Exploring the Chemical Space of Macro- and Micro-Algae Using Comparative Metabolomics.

With more than 156,000 described species, eukaryotic algae (both macro- and micro-algae) are a rich source of biological diversity, however their chemical diversity remains largely unexplored. Specialised metabolites with promising biological activities have been widely reported for seaweeds, and more recently extracts from microalgae have exhibited activity in anticancer, antimicrobial, and antioxidant screens. However, we are still missing critical information on the distinction of chemical profiles between macro- and microalgae, as well as the chemical space these metabolites cover. This study has used an untargeted comparative metabolomics approach to explore the chemical diversity of seven seaweeds and 36 microalgal strains. A total of 1390 liquid chromatography-mass spectrometry (LC-MS) features were detected, representing small organic algal metabolites, with no overlap between the seaweeds and microalgae. An in-depth analysis of four Dunaliella tertiolecta strains shows that environmental factors may play a larger role than phylogeny when classifying their metabolomic profiles.

Inhibitory Kappa B Kinase α (IKKα) Inhibitors That Recapitulate Their Selectivity in Cells against Isoform-Related Biomarkers.

IKKß plays a central role in the canonical NF-kB pathway, which has been extensively characterized. The role of IKKα in the noncanonical NF-kB pathway, and indeed in the canonical pathway as a complex with IKKß, is less well understood. One major reason for this is the absence of chemical tools designed as selective inhibitors for IKKα over IKKß. Herein, we report for the first time a series of novel, potent, and selective inhibitors of IKKα. We demonstrate effective target engagement and selectivity with IKKα in U2OS cells through inhibition of IKKα-driven p100 phosphorylation in the noncanonical NF-kB pathway without affecting IKKß-dependent IKappa-Bα loss in the canonical pathway. These compounds represent the first chemical tools that can be used to further characterize the role of IKKα in cellular signaling, to dissect this from IKKß and to validate it in its own right as a target in inflammatory diseases.

Formulation of Nonionic Surfactant Vesicles (NISV) Prepared by Microfluidics for Therapeutic Delivery of siRNA into Cancer Cells.

Small interfering RNAs (siRNA) have a broad potential as therapeutic agents to reversibly silence any target gene of interest. The clinical application of siRNA requires the use of safe and effective delivery systems. In this study, we investigated the use of nonionic surfactant vesicles (NISV) for the delivery of siRNA. Different types of NISV formulations were synthesized by microfluidic mixing and then evaluated for their physiochemical properties and cytotoxicity. The ability of the NISV to carry and transfect siRNA targeting green fluorescent protein (GFP) into A549 that stably express GFP (copGFP-A549) was evaluated. Flow cytometry and Western blotting were used to study the GFP expression knockdown, and significant knockdown was observed as a result of siRNA delivery to the cells by NISV. This occurred in particular when using Tween 85, which was able to achieve more than 70% GFP knockdown. NISV were thus demonstrated to provide a promising and effective platform for therapeutic delivery of siRNA.

Metabolomic Profiling of the Synergistic Effects of Melittin in Combination with Cisplatin on Ovarian Cancer Cells.

Melittin, the main peptide present in bee venom, has been proposed as having potential for anticancer therapy; the addition of melittin to cisplatin, a first line treatment for ovarian cancer, may increase the therapeutic response in cancer treatment via synergy, resulting in improved tolerability, reduced relapse, and decreased drug resistance. Thus, this study was designed to compare the metabolomic effects of melittin in combination with cisplatin in cisplatin-sensitive (A2780) and resistant (A2780CR) ovarian cancer cells. Liquid chromatography (LC) coupled with mass spectrometry (MS) was applied to identify metabolic changes in A2780 (combination treatment 5 µg/mL melittin + 2 µg/mL cisplatin) and A2780CR (combination treatment 2 µg/mL melittin + 10 µg/mL cisplatin) cells. Principal components analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) multivariate data analysis models were produced using SIMCA-P software. All models displayed good separation between experimental groups and high-quality goodness of fit (R²) and goodness of prediction (Q²), respectively. The combination treatment induced significant changes in both cell lines involving reduction in the levels of metabolites in the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, purine and pyrimidine metabolism, and the arginine/proline pathway. The combination of melittin with cisplatin that targets these pathways had a synergistic effect. The melittin-cisplatin combination had a stronger effect on the A2780 cell line in comparison with the A2780CR cell line. The metabolic effects of melittin and cisplatin in combination were very different from those of each agent alone.

