain 427 wild-type (427WT)) and various multi-drug resistant cell lines was assessed using a resazurin-based cell viability assay. Studies on mode of antitrypanosomal activity of some selected 3-aminosteroids against Tbb 427WT were also carried out. The tested compounds mostly showed moderate-to-low in vitro activities and low selectivity to mammalian cells. Interestingly, a certain aminosteroid, holarrhetine (10, IC50 = 0.045 ± 0.03 µM), was 2 times more potent against T. congolense than the standard veterinary drug, diminazene aceturate, and 10 times more potent than the control trypanocide, pentamidine, and displayed an excellent in vitro selectivity index of 2130 over L6 myoblasts. All multi-drug resistant strains of T. b. brucei tested were not significantly cross-resistant with the purified compounds. The growth pattern of Tbb 427WT on long and limited exposure time revealed gradual but irrecoverable growth arrest at ≥ IC50 concentrations of 3-aminosteroids. Trypanocidal action was not associated with membrane permeabilization of trypanosome cells but instead with mitochondrial membrane depolarization, reduced adenosine triphosphate (ATP) levels and G2/M cell cycle arrest which appear to be the result of mitochondrial accumulation of the aminosteroids. These findings provided insights for further development of this new and promising class of trypanocide against African trypanosomes.

Cholestane-3ß, 5α, 6ß-triol suppresses neuronal hyperexcitability via binding to voltage-gated sodium channels.

Neuronal hyperexcitability is identified as a critical pathological basis of epileptic seizures. Cholestane-3ß, 5α, 6ß-triol (Triol) is a major metabolic oxysterol of cholesterol. Although its neuroprotective effect on ischemia-induced neuronal injury and negative modulation of voltage-gated sodium (Nav) channels were well established, the physical binding site of triol to sodium channels and its effects on neuronal hyperexcitability have not yet been explored. In this study, we utilized molecular docking and molecular dynamics simulation to investigate the interaction between triol and Nav Channels. Our results demonstrated that triol binds to the indole ring of Trp122 of the Nav Channel in silico with a high biological affinity. We further found that triol negatively modulates the action potentials bursts of hippocampal neurons by cell-attached patch recording. Moreover, triol significantly inhibits low Mg2+-induced hyperexcitability in vitro. In addition, triol attenuates pentylenetetrazole (PTZ)-induced convulsive-form behavioral deficits in vivo. Together, our results suggest that triol suppresses neuronal hyperexcitability via binding to Nav channel, indicating that triol might be an attractive lead compound for the treatment of neuronal hyperexcitability-related neurological disorders, especially epileptic seizures.

Synthesis and characterization of a novel rhodamine labeled cholesterol reporter.

We introduce the novel fluorescent cholesterol probe RChol in which a sulforhodamine group is linked to the sixth carbon atom of the steroid backbone of cholesterol. The same position has recently been selected to generate the fluorescent reporter 6-dansyl-cholestanol (DChol) and the photoreactive 6-azi-cholestanol. In comparison with DChol, RChol is brighter, much more photostable, and requires less energy for excitation, i.e. favorable conditions for microscopical imaging. RChol easily incorporates into methyl-ß-cyclodextrin forming a water-soluble inclusion complex that acts as an efficient sterol donor for cells and membranes. Like cholesterol, RChol possesses a free 3'OH group, a prerequisite to undergo intracellular esterification. RChol was also able to support the growth of cholesterol auxotrophic cells and can therefore substitute for cholesterol as a major component of the plasma membrane. According to subcellular fractionation, slight amounts of RChol (~12%) were determined in low-density Triton-insoluble fractions whereas the majority of RChol was localized in non-rafts fractions. In phase-separated giant unilamellar vesicles, RChol preferentially partitions in liquid-disordered membrane domains. Intracellular RChol was transferred to extracellular sterol acceptors such as high density lipoproteins in a dose-dependent manner. Unlike DChol, RChol was not delivered to the cholesterol storage pathway. Instead, it translocated to endosomes/lysosomes with some transient contacts to peroxisomes. Thus, RChol is considered as a useful probe to study the endosomal/lysosomal pathway of cholesterol.

