The Best Brown Dwarf Yet?: A Companion to the Hyades Eclipsing Binary V471 Tau

We have carried out an analysis of about 160 eclipse timings spanning over 30 years of the Hyades eclipsing binary V471 Tauri that shows a long-term quasi-sinusoidal modulation of its observed eclipse arrival times. The O-Cs have been analyzed for the ``light-time'' effect that arises from the gravitational influence of a tertiary companion. The presence of a third body causes the relative distance of the eclipsing pair to the Earth to change as it orbits the barycenter of the triple system. The result of the analysis of the eclipse times yields a light-time semi-amplitude of 137.2+/-12.0 s, an orbital period of P_3 = 30.5+/-1.6 yr and an eccentricity of e_3 = 0.31+/-0.04. The mass of the tertiary component is M_3 sin i_3 = 0.0393+/-0.0038 Mo when a total mass of 1.61+/-0.06 Mo for V471 Tau is adopted. For orbital inclinations i_3 > 35 deg, the mass of the third body would be below the stable hydrogen burning limit of M = 0.07 Mo and it thus would be a brown dwarf. In the next several years (near maximum elongation), it should be feasible to obtain IR images and spectra of V471 Tau C that, when combined with the known mass, age, distance, and [Fe/H], will serve as a benchmark for understanding the physical properties and evolution of brown dwarfs.Comment: 9 pages, 3 figures, accepted for publication in ApJ Letter

Synthesis and preliminary mechanistic evaluation of 5-(p-tolyl)-1-(quinolin-2-yl)pyrazole-3-carboxylic acid amides with potent antiproliferative activity on human cancer cell lines

We synthesized a series of novel amide derivatives of 5-(p-tolyl)-1-(quinolin-2-yl)pyrazole-3-carboxylic acid and assessed their antiproliferative activities against three human cancer cell lines (Huh7, human liver; MCF7, breast and HCT116, colon carcinoma cell lines) with the sulforhodamine B assay. Compound 4j with 2-chloro-4-pyridinyl group in the amide part exhibited promising cytotoxic activity against all cell lines with IC50values of 1.6 μM, 3.3 μM and 1.1 μM for Huh7, MCF7 and HCT116 cells, respectively, and produced dramatic cell cycle arrest at SubG1/G1 phase as an indicator of apoptotic cell death induction. On the basis of their high potency in cellular environment, these straightforward pyrazole-3-carboxamide derivatives may possess potential in the design of more potent compounds for intervention with cancer cell proliferation. © 2014 Elsevier Masson SAS

The novel benzimidazole derivative BRP-7 inhibits leukotriene biosynthesis in vitro and in vivo by targeting 5-lipoxygenase-activating protein (FLAP).

BACKGROUND AND PURPOSE: Leukotrienes (LTs) are inflammatory mediators produced via the 5-lipoxygenase (5-LOX) pathway and are linked to diverse disorders, including asthma, allergic rhinitis and cardiovascular diseases. We recently identified the benzimidazole derivative BRP-7 as chemotype for anti-LT agents by virtual screening targeting 5-LOX-activating protein (FLAP). Here, we aimed to reveal the in vitro and in vivo pharmacology of BRP-7 as an inhibitor of LT biosynthesis. EXPERIMENTAL APPROACH: We analysed LT formation and performed mechanistic studies in human neutrophils and monocytes, in human whole blood (HWB) and in cell-free assays. The effectiveness of BRP-7 in vivo was evaluated in rat carrageenan-induced pleurisy and mouse zymosan-induced peritonitis. KEY RESULTS: BRP-7 potently suppressed LT formation in neutrophils and monocytes and this was accompanied by impaired 5-LOX co-localization with FLAP. Neither the cellular viability nor the activity of 5-LOX in cell-free assays was affected by BRP-7, indicating that a functional FLAP is needed for BRP-7 to inhibit LTs, and FLAP bound to BRP-7 linked to a solid matrix. Compared with the FLAP inhibitor MK-886, BRP-7 did not significantly inhibit COX-1 or microsomal prostaglandin E2 synthase-1, implying the selectivity of BRP-7 for FLAP. Finally, BRP-7 was effective in HWB and impaired inflammation in vivo, in rat pleurisy and mouse peritonitis, along with reducing LT levels. CONCLUSIONS AND IMPLICATIONS: BRP-7 potently suppresses LT biosynthesis by interacting with FLAP and exhibits anti-inflammatory effectiveness in vivo, with promising potential for further development

