Solvent-induced 7R-dioxygenase activity of soybean 15-lipoxygenase-1 in the formation of omega-3 DPA-derived resolvin analogs

The resolvin family contains important anti-inflammatory and pro-resolution compounds enzymatically derived in vivo from the polyunsaturated omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). More recently, docosapentaenoic acid (DPA) has emerged as another potentially important precursor in the biological production of resolvin compounds. In this work we have used medium engineering to develop a simple method for the controlled synthesis of two di-hydroxylated diastereomers of DPAn-3 catalyzed by soybean 15-lipoxygenase-1 (15-sLOX-1) in the presence of short chain n-alcohols, including methanol, ethanol and propan-1-ol. The complete structures of the two major products, 7S,17S-dihydroxydocosapenta-8Z,10E,13Z,15E,19Z-enoic acid (7S,17S-diHDPAn-3) and 7R,17S-dihydroxydocosapenta-8Z,10E,13Z,15E,19Z- enoic acid (7R,17S-diHDPAn-3), have been elucidated using spectroscopic analysis. The alcohol-dependent R-dioxygenase activity of soybean 15-lipoxygenase with mono-hydroperoxide intermediate substrates has also been demonstrated with other biologically relevant PUFAs, including DHA, EPA and ARA. The developed method has applications in the production of closely related isomers of naturally occurring resolvins and protectins, demonstrating the versatility of 15-sLOX-1 as a biocatalyst. © 2014 Elsevier B.V.

Design of new oxazaphosphorine anticancer drugs

The oxazaphosphorines including cyclophosphamide (CPA, Cytoxan, or Neosar), ifosfamide (IFO, Ifex) and trofosfamide (Ixoten) represent an important group of therapeutic agents due to their substantial antitumor and immunomodulating activity. However, several intrinsic limitations have been uncounted during the clinical use of these oxazaphosphorines, including substantial pharmacokinetic variability, resistance and severe host toxicity. To circumvent these problems, new oxazaphosphorines derivatives have been designed and evaluated with an attempt to improve the selectivity and response with reduced host toxicity. These include mafosfamide (NSC 345842), glufosfamide (D19575, β-Dglucosylisophosphoramide mustard), S-(-)-bromofosfamide (CBM-11), NSC 612567 (aldophosphamide perhydrothiazine) and NSC 613060 (aldophosphamide thiazolidine). Mafosfamide is an oxazaphosphorine analog that is a chemically stable 4-thioethane sulfonic acid salt of 4-hydroxy-CPA. Glufosfamide is IFO derivative in which the isophosphoramide mustard, the alkylating metabolite of IFO, is glycosidically linked to a β-D-glucose molecule. Phase II studies of glufosfamide in the treatment of pancreatic cancer, non-small cell lung cancer (NCSLC), and recurrent glioblastoma multiform (GBM) have recently completed and Phase III trials are ongoing, while Phase I studies of intrathecal mafosfamide have recently completed for the treatment of meningeal malignancy secondary to leukemia, lymphoma, or solid tumors. S-(-)- bromofosfamide is a bromine-substituted IFO analog being evaluated in a few Phase I clinical trials. The synthesis and development of novel oxazaphosphorine analogs with favourable pharmacokinetic and pharmacodynamic properties still constitutes a great challenge for medicinal chemists and cancer pharmacologists.

Chemiluminescence: liquid-phase

Article Outline
• Introduction
• Acidic Potassium Permanganate
• Acridinium Esters
• Diaryl Oxalates and Oxamides
• Dioxetanes
• Hypohalites
• Luminol and Its Analogs
• Tris(2,2′-bipyridyl)ruthenium(II)
• Practical Considerations
• HPLC and Flow Analysis
• Capillary Electrophoresis
• Enzyme Reaction Products
• Immunoassay and DNA Assays
• Further Reading

The presence of GABA in gastropod mucus and its role in inducing larval settlement

Chemical substances that induce larval settlement have been the focus of many gastropod studies due to the importance of wild stock recruitment and production within aquaculture facilities. Gamma-aminobutyric acid (GABA), GABA analogs, and GABA-mimetics associated with certain crustose coralline algae (CCA), are known to induce larval settlement in commercial abalone (Haliotis) species, and other gastropods. Furthermore, mucus secreted from these gastropods has been shown to induce larval settlement, but the stimulatory components of mucus have not been thoroughly investigated. We now present data confirming that GABA is the settlement-inducing effector molecule contained within abalone mucus. To do this, we initially generated anti-GABA for use in immunoenzyme and immunofluorescent microscopy. Using these techniques GABA was identified in the nerves and epithelial cells of the foot, including mucus. Dried mucus samples subject to HPLC analysis revealed a mean concentration of 0.68 mM GABA after sample rehydration. The presence of GABA in these samples was confirmed by time-of-flight mass spectroscopy (TOF-MS). In addition, GABA was detected in the mucus of several abalone species and other gastropods by immunocytochemistry. Subsequent bioassays using both dry and fresh mucus strongly promoted induction of larval settlement.