Structure-Activity Relationships of Small Molecule Autotaxin Inhibitors with a Discrete Binding Mode.

Autotaxin (ATX) is a secreted enzyme responsible for the hydrolysis of lysophosphatidylcholine (LPC) to the bioactive lysophosphatidic acid (LPA) and choline. The ATX-LPA signaling pathway is implicated in cell survival, migration, and proliferation; thus, the inhibition of ATX is a recognized therapeutic target for a number of diseases including fibrotic diseases, cancer, and inflammation, among others. Many of the developed synthetic inhibitors for ATX have resembled the lipid chemotype of the native ligand; however, a small number of inhibitors have been described that deviate from this common scaffold. Herein, we report the structure-activity relationships (SAR) of a previously reported small molecule ATX inhibitor. We show through enzyme kinetics studies that analogues of this chemotype are noncompetitive inhibitors, and by using a crystal structure with ATX we confirm the discrete binding mode.

Metabolomic Profiling of the Effects of Melittin on Cisplatin Resistant and Cisplatin Sensitive Ovarian Cancer Cells Using Mass Spectrometry and Biolog Microarray Technology.

In the present study, liquid chromatography-mass spectrometry (LC-MS) was employed to characterise the metabolic profiles of two human ovarian cancer cell lines A2780 (cisplatin-sensitive) and A2780CR (cisplatin-resistant) in response to their exposure to melittin, a cytotoxic peptide from bee venom. In addition, the metabolomics data were supported by application of Biolog microarray technology to examine the utilisation of carbon sources by the two cell lines. Data extraction with MZmine 2.14 and database searching were applied to provide metabolite lists. Principal component analysis (PCA) gave clear separation between the cisplatin-sensitive and resistant strains and their respective controls. The cisplatin-resistant cells were slightly more sensitive to melittin than the sensitive cells with IC50 values of 4.5 and 6.8 µg/mL respectively, although the latter cell line exhibited the greatest metabolic perturbation upon treatment. The changes induced by melittin in the cisplatin-sensitive cells led mostly to reduced levels of amino acids in the proline/glutamine/arginine pathway, as well as to decreased levels of carnitines, polyamines, adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD+). The effects on energy metabolism were supported by the data from the Biolog assays. The lipid compositions of the two cell lines were quite different with the A2780 cells having higher levels of several ether lipids than the A2780CR cells. Melittin also had some effect on the lipid composition of the cells. Overall, this study suggests that melittin might have some potential as an adjuvant therapy in cancer treatment.

Synthesis and Evaluation of a New Series of 8-(2-Nitroaryl)Xanthines as Adenosine Receptor Ligands.

Preclinical Research A new series of 1,3-dimethylxanthine derivatives bearing 8-(2-nitroaryl) residue was synthesized and evaluated for affinity for recombinant human adenosine receptors subtypes. Nitrate esters of 7-substituted-1,3-dimethyl-8-phenylxanthines were also synthesized and tested. Introducing a nitro substituent at the 2-position of the 8-substituted phenyl ring resulted in generally low affinity for adenosine receptors (ARs), selectivity toward the A2A subtype was enhanced in some of the compounds. 8-(4-Cyclopentyloxy-5-methoxy-2-nitrophenyl)-1,3-dimethylxanthine (9e) proved to be a potent compound among the 2-nitrophenyl substituted xanthines exhibiting a Ki = 1 µM at human A2A ARs with at least 30 fold selectivity versus human A1 and A2B ARs. Replacement of 8-chloropropoxy phenyl with 8-nitrooxypropoxy phenyl resulted in a negligible change in binding affinity of the 8-substituted xanthines for various AR subtypes. Drug Dev Res 77 : 241-250, 2016. © 2016 Wiley Periodicals, Inc.

Using Molecular Networking for Microbial Secondary Metabolite Bioprospecting.