BRP-187: A potent inhibitor of leukotriene biosynthesis that acts through impeding the dynamic 5-lipoxygenase/5-lipoxygenase-activating protein (FLAP) complex assembly

The pro-inflammatory leukotrienes (LTs) are formed from arachidonic acid (AA) in activated leukocytes, where 5-lipoxygenase (5-LO) translocates to the nuclear envelope to assemble a functional complex with the integral nuclear membrane protein 5-LO-activating protein (FLAP). FLAP, a MAPEG family member, facilitates AA transfer to 5-LO for efficient conversion, and LT biosynthesis critically depends on FLAP. Here we show that the novel LT biosynthesis inhibitor BRP-187 prevents the 5-LO/FLAP interaction at the nuclear envelope of human leukocytes without blocking 5-LO nuclear redistribution. BRP-187 inhibited 5-LO product formation in human monocytes and polymorphonuclear leukocytes stimulated by lipopolysaccharide plus N-formyl-methionyl-leucyl-phenylalanine (IC50=7-10nM), and upon activation by ionophore A23187 (IC50=10-60nM). Excess of exogenous AA markedly impaired the potency of BRP-187. Direct 5-LO inhibition in cell-free assays was evident only at >35-fold higher concentrations, which was reversible and not improved under reducing conditions. BRP-187 prevented A23187-induced 5-LO/FLAP complex assembly in leukocytes but failed to block 5-LO nuclear translocation, features that were shared with the FLAP inhibitor MK886. Whereas AA release, cyclooxygenases and related LOs were unaffected, BRP-187 also potently inhibited microsomal prostaglandin E2 synthase-1 (IC50=0.2μM), another MAPEG member. In vivo, BRP-187 (10mg/kg) exhibited significant effectiveness in zymosan-induced murine peritonitis, suppressing LT levels in peritoneal exudates as well as vascular permeability and neutrophil infiltration. Together, BRP-187 potently inhibits LT biosynthesis in vitro and in vivo, which seemingly is caused by preventing the 5-LO/FLAP complex assembly and warrants further preclinical evaluation

Identification of sulfation sites of metabolites and prediction of the compounds’ biological effects

Characterizing the biological effects of metabolic transformations (or biotransformation) is one of the key steps in developing safe and effective pharmaceuticals. Sulfate conjugation, one of the major phase II biotransformations, is the focus of this study. While this biotransformation typically facilitates excretion of metabolites by making the compounds more water soluble, sulfation may also lead to bioactivation, producing carcinogenic products. The end result, excretion or bioactivation, depends on the structural features of the sulfation sites, so obtaining the structure of the sulfated metabolites is critically important. We describe herein a very simple, high-throughput procedure for using mass spectrometry to identify the structure—and thus the biological fate—of sulfated metabolites. We have chemically synthesized and analyzed libraries of compounds representing all the biologically relevant types of sulfation products, and using the mass spectral data, the structural features present in these analytes can be reliably determined, with a 97% success rate. This work represents the first example of a high-throughput analysis that can identify the structure of sulfated metabolites and predict their biological effects

Consumption of pasteurized human lysozyme transgenic goats’ milk alters serum metabolite profile in young pigs

Nutrition, bacterial composition of the gastrointestinal tract, and general health status can all influence the metabolic profile of an organism. We previously demonstrated that feeding pasteurized transgenic goats’ milk expressing human lysozyme (hLZ) can positively impact intestinal morphology and modulate intestinal microbiota composition in young pigs. The objective of this study was to further examine the effect of consuming hLZ-containing milk on young pigs by profiling serum metabolites. Pigs were placed into two groups and fed a diet of solid food and either control (non-transgenic) goats’ milk or milk from hLZ-transgenic goats for 6 weeks. Serum samples were collected at the end of the feeding period and global metabolite profiling was performed. For a total of 225 metabolites (160 known, 65 unknown) semi-quantitative data was obtained. Levels of 18 known and 4 unknown metabolites differed significantly between the two groups with the direction of change in 13 of the 18 known metabolites being almost entirely congruent with improved health status, particularly in terms of the gastrointestinal tract health and immune response, with the effects of the other five being neutral or unknown. These results further support our hypothesis that consumption of hLZ-containing milk is beneficial to health

