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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.

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.

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. Cytometry Part A 55A:94–101, 2003. © 2003 Wiley-Liss, Inc.

We recently observed that specific inhibitors of post-proline cleaving aminodipeptidases cause apoptosis in quiescent lymphocytes in a process independent of CD26/dipeptidyl peptidase IV. These results led to the isolation and cloning of a new protease that we have termed quiescent cell proline dipeptidase (QPP). QPP activity was purified from CD26(-) Jurkat T cells. The protein was identified by labeling with [(3)H]diisopropylfluorophosphate and subjected to tryptic digestion and partial amino acid sequencing. The peptide sequences were used to identify expressed sequence tag clones. The cDNA of QPP contains an open reading frame of 1476 base pairs, coding for a protein of 492 amino acids. The amino acid sequence of QPP reveals similarity with prolylcarboxypeptidase. The putative active site residues serine, aspartic acid, and histidine of QPP show an ordering of the catalytic triad similar to that seen in the post-proline cleaving exopeptidases prolylcarboxypeptidase and CD26/dipeptidyl peptidase IV. The post-proline cleaving activity of QPP has an unusually broad pH range in that it is able to cleave substrate molecules at acidic pH as well as at neutral pH. QPP has also been detected in nonlymphocytic cell lines, indicating that this enzyme activity may play an important role in other tissues as well.

p56lck is a member of the src family of tyrosine kinases. Through modular binding units called SH2 domains, p56lck promotes phosphotyrosine-dependent protein-protein interactions and plays a critical role in signal transduction events that lead to T-cell activation. Starting from the phosphorylated dipeptide (2), a high-affinity ligand for the p56lck SH2 domain, we have designed novel dipeptides that contain monocharged, nonhydrolyzable phosphate group replacements and bind to the protein with KD's in the low micromolar range. Replacement of the phosphate group in phosphotyrosine-containing sequences by a (R/S)-hydroxyacetic (compound 8) or an oxamic acid (compound 10) moiety leads to hydrolytically stable, monocharged ligands, with 83- and 233-fold decreases in potency, respectively. This loss in binding affinity can be partially compensated for by incorporating large lipophilic groups at the inhibitor N-terminus. These groups provide up to 13-fold increases in potency depending on the nature of the phosphate replacement. The discovery of potent (2-3 microM), hydrolytically stable dipeptide derivatives, bearing only two charges at physiological pH, represents a significant step toward the discovery of compounds with cellular activity and the development of novel therapeutics for conditions associated with undesired T-cell proliferation.

Sec4, a GTP-binding protein of the ras superfamily, is required for exocytosis in the budding yeast Saccharomyces cerevisiae. To test the role of GTP hydrolysis in Sec4 function, we constructed a mutation, Q-79----L, analogous to the oncogenic mutation of Q-61----L in Ras, in a region of Sec4 predicted to interact with the phosphoryl group of GTP. The sec4-leu79 mutation lowers the intrinsic hydrolysis rate to unmeasurable levels. A component of a yeast lysate specifically stimulates the hydrolysis of GTP by Sec4, while the rate of hydrolysis of GTP by Sec4-Leu79 can be stimulated by this GAP activity to only 30% of the stimulated hydrolysis rate of the wild-type protein. The decreased rate of hydrolysis results in the accumulation of the Sec4-Leu79 protein in its GTP-bound form in an overproducing yeast strain. The sec4-leu79 allele can function as the sole copy of sec4 in yeast cells. However, it causes recessive, cold-sensitive growth, a slowing of invertase secretion, and accumulation of secretory vesicles and displays synthetic lethality with a subset of other secretory mutants, indicative of a partial loss of Sec4 function. While the level of Ras function reflects the absolute level of GTP-bound protein, our results suggest that the ability of Sec4 to cycle between its GTP and GDP bound forms is important for its function in vesicular transport, supporting a mechanism for Sec4 function which is distinct from that of the Ras protein.

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.

Sec4 is a Ras-like GTP-binding protein required for exocytosis in yeast. Unlike Ras, it is the ability of Sec4 to cycle between the GTP- and GDP-bound forms rather than the absolute levels of the GTP-bound form that is critical for function. This cycle may be coupled to an observed cycle of Sec4 localization within the cell. Sec4 binds to secretory vesicles which then fuse with the plasma membrane in exocytosis. Sec4 can recycle from the plasma membrane through a soluble pool to rebind to a new round of vesicles. We have found an activity in yeast (Saccharomyces cerevisiae) comparable to that of the GDP dissociation inhibitor protein isolated from mammalian cells that releases GDP-bound Sec4 from membranes. DSS4-1, a dominant suppressor of the sec4-8 temperature-sensitive mutation, encodes a nucleotide exchange protein. The cycle of Sec4 may function to allow the assembly and subsequent disassembly of a set of proteins necessary for exocytosis. Candidates for members of this set of proteins are encoded by sec genes which show strong genetic interactions with sec4-8. Two of these (SEC8 and SEC15) encode large proteins which form a complex that is peripherally associated with the plasma membrane.

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.

