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.

Establishment of Biosynthetic Pathways To Generate Castasterone as the Biologically Active Brassinosteroid in Brachypodium distachyon.

Gas chromatography-mass spectrometry (GC-MS) analysis revealed that castasterone and its biosynthetic precursors are found in Brachypodium distachyon. In vitro conversion experiments with crude enzyme solutions prepared from B. distachyon demonstrated the presence of the following biosynthetic sequences: campesterol → campesta-4-en-3-one → campesta-3-one → campestanol → 6-deoxocathasterone → 6-deoxoteasterone → teasterone ↔ 3-dehydroteasterone ↔ typhasterol → castasterone. campesterol → 22-hydroxycampesterol → 22-hydroxy-campesta-4-en-3-one → 22-hydroxy-campesta-3-one → 6-deoxo-3-dehydroteasterone → 3-dehydroteasterone. 6-deoxoteasterone ↔ 6-deoxo-3-dehydroteasterone ↔ 6-deoxotyphasterol → 6-deoxocastasterone → castasterone. This shows that there are campestanol-dependent and campestanol-independent pathway in B. distachyon that synthesize 24-methylated brassinosteroids (BRs). Biochemical analysis of BRs biosynthetic enzymes confirmed that BdDET2, BdCYP90B1, BdCYP90A1, BdCYP90D2, and BdCYP85A1 are orthologous to BR 5α-reductase, BR C-22 hydroxylase, BR C-3 oxidase, BR C-23 hydroxylase, and BR C-6 oxidase, respectively. Brassinolide was not identified in B. distachyon. Additionally, B. distachyon crude enzyme solutions could not catalyze the conversion of castasterone to brassinolide, and the gene encoding an ortholog of CYP85A2 (a brassinolide synthase) was not found in B. distachyon. These results strongly suggest that the end product for brassinosteroid biosynthesis which controls the growth and development of B. distachyon is not brassinolide but rather castasterone.

Molecular Mechanism of Brassinosteroid Perception by the Plant Growth Receptor BRI1.

Brassinosteroids (BRs) are essential phytohormones, which bind to the plant receptor, BRI1, to regulate various physiological processes. The molecular mechanism of the perception of BRs by the ectodomain of BRI1 remains not fully understood. It also remains elusive why a substantial difference in biological activity exists between the BRs. In this work, we study the binding mechanisms of the two most bioactive BRs, brassinolide (BLD) and castasterone (CAT), using molecular dynamics simulations. We report free-energy landscapes of the binding processes of both ligands, as well as detailed ligand binding pathways. Our results suggest that CAT has a lower binding affinity compared to BLD due to its inability to form hydrogen-bonding interactions with a tyrosine residue in the island domain of BRI1. We uncover a conserved nonproductive binding state for both BLD and CAT, which is more stable for CAT and may further contribute to the bioactivity difference. Finally, we validate past observations about the conformational restructuring and ordering of the island domain upon BLD binding. Overall, this study provides new insights into the fundamental mechanism of the perception of the two most bioactive BRs, which may create new avenues for genetic and agrochemical control of their signaling cascade.

Study on synthesis of key squalamine intermediate 7α, 24R-dihydroxy-5α-cholestan-3-one.

A facile novel strategy has been developed to obtain a key intermediate of squalamine, 7α, 24R -dihydroxy-5α-cholestan-3-one, starting from methyl Δ5-3ß-hydroxycholanate. The pure product was successfully synthesized and separated from the C-24 position epimers in good purity, d.e.% and yield.

LC-MSMS characterisations of scymnol and oxoscymnol biotransformations in incubation mixtures of rat liver microsomes.

The bile alcohol 5ß-scymnol ([24R]-(+)-5ß-cholestan-3α,7α,12α,24,26,27-hexol) is a therapeutic nutraceutical derived from marine sources, however very little is known about its potential for biotransformation as a xenobiotic in higher vertebrates. In this study, biotransformation products of scymnol catalysed by liver microsomes isolated from normal and streptozotocin (STZ)-treated male Wistar rats were characterised by liquid chromatography-tandem mass spectroscopy (LC-MSMS). In order of increasing polarity relative to the reversed phase sorbent, structural assignments were made for four biotransformation products, namely 3-oxoscymnol (5ß-cholestan-3-one-7α,12α,24,26,27-pentol); 7-oxoscymnol (5ß-cholestan-7-one-3α,12α,24,26,27-pentol); 3ß-scymnol (5ß-cholestan-3ß,7α,12α,24,26,27-hexol) and 6ß-hydroxyscymnol (5ß-cholestan-3α,6ß,7α,12α,24,26,27-heptol). In addition, a total of eight biotransformation products were characterised from microsomal incubations of crude oxoscymnol compounds, namely 7ß-scymnol; 3,12-dioxoscymnol; 3,7-dioxoscymnol; 7,12-dioxoscymnol; 12-oxo-3ß-scymnol; 7-oxo-3ß-scymnol; 6ß-hydroxy-12-oxoscymnol and 6ß-hydroxy-7-oxoscymnol. Collectively, the results indicate hepatic enzyme-catalysed hydroxylation, dehydrogenation and epimerisation reactions on the steroid nucleus of scymnol, and provide an insight into biotransformation pathways for scymnol use as a therapeutic nutraceutical in higher vertebrates.

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.