The oceans represent an understudied resource for the isolation of bacteria with the potential to produce novel secondary metabolites. In particular, actinomyces are well known to produce chemically diverse metabolites with a wide range of biological activities. This study characterised spore-forming bacteria from both Scottish and Antarctic sediments to assess the influence of isolation location on secondary metabolite production. Due to the selective isolation method used, all 85 isolates belonged to the phyla Firmicutes and Actinobacteria, with the majority of isolates belonging to the genera Bacillus and Streptomyces. Based on morphology, thirty-eight isolates were chosen for chemical investigation. Molecular networking based on chemical profiles (HR-MS/MS) of fermentation extracts was used to compare complex metabolite extracts. The results revealed 40% and 42% of parent ions were produced by Antarctic and Scottish isolated bacteria, respectively, and only 8% of networked metabolites were shared between these locations, implying a high degree of biogeographic influence upon secondary metabolite production. The resulting molecular network contained over 3500 parent ions with a mass range of m/z 149-2558 illustrating the wealth of metabolites produced. Furthermore, seven fermentation extracts showed bioactivity against epithelial colon adenocarcinoma cells, demonstrating the potential for the discovery of novel bioactive compounds from these understudied locations.

Efecto inhibitorio de un extracto acuoso de las hojas de Allophylus cominia (L. ) Sw sobre las proteínas tirosina fosfatasa 1B y dipeptidil peptidasa IV / Inhibitory effect of Allophylus cominia (L. ) Sw leaves aqueous extract on tyrosine phosphatase 1B and dipeptidyl peptidase IV proteins

Introducción: Allophylus cominia (L.) Sw es una planta medicinal cubana usada por la medicina tradicional para el tratamiento de la diabetes, cuyo mecanismo de acción es desconocido. Objetivo: evaluar el efecto del extracto acuoso de hojas de A. cominia (L.) Sw y sus fracciones sobre la proteína tirosina fosfatasa 1B (PTP1B) y dipeptidil peptidasa IV (DPPIV) como diana terapéuticas para el tratamiento de la diabetes tipo 2. Métodos: el extracto acuoso de hojas de A. cominia fue fraccionado sucesivamente con mezclas de solventes orgánicos, incrementando la polaridad, para obtener diez fracciones. El extracto y sus fracciones fueron evaluados para su posible actividad antidiabética sobre diana terapéuticas de diabetes tipo 2: PTP1B y DPPIV. Se realizaron ensayos de inhibición enzimática y la actividad inhibitoria se calculó a partir de los valores de fluorescencia, empleando longitudes de onda de excitación y de emisión de 360 nm y 460 nm respectivamente. Resultados: el extracto acuoso de A. cominia inhibió la actividad enzimática de PTP1B y DPPIV de manera dependiente de la concentración, con valores de CI50 de 0,69 μg/mL y 344,3 μg/mL respectivamente. Varias fracciones se detectaron como potentes inhibidores de PTP1B. Las fracciones más polares AcF9 y AcF10 fueron las más activas, y mostraron valores de CI50 de 4,4 µg/mL y 3,8 µg/mL respectivamente. Las fracciones mostraron una ligera inhibición de DPPIV, y las más activas resultaron AcF6, AcF9 y AcF10, con valores de porcentajes de inhibición de 52,0 por ciento, 39,0 por ciento y 40,0 por ciento respectivamente. Conclusiones: el extracto acuoso de A. cominia y sus fracciones polares (AcF9 y AcF10) tienen propiedades antidiabéticas in vitro y son candidatos promisorios para el desarrollo de nuevos medicamentos con actividad inhibidora de PTP1B y DPPIV para el tratamiento de la diabetes tipo 2(AU)