Substrates and inhibitors of human multidrug resistance associated proteins and the implications in drug development

Human contains 49 ATP-binding cassette (ABC) transporter genes and the multidrug resistance associated proteins (MRP1/ABCC1, MRP2/ABCC2, MRP3/ABCC3, MRP4/ABCC4, MRP5/ABCC5, MRP6/ABCC6, MRP7/ABCC10, MRP8/ABCC11 and MRP9/ABCC12) belong to the ABCC family which contains 13 members. ABCC7 is cystic fibrosis transmembrane conductance regulator; ABCC8 and ABCC9 are the sulfonylurea receptors which constitute the ATP-sensing subunits of a complex potassium channel. MRP10/ABCC13 is clearly a pseudo-gene which encodes a truncated protein that is highly expressed in fetal human liver with the highest similarity to MRP2/ABCC2 but without transporting activity. These transporters are localized to the apical and/or basolateral membrane of the hepatocytes, enterocytes, renal proximal tubule cells and endothelial cells of the blood-brain barrier. MRP/ABCC members transport a structurally diverse array of important endogenous substances and xenobiotics and their metabolites (in particular conjugates) with different substrate specificity and transport kinetics. The human MRP/ABCC transporters except MRP9/ABCC12 are all able to transport organic anions, such as drugs conjugated to glutathione, sulphate or glucuronate. In addition, selected MRP/ABCC members may transport a variety of endogenous compounds, such as leukotriene C(4) (LTC(4) by MRP1/ABCC1), bilirubin glucuronides (MRP2/ABCC2, and MRP3/ABCC3), prostaglandins E1 and E2 (MRP4/ABCC4), cGMP (MRP4/ABCC4, MRP5/ABCC5, and MRP8/ABCC11), and several glucuronosyl-, or sulfatidyl steroids. In vitro, the MRP/ABCC transporters can collectively confer resistance to natural product anticancer drugs and their conjugated metabolites, platinum compounds, folate antimetabolites, nucleoside and nucleotide analogs, arsenical and antimonial oxyanions, peptide-based agents, and in concert with alterations in phase II conjugating or biosynthetic enzymes, classical alkylating agents, alkylating agents. Several MRP/ABCC members (MRPs 1-3) are associated with tumor resistance which is often caused by an increased efflux and decreased intracellular accumulation of natural product anticancer drugs and other anticancer agents. Drug targeting of these transporters to overcome MRP/ABCC-mediated multidrug resistance may play a role in cancer chemotherapy. Most MRP/ABCC transporters are subject to inhibition by a variety of compounds. Based on currently available preclinical and limited clinical data, it can be expected that modulation of MRP members may represent a useful approach in the management of anticancer and antimicrobial drug resistance and possibly of inflammatory diseases and other diseases. A better understanding of their substrates and inhibitors has important implications in development of drugs for treatment of cancer and inflammation.

Differentially expressed genes associated with obesity and type 2 diabetes

The present invention relates to nucleic acid molecules which encode expression products associated with the modulation of obesity, anorexia, weight maintenance, diabetes and/or metabolic energy levels. The nucleic acid molecules and expression products of the present invention are produced by recombinant means or isolated from natural resources. The subject nucleic acid molecules and expression products and their derivatives, homologs, analogs and mimetics are proposed to be useful as therapeutic and diagnostic agents for obesity, anorexia, weight maintenance, diabetes and/or energy imbalance or as targets for the design and/or identification of modulators of their activity and/or function. The subject nucleic acid molecules and expression products are identified using differential display techniques.