Brassinolide-like activity of castasterone analogs with varied side chains against rice lamina inclination.

Brassinolide (BL) and castasterone (CS) are the representative members of brassinosteroid class of plant steroid hormone having plant growth promoting activity. In this study, eleven CS analogs bearing a variety of side chains were synthesized to determine the effect of the side chain structures on the BL-like activity. The plant hormonal activity was evaluated in a dwarf rice lamina inclination assay, and the potency was determined as the reciprocal logarithm of the 50% effective dose (ED50) from each dose-response curve. The reciprocal logarithm of ED50 (pED50) was decreased dramatically upon deletion of the C-28 methyl group of CS. The introduction of oxygen-containing groups such as hydroxy, methoxy, and ethoxycarbonyl was also unfavorable to the activity. The pED50 was influenced by the geometry of carbon-carbon double bond between C-24 and C-25 (cis and trans), but the introduction of a fluorine atom at the C-25 position of the double bond did not significantly change the activity. The binding free energy (ΔG) was calculated for all ligand-receptor binding interactions using molecular dynamics, resulting that ΔG is linearly correlated with the pED50.

Three Novel Bile Alcohols of Mature Male Sea Lamprey (Petromyzon marinus) Act as Chemical Cues for Conspecifics.

Sea lamprey, Petromyzon marinus, rely heavily on chemical cues that mediate their life history events, such as migration and reproduction. Here, we describe petromyzone A-C (1-3), three novel bile alcohols that are highly oxidized and sulfated, isolated from water conditioned with spermiated male sea lamprey. Structures of these compounds were unequivocally established by spectroscopic analyses and by comparison with spectra of known compounds. Electro-olfactogram recordings showed that 1 at 10-11 M was stimulatory to the adult sea lamprey olfactory epithelium, while 2 and 3 were stimulatory at 10-13 M. Behavioral assays indicated that 1 is attractive, 2 is not attractive or repulsive, and 3 is repulsive to ovulated female sea lamprey. The results suggest that 1 and 2 may be putative pheromones that mediate chemical communication in sea lamprey. The identification of these three components enhances our understanding of the structures and functions of sex pheromone components in this species and may provide useful behavioral manipulation tools for the integrated management of sea lamprey, a destructive invader in the Laurentian Great Lakes.

Structural insight into brassinosteroid perception by BRI1.

Brassinosteroids are essential phytohormones that have crucial roles in plant growth and development. Perception of brassinosteroids requires an active complex of BRASSINOSTEROID-INSENSITIVE 1 (BRI1) and BRI1-ASSOCIATED KINASE 1 (BAK1). Recognized by the extracellular leucine-rich repeat (LRR) domain of BRI1, brassinosteroids induce a phosphorylation-mediated cascade to regulate gene expression. Here we present the crystal structures of BRI1(LRR) in free and brassinolide-bound forms. BRI1(LRR) exists as a monomer in crystals and solution independent of brassinolide. It comprises a helical solenoid structure that accommodates a separate insertion domain at its concave surface. Sandwiched between them, brassinolide binds to a hydrophobicity-dominating surface groove on BRI1(LRR). Brassinolide recognition by BRI1(LRR) is through an induced-fit mechanism involving stabilization of two interdomain loops that creates a pronounced non-polar surface groove for the hormone binding. Together, our results define the molecular mechanisms by which BRI1 recognizes brassinosteroids and provide insight into brassinosteroid-induced BRI1 activation.

Structural basis of steroid hormone perception by the receptor kinase BRI1.

