Synthesis and inhibitory activity against COX-2 catalyzed prostaglandin production of chrysin derivatives

A series of chrysin derivatives were prepared and evaluated for their inhibitory activities of cyclooxygenase-2 catalyzed prostaglandin production. Chrysin derivatives were prepared from 2-hydroxyacetophenone, 2,4-dihydroxyacetophenone and 2,6-dihydroxyacetophenone in 2 to 4 steps, respectively. Methxoylated chrysin derivatives were converted to the corresponding hydroxylated chrysin derivatives by the reaction with BBr(3) in good yields. The inhibitory activity of the chrysin derivatives against prostaglandin production from lipopolysaccharide-treated RAW 264.7 cells was measured. We found that chrysin derivatives with 3',4'-dichloro substituents (5e, 6e and 7e) exhibited good inhibitory activity of prostaglandin production.     

Synthesis and structural-activity relationships of 3-hydroxyquinazoline-2,4-dione antibacterial agents

A series of 3-hydroxyquinazoline-2,4-diones was synthesized and evaluated for antibacterial activity. This series represents a novel addition to the DNA gyrase inhibitor class of antibacterials. Appropriate substitutions onto the core template yielded compounds with excellent potency against E. coli gyrase and significant in vitro Gram-negative and Gram-positive antibacterial activity.     

Piperazinebenzylamines as potent and selective antagonists of the human melanocortin-4 receptor

SAR studies of a series of piperazinebenzylamines resulted in the discovery of potent antagonists of the human melanocortin-4 receptor. Compounds 11c, 11d, and 11l, which had K(i) values of 21, 14, and 15 nM, respectively, possessed low efficacy in cAMP stimulation ( approximately 15% of alpha-MSH maximal level) mediated by MC4R, and functioned as antagonists in inhibition of alpha-MSH-stimulated cAMP release in a dose-dependent manner (11l, IC(50)=36 nM).     

Two PAR6 proteins become asymmetrically localized during establishment of polarity in mouse oocytes

Meiotic maturation in mammals is characterized by two asymmetric divisions, leading to the formation of two polar bodies and the female gamete. Whereas the mouse oocyte is a polarized cell, molecules implicated in the establishment of this polarity are still unknown. PAR proteins have been demonstrated to play an important role in cell polarity in many cell types, where they control spindle positioning and asymmetric distribution of determinants. Here we show that two PAR6-related proteins have distinct polarized distributions in mouse oocytes. mPARD6a is first localized on the spindle and then accumulates at the pole nearest the cortex during spindle migration. In the absence of microtubules, the chromosomes still migrate to the cortex, and mPARD6a was found associated with the chromosomes and was facing the cortex. mPARD6a is the first identified protein to associate with the spindle during spindle migration and to relocalize to the chromosomes in the absence of microtubule behavior, suggesting a role in spindle migration. The other protein, mPARD6b, was found on spindle microtubules until entry into meiosis II and relocalized to the cortex at the animal pole during metaphase II arrest. mPARD6b is the first identified protein to localize to the animal pole of the mouse oocyte and likely contributes to the polarization of the cortex.     

Essential roles of receptor-interacting protein and TRAF2 in oxidative stress-induced cell death

Oxidative stress and reactive oxygen species (ROS) can elicit and modulate various physiological and pathological processes, including cell death. However, the mechanisms controlling ROS-induced cell death are largely unknown. Data from this study suggest that receptor-interacting protein (RIP) and tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2), two key effector molecules of TNF signaling, are essential for ROS-induced cell death. We found that RIP(-/-) or TRAF2(-/-) mouse embryonic fibroblasts (MEF) are resistant to ROS-induced cell death when compared to wild-type cells, and reconstitution of RIP and TRAF2 gene expression in their respective deficient MEF cells restored their sensitivity to H(2)O(2)-induced cell death. We also found that RIP and TRAF2 form a complex upon H(2)O(2) exposure, but without the participation of TNFR1. The colocalization of RIP with a membrane lipid raft marker revealed a possible role of lipid rafts in the transduction of cell death signal initiated by H(2)O(2). Finally, our results demonstrate that activation of c-Jun NH(2)-terminal kinase 1 is a critical event downstream of RIP and TRAF2 in mediating ROS-induced cell death. Therefore, our study uncovers a novel signaling pathway regulating oxidative stress-induced cell death.     

Suppression of the induction of alpha, beta, and lambda interferons by the NS1 and NS2 proteins of human respiratory syncytial virus in human epithelial cells and macrophages [corrected

Wild-type human respiratory syncytial virus (HRSV) is a poor inducer of alpha/beta interferons (IFN-alpha/beta). However, recombinant HRSV lacking the NS1 and NS2 genes (Delta NS1/2) induced high levels of IFN-alpha and -beta in human pulmonary epithelial cells (A549) as well as in macrophages derived from primary human peripheral blood monocytes. Results with NS1 and NS2 single- and double-gene-deletion viruses indicated that the two proteins function independently as well as coordinately to achieve the full inhibitory effect, with NS1 having a greater independent role. The relative contributions of the individual NS proteins were the converse of that recently described for bovine RSV (J. F. Valarcher, J. Furze, S. Wyld, R. Cook, K. K. Conzelmann, and G. Taylor, J. Virol. 77:8426-8439, 2003). This pattern of inhibition by HRSV NS1 and NS2 also extended to the newly described antiviral cytokines IFN-lambda 1, -2 and -3.     