Cholesterol is necessary both for the toxic effect of Abeta peptides on vascular smooth muscle cells and for Abeta binding to vascular smooth muscle cell membranes

Accumulation of beta amyloid (Aβ) in the brain is central to the pathogenesis of Alzheimer's disease. Aβ can bind to membrane lipids and this binding may have detrimental effects on cell function. In this study, surface plasmon resonance technology was used to study Aβ binding to membranes. Aβ peptides bound to synthetic lipid mixtures and to an intact plasma membrane preparation isolated from vascular smooth muscle cells. Aβ peptides were also toxic to vascular smooth muscle cells. There was a good correlation between the toxic effect of Aβ peptides and their membrane binding. 'Ageing' the Aβ peptides by incubation for 5 days increased the proportion of oligomeric species, and also increased toxicity and the amount of binding to lipids. The toxicities of various Aβ analogs correlated with their lipid binding. Significantly, binding was influenced by the concentration of cholesterol in the lipid mixture. Reduction of cholesterol in vascular smooth muscle cells not only reduced the binding of Aβ to purified plasma membrane preparations but also reduced Aβ toxicity. The results support the view that Aβ toxicity is a direct consequence of binding to lipids in the membrane. Reduction of membrane cholesterol using cholesterol-lowering drugs may be of therapeutic benefit because it reduces Aβ-membrane binding.

Agonist allosteric enhancers at A1 adenosine receptors

Pharmaceutical agents that may be protective to the heart during ischemia were targeted in this project. Analogs to a lead compound containing potential cardioprotective properties were prepared. In the pharmacological evaluation five compounds were found to be more potent than the lead compound.

A gel-capture assay for characterizing the sialyl-glycan selectivity of influenza viruses

Sialic acids (SA) usually linked to galactose (Gal) in an α2,6- or α2,3-configuration are considered the main cell receptors for influenza viruses, in particular for their hemagglutinins (HA). The typing of influenza virus HA receptor selectivity is relevant for understanding the transmissibility of avian and swine viruses to the human population. In this study we developed a simple and inexpensive gel-capture assay (GCA) of the influenza virus HA receptor-binding selectivity. Its principle is the binding of soluble influenza virus to pentasaccharide analogs, representatives of receptors of human and avian influenza viruses, immobilized on a gel resin. The human and avian analogs consisted of a sialyllactose-N-tetraose c (LSTc) [Neu5Ac(α2,6)Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc] and a sialyllactose-N-tetraose a (LSTa) [Neu5Ac(α2,3)Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc], respectively. Following equilibration, the unbound virus is washed away and the bound one is assayed via HA by densitometry as a function of the analog concentration. Using GCA, the receptor selectivity of three influenza viruses of different HA subtype was investigated. The results showed that the egg-adapted A/California/07/2009 (H1N1) virus exhibited an avian α2,3-linked LSTa selectivity, however, it retained the ability to bind to the α2,6-linked LSTc human receptor analog. Influenza B virus B/Florida/4/2006 showed α2,6-linked LSTc selectivity and a poor α2,3-linked LSTa avidity. The H3N2 virus A/Wisconsin/15/2009 displayed almost comparable avidity for both receptor analogs with a marginally greater α2,3-linked LSTa avidity. The described assay protocol provides a simple and rapid method for the characterization of influenza virus HA receptor binding selectivity. Keywords: influenza virus; hemagglutinin; receptor; sialyllactose-N-tetraose; gel-capture assay.

Identification and characterization of Kava-derived compounds mediating TNF-alpha suppression

There is a substantial unmet need for new classes of drugs that block TNF-α-mediated inflammation, and particularly for small molecule agents that can be taken orally. We have screened a library of natural products against an assay measuring TNF-α secretion in lipopolysaccharide-stimulated THP-1 cells, seeking compounds capable of interfering with the TNF-α-inducing transcription factor lipopolysaccharide-induced TNF-α factor. Among the active compounds were several produced by the kava plant (Piper mysticum), extracts of which have previously been linked to a range of therapeutic effects. When tested in vivo, a representative of these compounds, kavain, was found to render mice immune to lethal doses of lipopolysaccharide. Kavain displays promising pharmaceutical properties, including good solubility and high cell permeability, but pharmacokinetic experiments in mice showed relatively rapid clearance. A small set of kavain analogs was synthesized, resulting in compounds of similar or greater potency in vitro compared with kavain. Interestingly, a ring-opened analog of kavain inhibited TNF-α secretion in the cell-based assay and suppressed lipopolysaccharide-induced TNF-α factor expression in the same cells, whereas the other compounds inhibited TNF-α secretion without affecting lipopolysaccharide-induced TNF-α factor levels, indicating a potential divergence in mechanism of action.