Introduction: Allophylus cominia (L.) Sw is a Cuban medicinal plant used by traditional medicine for the treatment of diabetes with unknown mechanisms of action. Objective: to evaluate the effect of Allophylus cominia (L.) Sw leaves aqueous extract and its fractions on protein tyrosine phosphatase 1B (PTP1B) and dipeptidyl peptidase IV (DPPIV) enzymatic activity, as therapeutic targets of type 2 diabetes. Methods: the aqueous extract of A. cominia leaves was successively partitioned with organic solvents mixtures, thus increasing polarity in order to obtain ten fractions. The extract and its fractions were tested for their possible antidiabetic activity on therapeutic targets of type 2 diabetes: PTP1B and DPPIV. The enzymatic inhibition assays were performed and the inhibitory activity was calculated with the fluorescence values using an excitation wavelength of 360 nm and an emission wavelength of 460 nm. Results: the aqueous extract from A. cominia inhibited the enzymatic activity of PTP1B and DPPIV according to the concentration, being IC50 values equal to 0.69 μg/mL and 344.3 μg/mL, respectively. Several fractions were detected as potent PTP1B inhibitors. The most polar fractions AcF9 and AcF10 were more active, showing IC50 values of 4.4 µg/mL and 3.8 µg/mL respectively. The fractions showed a slight DPPIV inhibition, being fractions AcF6, AcF9 and AcF10 the most active, exhibiting inhibition percentages of 52.0 percent, 39.0 percent and 40.0 percent respectively. Conclusions: A. cominia aqueous extract and its polar fractions (AcF9 and AcF10) have antidiabetic properties in vitro and are promissory candidates for development of new drugs with inhibitory activity of PTP1B and DPPIV for type 2 diabetes treatment(AU)

Efecto inhibitorio de un extracto acuoso de las hojas de Allophylus cominia (L) Sw sobre las proteínas tirosina fosfatasa 1B y dipeptidil peptidasa IV / Inhibitory effect of Allophylus cominia (L) Sw leaves aqueous extract on tyrosine phosphatase 1B and dipeptidyl peptidase IV proteins

Introducción: Allophylus cominia (L.) Sw es una planta medicinal cubana usada por la medicina tradicional para el tratamiento de la diabetes, cuyo mecanismo de acción es desconocido. Objetivo: evaluar el efecto del extracto acuoso de hojas de A. cominia (L.) Sw y sus fracciones sobre la proteína tirosina fosfatasa 1B (PTP1B) y dipeptidil peptidasa IV (DPPIV) como diana terapéuticas para el tratamiento de la diabetes tipo 2. Métodos: el extracto acuoso de hojas de A. cominia fue fraccionado sucesivamente con mezclas de solventes orgánicos, incrementando la polaridad, para obtener diez fracciones. El extracto y sus fracciones fueron evaluados para su posible actividad antidiabética sobre diana terapéuticas de diabetes tipo 2: PTP1B y DPPIV. Se realizaron ensayos de inhibición enzimática y la actividad inhibitoria se calculó a partir de los valores de fluorescencia, empleando longitudes de onda de excitación y de emisión de 360 nm y 460 nm respectivamente. Resultados: el extracto acuoso de A. cominia inhibió la actividad enzimática de PTP1B y DPPIV de manera dependiente de la concentración, con valores de CI50 de 0,69 µg/mL y 344,3 µg/mL respectivamente. Varias fracciones se detectaron como potentes inhibidores de PTP1B. Las fracciones más polares AcF9 y AcF10 fueron las más activas, y mostraron valores de CI50 de 4,4 µg/mL y 3,8 µg/mL respectivamente. Las fracciones mostraron una ligera inhibición de DPPIV, y las más activas resultaron AcF6, AcF9 y AcF10, con valores de porcentajes de inhibición de 52,0 por ciento, 39,0 por ciento y 40,0 por ciento respectivamente. Conclusiones: el extracto acuoso de A. cominia y sus fracciones polares (AcF9 y AcF10) tienen propiedades antidiabéticas in vitro y son candidatos promisorios para el desarrollo de nuevos medicamentos con actividad inhibidora de PTP1B y DPPIV para el tratamiento de la diabetes tipo 2(AU)