Polyhydroxylated steroids are regulators of body shape and size in higher organisms. In metazoans, intracellular receptors recognize these molecules. Plants, however, perceive steroids at membranes, using the membrane-integral receptor kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1). Here we report the structure of the Arabidopsis thaliana BRI1 ligand-binding domain, determined by X-ray diffraction at 2.5 Å resolution. We find a superhelix of 25 twisted leucine-rich repeats (LRRs), an architecture that is strikingly different from the assembly of LRRs in animal Toll-like receptors. A 70-amino-acid island domain between LRRs 21 and 22 folds back into the interior of the superhelix to create a surface pocket for binding the plant hormone brassinolide. Known loss- and gain-of-function mutations map closely to the hormone-binding site. We propose that steroid binding to BRI1 generates a docking platform for a co-receptor that is required for receptor activation. Our findings provide insight into the activation mechanism of this highly expanded family of plant receptors that have essential roles in hormone, developmental and innate immunity signalling.

Synthesis and cytotoxic activity of two steroids: icogenin aglycone analogs.

During the process of icogenin analog research, we obtained two cytotoxic steroids: compound 4 and compound 6 casually. Their in vitro antitumor activities were tested by the standard MTT assay. The results disclosed that compound 4 (IC50 = 3.65-6.90 µM) showed potential antitumor activities against HELA, KB cell lines and compound 6 (IC50 = 2.40-9.05 µM) showed potential antitumor activities against HELA, BGC-823, KB, A549, HCT-8 cell lines.

A new bioactive steroidal ketone from the South China Sea sponge Xestospongia testudinaria.

A new steroidal ketone (1), with an ergosta-22,25-diene side chain, was obtained from the South China Sea marine sponge Xestospongia testudinaria. The structure of 1 was determined on the basis of detailed spectroscopic analysis and by comparison with literature. Compound 1 exhibited significant inhibitory activity against protein tyrosine phosphatase 1B (PTP1B), a key target for the treatment of type II diabetes and obesity, with an IC50 value of 4.27 ± 0.55 µM, which is comparable with the positive control oleanolic acid (IC50 = 2.63 ± 0.22 µM).

Quantification of Oxidized and Unsaturated Bile Alcohols in Sea Lamprey Tissues by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry.

A sensitive and reliable method was developed and validated for the determination of unsaturated bile alcohols in sea lamprey tissues using liquid-liquid extraction and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The liver, kidney, and intestine samples were extracted with acetonitrile and defatted by n-hexane. Gradient UHPLC separation was performed using an Acquity BEH C18 column with a mobile phase of water and methanol containing 20 mM triethylamine. Multiple reaction monitoring modes of precursor-product ion transitions for each analyte was used. This method displayed good linearity, with correlation coefficients greater than 0.99, and was validated. Precision and accuracy (RSD %) were in the range of 0.31%-5.28%, while mean recoveries were between 84.3%-96.3%. With this technique, sea lamprey tissue samples were analyzed for unsaturated bile alcohol analytes. This method is practical and particularly suitable for widespread putative pheromone residue analysis.

Occurrence of phosphorylated castasterone in Arabidopsis thaliana and Lycopersicum esculentum.

An in vitro enzyme assay using radioisotope-labeled (3) H-castasterone ((3) H-CS) or (32) P-ATP showed that CS can be phosphorylated by ATP in Arabidopsis and tomato plants. Gas chromatography-mass spectrometry (GC-MS) analysis using non-isotope-labeled CS and ATP revealed that the phosphorylation of CS occurs at the side chain, most likely at the C-23 hydroxyl. The polar fractions than free brassinosteroids (BRs) obtained from extracts of Arabidopsis and tomato showed almost no BRs activity in a rice lamina inclination bioassay. However, the fractions showed increased bioactivity after treatment with wheat germ acidic phosphatase (WGAP). Additionally, CS was identified from the hydrolysate by WGAP using GC-MS analysis in both plants. In contrast, the polar fractions obtained from BR-deficient mutants, Arabidopsis cyp85a2 and tomato d(x) , did not show an increase in biological activity with WGAP treatment, and no free BRs, including CS, were detected in the hydrolysate. This suggests that CS phosphate is a naturally occurring biologically inactive conjugate that is generated when CS is normally synthesized in Arabidopsis and tomato plants. Taken together, these results suggest that phosphorylation of CS is an important conjugation process for the maintenance of the homeostatic level of an active BR and thus the regulation of the growth and development of plants.

