Substituted pyrazoles as novel sEH antagonist: investigation of key binding interactions within the catalytic domain.

A novel series of pyrazole sEH inhibitors is reported. Lead optimization efforts to replace the aniline core are also described. In particular, 2-pyridine, 3-pyridine and pyridazine analogs are potent sEH inhibitors with favorable CYP3A4 inhibitory and microsomal stability profiles.

Design and synthesis of substituted nicotinamides as inhibitors of soluble epoxide hydrolase.

A series of potent nicotinamide inhibitors of soluble epoxides hydrolase (sEH) is disclosed. This series was designed using structure-based deconstruction and a combination of two HTS hit series, resulting in hybrid analogs that retained the optimal potency from one series, and acceptable in vitro metabolic stability from the other. Structure-guided optimization of these analogs gave rise to nanomolar inhibitors of human sEH that had acceptable plasma exposure to qualify them as probes to determine the in vivo phenotypic consequences of sEH inhibition.

Compensatory mechanism for homeostatic blood pressure regulation in Ephx2 gene-disrupted mice.

Arachidonic acid-derived epoxides, epoxyeicosatrienoic acids, are important regulators of vascular homeostasis and inflammation, and therefore manipulation of their levels is a potentially useful pharmacological strategy. Soluble epoxide hydrolase converts epoxyeicosatrienoic acids to their corresponding diols, dihydroxyeicosatrienoic acids, modifying or eliminating the function of these oxylipins. To better understand the phenotypic impact of Ephx2 disruption, two independently derived colonies of soluble epoxide hydrolase-null mice were compared. We examined this genotype evaluating protein expression, biofluid oxylipin profile, tissue oxylipin production capacity, and blood pressure. Ephx2 gene disruption eliminated soluble epoxide hydrolase protein expression and activity in liver, kidney, and heart from each colony. Plasma levels of epoxy fatty acids were increased, and fatty acid diols levels were decreased, while measured levels of lipoxygenase- and cyclooxygenase-dependent oxylipins were unchanged. Liver and kidney homogenates also show elevated epoxide fatty acids. However, in whole kidney homogenate a 4-fold increase in the formation of 20-hydroxyeicosatetraenoic acid was measured along with a 3-fold increase in lipoxygenase-derived hydroxylation and prostanoid production. Unlike previous reports, however, neither Ephx2-null colony showed alterations in basal blood pressure. Finally, the soluble epoxide hydrolase-null mice show a survival advantage following acute systemic inflammation. The data suggest that blood pressure homeostasis may be achieved by increasing production of the vasoconstrictor, 20-hydroxyeicosatetraenoic acid in the kidney of the Ephx2-null mice. This shift in renal metabolism is likely a metabolic compensation for the loss of the soluble epoxide hydrolase gene.

Development of a high-throughput assay to measure histidine decarboxylase activity.

Histamine is a well-known mediator of allergic, inflammatory, and neurological responses. More recent studies suggest a role for histamine and its receptors in a wide range of biological processes, including T-cell maturation and bone remodeling. Histamine serum levels are regulated mainly by the activity of the histamine-synthesizing enzyme histidine decarboxylase (HDC). Despite the importance of this enzyme in many physiological processes, very few potent HDC inhibitors have been identified. HDC assays suitable for high-throughput screening have not been reported. The authors describe the development of a fluorescence polarization assay to measure HDC enzymatic activity. They used a fluorescein-histamine probe that binds with high affinity to an antihistamine antibody for detection. Importantly, they show that probe binding is fully competed by histamine, but no competition by the HDC substrate histidine was observed. The automated assay was performed in a total volume of 60 muL, had an assay window of 80 to 100 mP, and had a Z' factor of 0.6 to 0.7. This assay provides new tools to study HDC activity and pharmacological modulation of histamine levels.

Isolation of mast cell secretory lysosomes using flow cytometry.

BACKGROUND: Mast cells are specialized secretory cells of the immune system. Through exocytosis of their secretory lysosomes and secretory granules, mast cells release biologically active substances such as histamine and proteases. Mast cell secretory granules have been studied extensively but much less attention has been given to secretory lysosomes. Studies on mast cell secretory lysosomes are limited by the lack of selective markers and the difficulty to isolate this organelle from conventional lysosomes. Our goal was to develop better tools to study secretory lysosomes. METHODS: We engineered a rat mast cell line over expressing a rat mast cell protease (RMCP) tagged with a red fluorescent protein (RMCP-DsRed). We used single organelle flow analysis (SOFA) to detect fluorescently labeled secretory lysosomes. The labeled organelles were then sorted using the fluorescence-assisted organelle sorting (FAOS) method. RESULTS: We show that the RMCP-DsRed fusion protein selectively localizes to the lysosomal compartment and is exocytosed upon activation, confirming its localization in secretory lysosomes. Lysosomal fractions from cells expressing the RMCP-DsRed fusion were analyzed by SOFA and a specific population of secretory lysosome was identified. Finally, we sorted secretory lysosomes and showed that the sorted material had a higher specific activity for the compartment marker hexosaminidase than a sample obtained by conventional methods. CONCLUSIONS: Our work further demonstrates the usefulness of flow cytometry to study cellular organelles, and provides new tools to better understand the physiology of secretory lysosomes.