Management of advanced neuroendocrine tumors with hepatic metastasis

Neuroendocrine tumors (NETs) in general and specifically these gastroenteropancreatic-neuroendocrine tumors often present a considerable diagnostic and therapeutic challenge, especially those that behave in an aggressive fashion. The majority of tumors are diagnosed at a stage that the only curative treatment, radical surgical intervention, is no longer an option and thus long-term therapy with somatostatin analogs is focused on symptom amelioration and in the improvement of quality of life. Although biotherapy is currently the most efficient treatment to achieve palliation, conventional chemotherapy may have some utility in undifferentiated or highly proliferating neuroendocrine carcinomas and pancreatic NETs. Hepatic metastases, depending on size, location, and number may be amenable to surgical resection or radiofrequency ablation. If surgery is not feasible, embolization either alone (bland), in combination with chemotherapeutic agents, or using radioactive microspheres can be used. Peptide receptor targeted radiotherapy using radiolabeled octapeptide analogs (⁹⁰Yttrium or ¹⁷⁷Lutetium-octreotide) may lead to reduction in tumor size, but in most circumstances has a tumor stabilizing effect. A variety of antiangiogenesis and growth factor-targeted agents have been evaluated, but to date, the results have failed to meet our expectations.

An in vitro comparative assessment with a series of new triphenyltin(IV) 2-/4-[(E)-2-(aryl)-1-diazenyl]benzoates endowed with anticancer activities: Structural modifications, analysis of efficacy and cytotoxicity involving human tumor cell lines

Four new triphenyltin(IV) complexes of composition Ph₃SnLH (where LH = 2-/4-[(E)-2-(aryl)-1-diazenyl]benzoate) (1–4) were synthesized and characterized by spectroscopic (¹H, ¹³C and ¹¹⁹Sn NMR, IR, ¹¹⁹Sn Mössbauer) techniques in combination with elemental analysis. The ¹¹⁹Sn NMR spectroscopic data indicate a tetrahedral coordination geometry in non-coordinating solvents. The crystal structures of three complexes, Ph₃SnL¹H (1), Ph₃SnL³H (3), Ph₃SnL⁴H (4), were determined. All display an essentially tetrahedral geometry with angles ranging from 93.50(8) to 124.5(2)°; ¹¹⁹Sn Mössbauer spectral data support this assignment. The cytotoxicity studies were performed with complexes 1–4, along with a previously reported complex (5) in vitro across a panel of human tumor cell lines viz., A498, EVSA-T, H226, IGROV, M19 MEL, MCF-7 and WIDR. The screening results were compared with the results from other related triphenyltin(IV) complexes (6–7) and tributyltin(IV) complexes (8–11) having 2-/4-[(E)-2-(aryl)-1-diazenyl]benzoates framework. In general, the complexes exhibit stronger cytotoxic activity. The results obtained for 1–3 are also comparable to those of its o-analogs i.e. 4–7, except 5, but the advantage is the former set of complexes demonstrated two folds more cytotoxic activity for the cell line MCF-7 with ID₅₀ values in the range 41–53 ng/ml. Undoubtedly, the cytotoxic results of complexes 1–3 are far superior to CDDP, 5-FU and ETO, and related tributyltin(IV) complexes 8–11. The quantitative structure-activity relationship (QSAR) studies for the cytotoxicity of triphenyltin(IV) complexes 1–7 and tributyltin(IV) complexes 8–11 is also discussed against a panel of human tumor cell lines.

Preliminary investigations into triazole derived androgen receptor antagonists

A range of 1,4-substituted-1,2,3-N-phenyltriazoles were synthesized and evaluated as non-steroidal androgen receptor (AR) antagonists. The motivation for this study was to replace the N-phenyl amide portion of small molecule antiandrogens with a 1,2,3-triazole and determine effects, if any, on biological activity. The synthetic methodology presented herein is robust, high yielding and extremely rapid. Using this methodology a series of 17 N-aryl triazoles were synthesized from commercially available starting materials in less than 3h. After preliminary biological screening at 20 and 40 μM, the most promising three compounds were found to display IC50 values of 40-50 μM against androgen dependent (LNCaP) cells and serve as a starting point for further structure-activity investigations. All compounds in this work were the focus of an in silico study to dock the compounds into the human androgen receptor ligand binding domain (hARLBD) and compare their predicted binding affinity with known antiandrogens. A comparison of receptor-ligand interactions for the wild type and T877A mutant AR revealed two novel polar interactions. One with Q738 of the wild type site and the second with the mutated A877 residue.

Targeting VEGF with LNA-stabilized G-rich oligonucleotide for efficient breast cancer inhibition

In this study, we investigated the efficacy of an LNA (locked nucleic acid)-modified DNA aptamer named RNV66 targeting VEGF against various breast cancer cell lines. Our results demonstrate that RNV66 efficiently inhibits breast cancer cell proliferation both in vitro and in vivo. Introduction of LNA nucleotides were crucial for higher efficacy. Furthermore, the binding interaction of RNV66 with VEGF was investigated using molecular dynamic simulations leading to the first computational model of the LNA aptamer-VEGF complex blocking its interaction with VEGF-receptor.