INTRODUCTION: Allophylus cominia (L.) Sw is a Cuban medicinal plant used by traditional medicine for the treatment of diabetes with unknown mechanisms of action. OBJECTIVE: to evaluate the effect of Allophylus cominia (L.) Sw leaves aqueous extract and its fractions on protein tyrosine phosphatase 1B (PTP1B) and dipeptidyl peptidase IV (DPPIV) enzymatic activity, as therapeutic targets of type 2 diabetes. METHODS: the aqueous extract of A. cominia leaves was successively partitioned with organic solvents mixtures, thus increasing polarity in order to obtain ten fractions. The extract and its fractions were tested for their possible antidiabetic activity on therapeutic targets of type 2 diabetes: PTP1B and DPPIV. The enzymatic inhibition assays were performed and the inhibitory activity was calculated with the fluorescence values using an excitation wavelength of 360 nm and an emission wavelength of 460 nm. RESULTS: the aqueous extract from A. cominia inhibited the enzymatic activity of PTP1B and DPPIV according to the concentration, being IC50 values equal to 0.69 µg/mL and 344.3 µg/mL, respectively. Several fractions were detected as potent PTP1B inhibitors. The most polar fractions AcF9 and AcF10 were more active, showing IC50values of 4.4 µg/mL and 3.8 µg/mL respectively. The fractions showed a slight DPPIV inhibition, being fractions AcF6, AcF9 and AcF10 the most active, exhibiting inhibition percentages of 52.0 percent, 39.0 percent and 40.0 percent respectively. CONCLUSIONS: A. cominia aqueous extract and its polar fractions (AcF9 and AcF10) have antidiabetic properties in vitro and are promissory candidates for development of new drugs with inhibitory activity of PTP1B and DPPIV for type 2 diabetes treatment(AU)

A novel dihydro-pyrazolo(3,4d)(1,2,4)triazolo(1,5a)pyrimidin-4-one (AJ23) is an antagonist at adenosine A(1) receptors and enhances consolidation of step-down avoidance.

Adenosine A(1) receptor antagonists are of potential value in the treatment of cognitive dysfunction. We have developed compound AJ23 (7-methyl-1-phenyl-1,8-dihydro-pyrazolo-(3,4d)(1,2,4)-triazolo(1,5a)-pyrimidin-4-one) as a novel, non-xanthine based antagonist at A(1) receptors. It has micromolar affinity at human A(1) receptors with a 45-fold selectivity for A(1) over A(2A) receptors and little affinity for many other receptors and transporters tested in a screening panel. AJ23 blocks A(1) receptors in the rat hippocampus, increasing the baseline size of excitatory post-synaptic potentials and blocking the inhibitory effects of adenosine. When administered directly into the rodent hippocampus this compound improves consolidation in a step-down avoidance learning task. The results suggest that AJ23 or derivatives may represent possible leads for further chemical development towards a chemically novel group of antagonists at A(1) receptors with potential value as cognitive enhancers.

Pharmacological actions of the South African medicinal and functional food plant Sceletium tortuosum and its principal alkaloids.

ETHNOPHARMACOLOGICAL RELEVANCE: The South African plant Sceletium tortuosum has been known for centuries for a variety of traditional uses, and, more recently, as a possible source of anti-anxiety or anti-depressant effects. A standardised extract Zembrin(®) was used to test for pharmacological activities that might be relevant to the ethnopharmacological uses, and three of the main alkaloids were also tested. MATERIALS AND METHODS: A standardised ethanolic extract was prepared from dried plant material, along with the purified alkaloids mesembrine, mesembrenone and mesembrenol. These were tested on a panel of receptors, enzymes and other drug targets, and for cytotoxic effects on mammalian cells. RESULTS: The extract was a potent blocker in 5-HT transporter binding assays (IC(50) 4.3 µg/ml) and had powerful inhibitory effects on phosphodiesterase 4 (PDE4) (IC(50) 8.5 µg/ml), but not other phosphodiesterases. There were no cytotoxic effects. Mesembrine was the most active alkaloid against the 5-HT transporter (K(i) 1.4 nM), while mesembrenone was active against the 5-HT transporter and PDE4 (IC(50)'s<1 µM). CONCLUSIONS: The activity of the Sceletium tortuosum extract on the 5-HT transporter and PDE4 may explain the clinical effects of preparations made from this plant. The activities relate to the presence of alkaloids, particularly mesembrine and mesembrenone.

Synthesis of a new series of 1H-imidazol-1-yl substituted 8-phenylxanthines as adenosine receptor ligands.