Active site mutations and substrate inhibition in human sulfotransferase 1A1 and 1A3

Human SULT1A1 is primarily responsible for sulfonation of xenobiotics, including the activation of promutagens, and it has been implicated in several forms of cancer. Human SULT1A3 has been shown to be the major sulfotransferase that sulfonates dopamine. These two enzymes shares 93% amino acid sequence identity and have distinct but overlapping substrate preferences. The resolution of the crystal structures of these two enzymes has enabled us to elucidate the mechanisms controlling their substrate preferences and inhibition. The presence of two p-nitrophenol (pNP) molecules in the crystal structure of SULT1A1 was postulated to explain cooperativity at low and inhibition at high substrate concentrations, respectively. In SULT1A1, substrate inhibition occurs with pNP as the substrate but not with dopamine. For SULT1A3, substrate inhibition is found for dopamine but not with pNP. We investigated how substrate inhibition occurs in these two enzymes using molecular modeling, site-directed mutagenesis, and kinetic analysis. The results show that residue Phe-247 of SULT1A1, which interacts with both p-nitrophenol molecules in the active site, is important for substrate inhibition. Mutation of phenylalanine to leucine at this position in SULT1A1 results in substrate inhibition by dopamine. We also propose, based on modeling and kinetic studies, that substrate inhibition by dopamine in SULT1A3 is caused by binding of two dopamine molecules in the active site. © 2004 by The American Society for Biochemistry and Molecular Biology, Inc

Sulfation of indoxyl by human and rat aryl (phenol) sulfotransferases to form indoxyl sulfate

The aim of this study was to identify sulfotransferase (SULT) isoform(s) responsible for the formation of indoxyl sulfate from indoxyl (3-hydroxyindole). Indoxyl was incubated together with the co-substrate 3′-phosphoadenosine 5′-phosphosulfate (PAPS) and either human or rat liver cytosol or recombinant sulfotransferase enzymes. Formation of indoxyl sulfate from indoxyl was measured by HPLC and used for determination of sulfonation rates. Both cytosols sulfonated indoxyl with apparent Km values of 6.8 ± 0.9 μM for human and 3.2 ± 0.6 μM for rat cytosol. To help identify the isoform(s) of SULT responsible for indoxyl sulfate formation, indoxyl was incubated with human and rat liver cytosols and PAPS in the presence of isoform-specific SULT inhibitors. No inhibition was observed by DHEA, a specific hydroxysteroid sulfotransferase inhibitor, nor by oestrone, an inhibitor of oestrogen sulfotransferase. However, an aryl (phenol) sulfotransferase inhibitor, 2,6-dichloro-4-nitrophenol (DCNP), inhibited the formation of indoxyl sulfate with a IC50 values of 3.2 μM for human and 1.0 μM for rat cytosol indicating that human and rat aryl (phenol) sulfotransferases are responsible for the formation of indoxyl sulfate. When indoxyl was incubated with SULT1A1*2, a human recombinant aryl SULT, an apparent Km value of 5.6 ± 1.8 μM was obtained. Kinetic studies with human and rat cytosols and human recombinant SULT1A1*2 gave similar kinetic values indicating that human and rat aryl sulfotransferases efficiently catalyze the formation of indoxyl sulfate, an important uremic toxin metabolite

Optimisation by design of experiment of benzimidazol-2-one synthesis under flow conditions

A novel flow-based approach for the preparation of benzimidazol-2-one (1) scaffold by the 1,1'-carbonyldiimidazole (CDI)-promoted cyclocarbonylation of o-phenylenediamine (2) is reported. Starting from a preliminary batch screening, the model reaction was successfully translated under flow conditions and optimised by means of design of experiment (DoE). The method allowed the efficient preparation of this privileged scaffold and to set up a general protocol for the multigram-scale preparation in high yield, purity, and productivity, and was successfully applied for the multigram flow synthesis of N-(2-chlorobenzyl)-5-cyano-benzimidazol-2-one, which is a key synthon for hit-to-lead explorations in our anti-inflammatory drug discovery program