An improved synthesis of [26-(2)H3]castasterone.

Commercially available epicastasterone has been employed as a starting material for the preparation of [26-(2)H3 ]castasterone. The chemical synthesis has been realized in 13 chemical steps and 4.6% total yield. The target compound is intended to be used as internal standard for the quantitative analysis of brassinosteroids.

Microdiplanol and microdiplane: a new m-anisaldehyde and a new 24-methylcholestanol derivative from the endophytic fungus Microdiplodia sp.

Phytochemical investigation of the endophytic fungus Microdiplodia sp. afforded a new m-anisaldehyde derivative named microdiplanol (1) and a new 24-methylcholestanol derivative named microdiplane (2). Their structures were confirmed by a comprehensive analysis of 1D and 2D NMR and mass spectrometric data.

Bufadienolides and polyhydroxycholestane derivatives from Bufo bufo gargarizans.

A new polyhydroxycholestane sulfate ester, 3α,12ß,25,26-tetrahydroxy-7-oxo-5ß-cholestane 26-O-sulfate (1), was isolated from dried skin of Bufo bufo gargarizans Cantor and its structure was elucidated on the basis of extensive 1D and 2D NMR as well as HR-ESI-MS analysis. A comparison of steroidal metabolite profiles, based on HPLC and LC-MS analyses, indicates that the chemical compositions of the various parts of toads, such as venom, skin and stratum corneum, are significantly different.

Improved cross validation of a static ubiquitin structure derived from high precision residual dipolar couplings measured in a drug-based liquid crystalline phase.

The antibiotic squalamine forms a lyotropic liquid crystal at very low concentrations in water (0.3-3.5% w/v), which remains stable over a wide range of temperature (1-40 °C) and pH (4-8). Squalamine is positively charged, and comparison of the alignment of ubiquitin relative to 36 previously reported alignment conditions shows that it differs substantially from most of these, but is closest to liquid crystalline cetyl pyridinium bromide. High precision residual dipolar couplings (RDCs) measured for the backbone (1)H-(15)N, (15)N-(13)C', (1)H(α)-(13)C(α), and (13)C'-(13)C(α) one-bond interactions in the squalamine medium fit well to the static structural model previously derived from NMR data. Inclusion into the structure refinement procedure of these RDCs, together with (1)H-(15)N and (1)H(α)-(13)C(α) RDCs newly measured in Pf1, results in improved agreement between alignment-induced changes in (13)C' chemical shift, (3)JHNHα values, and (13)C(α)-(13)C(ß) RDCs and corresponding values predicted by the structure, thereby validating the high quality of the single-conformer structural model. This result indicates that fitting of a single model to experimental data provides a better description of the average conformation than does averaging over previously reported NMR-derived ensemble representations. The latter can capture dynamic aspects of a protein, thus making the two representations valuable complements to one another.

Fluorescent castasterone reveals BRI1 signaling from the plasma membrane.

Receptor-mediated endocytosis is an integral part of signal transduction as it mediates signal attenuation and provides spatial and temporal dimensions to signaling events. One of the best-studied leucine-rich repeat receptor-like kinases in plants, BRASSINOSTEROID INSENSITIVE 1 (BRI1), perceives its ligand, the brassinosteroid (BR) hormone, at the cell surface and is constitutively endocytosed. However, the importance of endocytosis for BR signaling remains unclear. Here we developed a bioactive, fluorescent BR analog, Alexa Fluor 647-castasterone (AFCS), and visualized the endocytosis of BRI1-AFCS complexes in living Arabidopsis thaliana cells. Impairment of endocytosis dependent on clathrin and the guanine nucleotide exchange factor for ARF GTPases (ARF-GEF) GNOM enhanced BR signaling by retaining active BRI1-ligand complexes at the plasma membrane. Increasing the trans-Golgi network/early endosome pool of BRI1-BR complexes did not affect BR signaling. Our findings provide what is to our knowledge the first visualization of receptor-ligand complexes in plants and reveal clathrin- and ARF-GEF-dependent endocytic regulation of BR signaling from the plasma membrane.