Solvent-free synthesis of 6ß-phenylamino-cholestan-3ß,5α-diol and (25R)-6ß-phenylaminospirostan-3ß,5α-diol as potential antiproliferative agents.

In this paper is described a synthetic route to 6ß-phenylamino-cholestan-3ß,5α-diol and (25R)-6ß-phenylaminospirostan-3ß,5α-diol, starting from cholesterol and diosgenin, respectively. The products were obtained in two steps by epoxidation followed by aminolysis, through an environmentally friendly and solvent-free method mediated by SZ (sulfated zirconia) as catalyst. The use of SZ allows chemo- and regioselective ring opening of the 5,6α-epoxide during the aminolysis reaction eliminating the required separation of the epoxide mixture. The products obtained were spectroscopically characterized by 1H, PENDANT 13C NMR and HETCOR experiments, and complemented with FTIR-ATR and HRMS. The antiproliferative effect of the ß-aminoalcohols was evaluated on MCF-7 cells after 48h of incubation, by MTT and CVS assays. These methodologies showed that both compounds have antiproliferative activity, being more active the cholesterol analogue. Additionally, the cell images obtained by Harris' Hematoxylin and Eosin (H&E) staining protocol, evidenced formation of apoptotic bodies due to the presence of the obtained ß-aminoalcohols in a dose-dependent manner.

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.

Quantitative analysis of the tumor suppressor dendrogenin A using liquid chromatography tandem mass spectrometry.

Dendrogenin A (DDA) was recently identified as a mammalian cholesterol metabolite that displays tumor suppressor and neurostimulating properties at low doses. In breast tumors, DDA levels were found to be decreased compared to normal tissues, evidencing a metabolic deregulation of DDA production in cancers. DDA is an amino-oxysterol that contains three protonatable nitrogen atoms. This makes it physico-chemically different from other oxysterols and it therefore requires specific analytical methods We have previously used a two-step method for the quantification of DDA in biological samples: 1) DDA purification from a Bligh and Dyer extract by RP-HPLC using a 250×4.6mm column, followed by 2) nano-electrospray ionization mass spectrometry (MS) fragmentation to analyze the HPLC fraction of interest. We report here the development a liquid chromatography tandem mass spectrometry method for the analysis of DDA and its analogues. This new method is fast (10min), resolving (peak width <4s) and has a weak carryover (<0.01%). We show that this technique efficiently separates DDA from its C17 isomer and other steroidal alkaloids from the same family establishing a proof of concept for the analysis of this family of amino-oxysterols.

Mimicking natural phytohormones. 26-Hydroxycholestan-22-one derivatives as plant growth promoters.

26-Hydroxycholestan-22-one derivatives with oxygenated functions in the rings A and/or B were successfully synthesized from diosgenin. After the modifications of rings A and B, the spiroketal side chain was selectively opened through a Lewis acid mediated acetolysis to afford the cholestane derivatives. These compounds incorporate pharmacophores, which mimic the activity of natural phytohormones and show high growth promoting activity in Mexican rice cultivars using the rice lamina inclination test.

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.

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.

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.

Synthetic Protocol for AFCS: A Biologically Active Fluorescent Castasterone Analog Conjugated to an Alexa Fluor 647 Dye.

Synthetic derivatization of hormonally active brassinosteroids (BRs) can provide useful small molecule tools to probe BR signaling pathways, such as fluorescent analogs. However, most biologically active BRs are not suitable for direct chemical conjugation techniques because their derivatization typically requires extensive synthetic work and chemistry expertise. Here, we describe an operationally simple, two-step procedure to prepare and purify an Alexa Fluor 647-castasterone (AFCS) from commercially available materials. The reported strategy is also amenable to the introduction of various other amine-based labeling groups.

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.

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.

Novel, major 2α- and 2ß-hydroxy bile alcohols and bile acids in the bile of Arapaima gigas, a large South American river fish.

Bile alcohols and bile acids from gallbladder bile of the Arapaima gigas, a large South American freshwater fish, were isolated by reversed-phase high-performance liquid chromatography. The structures of the major isolated compounds were determined by electrospray-tandem mass spectrometry and nuclear magnetic resonance using (1)H- and (13)C-NMR spectra. The novel bile salts identified were six variants of 2-hydroxy bile acids and bile alcohols in the 5α- and 5ß-series, with 29% of all compounds having hydroxylation at C-2. Three C27 bile alcohols were present (as ester sulfates): (24ξ,25ξ)-5α-cholestan-2α,3α,7α,12α,24,26-hexol; (25ξ)-5ß-cholestan-2ß,3α,7α,12α,26,27-hexol, and (25ξ)-5α-cholestan-2α,3α,7α,12α,26,27-hexol. A single C27 bile acid was identified: (25ξ)-2α,3α,7α,12α-tetrahydroxy-5α-cholestan-26-oic acid, present as its taurine conjugate. Two novel C24 bile acids were identified: the 2α-hydroxy derivative of allochenodeoxycholic acid and the 2ß-hydroxy derivative of cholic acid, both occurring as taurine conjugates. These studies extend previous work in establishing the natural occurrence of novel 2α- and 2ß-hydroxy-C24 and C27 bile acids as well as C27 bile alcohols in both the normal (5ß) as well as the (5α) "allo" A/B-ring juncture. The bile salt profile of A. gigas appears to be unique among vertebrates.