A novel 3-deoxy-D-manno-octulosonic acid (Kdo) hydrolase that removes the outer Kdo sugar of Helicobacter pylori lipopolysaccharide

The lipid A domain anchors lipopolysaccharide (LPS) to the outer membrane and is typically a disaccharide of glucosamine that is both acylated and phosphorylated. The core and O-antigen carbohydrate domains are linked to the lipid A moiety through the eight-carbon sugar 3-deoxy-D-manno-octulosonic acid known as Kdo. Helicobacter pylori LPS has been characterized as having a single Kdo residue attached to lipid A, predicting in vivo a monofunctional Kdo transferase (WaaA). However, using an in vitro assay system we demonstrate that H. pylori WaaA is a bifunctional enzyme transferring two Kdo sugars to the tetra-acylated lipid A precursor lipid IV(A). In the present work we report the discovery of a Kdo hydrolase in membranes of H. pylori capable of removing the outer Kdo sugar from Kdo2-lipid A. Enzymatic removal of the Kdo group was dependent upon prior removal of the 1-phosphate group from the lipid A domain, and mass spectrometric analysis of the reaction product confirmed the enzymatic removal of a single Kdo residue by the Kdo-trimming enzyme. This is the first characterization of a Kdo hydrolase involved in the modification of gram-negative bacterial LPS.     

Lipid sulfates and sulfonates are allosteric competitive inhibitors of the N-terminal phosphatase activity of the mammalian soluble epoxide hydrolase

The EPXH2 gene encodes for the soluble epoxide hydrolase (sEH), a homodimeric enzyme with each monomer containing two domains with distinct activities. The C-terminal domain, containing the epoxide hydrolase activity (Cterm-EH), is involved in the metabolism of arachidonic acid epoxides, endogenous chemical mediators that play important roles in blood pressure regulation, cell growth, and inflammation. We recently demonstrated that the N-terminal domain contains a Mg2+-dependent lipid phosphate phosphatase activity (Nterm-phos). However, the biological role of this activity is unknown. The inability of known phosphatase inhibitors to inhibit the Nterm-phos constitutes a significant barrier to the elucidation of its function. We describe herein sulfate, sulfonate, and phosphonate lipids as novel potent inhibitors of Nterm-phos. These compounds are allosteric competitive inhibitors with K(I) in the hundred nanomolar range. These inhibitors may provide a valuable tool to investigate the biological role of the Nterm-phos. We found that polyisoprenyl phosphates are substrates of Nterm-phos, suggesting a possible role in sterol synthesis or inflammation. Furthermore, some of these compounds inhibit the C-terminal sEH activity through a noncompetitive inhibition mechanism involving a new binding site on the C-terminal domain. This novel site may play a role in the natural in vivo regulation of epoxide hydrolysis by sEH.     

Phosphorylation of Grb10 by mitogen-activated protein kinase: identification of Ser150 and Ser476 of human Grb10zeta as major phosphorylation sites

Grb10 is a Src-homology 2 (SH2) and Pleckstrin-homology (PH) domain-containing protein that binds to several autophosphorylated receptor tyrosine kinases including the insulin receptor (IR). Our previous studies showed that Grb10 underwent insulin-stimulated serine phosphorylation, yet the kinase(s) responsible for phosphorylation and the sites of the phosphorylation remain unknown. In this report, we show that Grb10 is a direct substrate of the p42/44 mitogen-activated protein kinase (MAPK). In addition, we found that inhibition of the MAPK signaling pathway reduced Grb10 phosphorylation in cells. Using site-directed mutagenesis, phosphopeptide mapping, and capillary HPLC-electrospray-tandem mass spectrometry analysis, we identified Ser(150), Ser(418), and Ser(476) of human Grb10zeta as MAPK-mediated in vitro phosphorylation sites. In vivo labeling and two-dimensional phosphopeptide mapping studies revealed that Ser(150) and Ser(476) of human Grb10zeta are phosphorylated in intact cells. Replacing Ser(150) and Ser(476) with alanines reduced the inhibitory effect of human Grb10zeta on insulin-stimulated IRS1 tyrosine phosphorylation. Taken together, our findings suggest that phosphorylation of the adaptor protein may provide a feedback inhibitory mechanism by which Grb10 regulates insulin signaling.     

The elementary mass action rate constants of P-gp transport for a confluent monolayer of MDCKII-hMDR1 cells

The human multi-drug resistance membrane transporter, P-glycoprotein, or P-gp, has been extensively studied due to its importance to human health and disease. Thus far, the kinetic analysis of P-gp transport has been limited to steady-state Michaelis-Menten approaches or to compartmental models, neither of which can prove molecular mechanisms. Determination of the elementary kinetic rate constants of transport will be essential to understanding how P-gp works. The experimental system we use is a confluent monolayer of MDCKII-hMDR1 cells that overexpress P-gp. It is a physiologically relevant model system, and transport is measured without biochemical manipulations of P-gp. The Michaelis-Menten mass action reaction is used to model P-gp transport. Without imposing the steady-state assumptions, this reaction depends upon several parameters that must be simultaneously fitted. An exhaustive fitting of transport data to find all possible parameter vectors that best fit the data was accomplished with a reasonable computation time using a hierarchical algorithm. For three P-gp substrates (amprenavir, loperamide, and quinidine), we have successfully fitted the elementary rate constants, i.e., drug association to P-gp from the apical membrane inner monolayer, drug dissociation back into the apical membrane inner monolayer, and drug efflux from P-gp into the apical chamber, as well as the density of efflux active P-gp. All three drugs had overlapping ranges for the efflux active P-gp, which was a benchmark for the validity of the fitting process. One novel finding was that the association to P-gp appears to be rate-limited solely by drug lateral diffusion within the inner monolayer of the plasma membrane for all three drugs. This would be expected if P-gp structure were open to the lipids of the apical membrane inner monolayer, as has been suggested by recent structural studies. The fitted kinetic parameters show how P-gp efflux of a wide range of xenobiotics has been maximized.