A new series of 1H-imidazol-1-yl substituted 8-phenylxanthine analogs has been synthesized to study the effects of the imidazole group on the binding affinity of compounds for adenosine receptors. Competition binding studies of these compounds were carried out in vitro with human cloned receptors using [(3) H]DPCPX and [(3) H]ZM 241385 as radioligands at A(1) and A(2A) adenosine receptors, respectively. The effect of the substitution pattern of the (imidazolyl)alkoxy group on various positions of the phenyl ring at C(8) was also studied. The xanthine derivatives displayed varying degrees of affinity and selectivity towards A(1) and A(2A) receptor subtypes despite a common but variedly substituted Ar-C(8).

Synthesis of quaternary ammonium salts of 16E-[4-(2-alkylaminoethoxy)-3-methoxybenzylidene]androstene derivatives as skeletal muscle relaxants.

Synthesis of eighteen new quaternary ammonium salts of 16E-arylidene androstene derivatives as skeletal muscle relaxants is reported in the present study. The effects of possibly extended interonium distances on muscle relaxant activity are discussed. All the quaternary ammonium steroids produced reduction in the twitch responses, when screened for in vitro neuromuscular blocking activity using isolated chick biventer cervicis muscle preparation. However, the variable interonium distance, which is believed to range from 11 to 17 Å in these quaternary compounds and is associated with the built in flexibility of these structures about the single bonds on the moieties linked to ring D of the steroid skeleton, resulted in varied degrees of muscle relaxant activity. Some of the compounds also inhibited acetylcholinesterase activity in low concentrations so that they would not be directly suitable for use as muscle relaxants.

Synthesis of 8-(cyclopentyloxy)phenyl substituted xanthine derivatives as adenosine A2A ligands.

The present paper describes the synthesis of a series of 8-(cyclopentyloxy)phenyl-xanthines and their evaluation for affinity for A1 and A2 adenosine receptors using radioligand binding assays. The effects of moving the cyclopentyloxy substituent with or without an ortho methoxy group on the various positions of the 8-phenyl ring have been studied. The vanilloid based xanthines 8-[4-(cyclopentyloxy)-3-methoxyphenyl]-1,3-dimethylxanthine (6a) (K(i)= 100 nM) and 8-[(4-cyclopentyloxy)-3-methoxyphenyl] -3-methyl-1-propylxanthine (12) (K(i) = 150 nM) displayed the highest affinity at A2A receptors as well as over 1000 fold selectivity over the A1 adenosine receptor subtype.

Synthesis of a series of 8-(substituted-phenyl)xanthines and a study on the effects of substitution pattern of phenyl substituents on affinity for adenosine A(1) and A(2A) receptors.

A new series of 8-(substituted-phenyl)xanthines have been synthesized and compounds were evaluated for their affinity for A(1) and A(2) adenosine receptors (AR) using radioligand binding assays. The effects of varying the positions of 8-phenyl substituents on affinity and selectivity at A(1) and A(2A) adenosine receptors have been studied. Isovanilloid 1,3-dimethyl-8-[4-methoxy-3-(2-morpholin-4-ylethoxy)phenylxanthine (9d) displayed the highest affinity and selectivity towards A(2A) AR subtypes with K(i)=100nM over A(1) receptors (Ki>100mM). It has been observed that substitution pattern on 8-phenyl group greatly affects the affinity and selectivity at adenosine receptors, with A(2A) tolerating bulkier substituents than did A(1) receptors.

M4 agonists/5HT7 antagonists with potential as antischizophrenic drugs: serominic compounds.

Chronic low-dose treatment of rats with the psychomimetic drug, phencyclidine, induces regionally specific metabolic and neurochemical changes in the CNS that mirror those observed in the brains of schizophrenic patients. Recent evidence suggests that drugs targeting serotoninergic and muscarinic receptors, and in particular 5-HT(7) antagonists and M(4) agonists, exert beneficial effects in this model of schizophrenia. Compounds that display this combined pattern of activity we refer to as serominic compounds. Based upon leads from natural product screening, we have designed and synthesised such serominic compounds, which are principally arylamidine derivatives of tetrahydroisoquinolines, and shown that they have the required serominic profile in ligand binding assays and show potential antipsychotic activity in functional assays.