Introduction: M cells are located in the epithelial layer covering the gut-associated lymphoid tissue and are responsible for delivery of macromolecules and microorganisms to the underlying lymphoid cells. It has been shown that the human colonic cell line Caco-2 can be converted to M cells in vitro following coculture with isolated lymphocytes from murine Peyer's patches. Studies were undertaken to evaluate and characterize the transepithelial transport of select macromolecules across these in vitro derived M cells. Methods: Caco-2 cells were converted to M cells as reported previously. The morphology of Caco-2 cells and M cells was compared by transmission electron microscopy (TEM). The transport properties of macromolecules such as horseradish peroxidase, FITC-conjugated polystyrene beads, and radiolabeled dextrans were examined. The activation of murine antigen-specific T cells following transport of the antigen ovalbumin across the M-cell barrier was assessed by measuring cytokine production. Results: M cells were shown to be irregular in shape and have fewer and shorter microvilli compared to the Caco-2 cell progenitors. These cells were still able to form tight junctions and monolayers on polycarbonate membranes. Time-course studies demonstrated that the transport of polystyrene beads and large-molecular-weight dextrans at physiological temperature across M-cell-containing monolayers was size dependent and more rapid than across Caco-2 cell monolayers. The transport of dextrans was also shown to be temperature and concentration dependent. Befitting the role of the M cell in mucosal defense, protein antigen could be delivered by these cells in order to be processed and presented to antigen-specific CD4+ T lymphocytes. Discussion: The M-cell permeability model is a functional and practical system for evaluating the transport properties of macromolecules and assessing the potential for intestinal mucosal antigen sampling to elicit immunological responses.

p56lck is a member of the src family of tyrosine kinases and plays a critical role in the signal transduction events that lead to T cell activation. Ligands for the p56lck SH2 domain have the potential to disrupt the interaction of p56lck with its substrates and derail the signaling cascade that leads to the production of cytokines such as interleukin-2. Starting from the quintuply charged (at physiological pH) phosphorylated tetrapeptide, AcpYEEI, we recently disclosed (J. Med. Chem. 1999, 42, 722 and J. Med. Chem. 1999, 42, 1757) the design of the modified dipeptide 3, which carries just two charges at physiological pH. Here we present the elaboration of 3 to the nonpeptidic, monocharged compound, 9S. This molecule displays good binding affinity for the p56lck SH2 domain (K(d) 1 microM) and good cell permeation, and this combination of properties allowed us to demonstrate clear-cut inhibitory effects on a very early event in T cell activation, namely calcium mobilization.

Rab GDP dissociation inhibitor (Rab GDI), will induce the dissociation of GDP-bound rab3A from synaptic membranes and will inhibit GDP dissociation from Sec4, a member of the Rab subgroup of the Ras GTPase superfamily which is required for exocytosis in Saccharomyces cerevisiae. We report that Rab GDI releases GDP-bound Sec4 from yeast membranes. dGDI, a Drosophila homologue can similarly inhibit GDP dissociation from Sec4 and release GDP-bound Sec4 from yeast membranes. An activity partially purified from yeast cytosol dissociates GDP-bound Sec4 from yeast membranes, suggesting that yeast also possess a GDI protein that functions to recycle Sec4 from its target membrane.

Sec4, a GTP-binding protein of the ras superfamily, is required for exocytosis in the budding yeast Saccharomyces cerevisiae. To test the role of GTP hydrolysis in Sec4 function, we constructed a mutation, Q-79----L, analogous to the oncogenic mutation of Q-61----L in Ras, in a region of Sec4 predicted to interact with the phosphoryl group of GTP. The sec4-leu79 mutation lowers the intrinsic hydrolysis rate to unmeasurable levels. A component of a yeast lysate specifically stimulates the hydrolysis of GTP by Sec4, while the rate of hydrolysis of GTP by Sec4-Leu79 can be stimulated by this GAP activity to only 30% of the stimulated hydrolysis rate of the wild-type protein. The decreased rate of hydrolysis results in the accumulation of the Sec4-Leu79 protein in its GTP-bound form in an overproducing yeast strain. The sec4-leu79 allele can function as the sole copy of sec4 in yeast cells. However, it causes recessive, cold-sensitive growth, a slowing of invertase secretion, and accumulation of secretory vesicles and displays synthetic lethality with a subset of other secretory mutants, indicative of a partial loss of Sec4 function. While the level of Ras function reflects the absolute level of GTP-bound protein, our results suggest that the ability of Sec4 to cycle between its GTP and GDP bound forms is important for its function in vesicular transport, supporting a mechanism for Sec4 function which is distinct from that of the Ras protein.

Publisher Summary This chapter explains the purification of sec4 protein from saccharomyces cerevisiae and Escherichia coli. Sec4p undergoes a cycle of GTP binding and hydrolysis that may be coupled to a cycle of subcellular localization in which Sec4p first attaches to post-Golgi vesicles that go on to fuse with the plasma membrane. Sec4p then recycles through a soluble pool to reassociate with a new round of vesicles. Membrane attachment of Sec4p, Ypt1p, and the many Rab homologs requires prior modification of carboxy-terminal cysteine moieties by the addition of isoprenyl lipids. The chapter discusses procedures for producing and purifying large amounts of native Sec4p from either yeast or bacteria. The purification of Sec4p from yeast is described and the purification of Sec4 from bacteria is adapted from the yeast protocol. As bacteria lack prenylation enzymes, bacterially produced Sec4p is a convenient substrate for modification studies.

Mutant alleles of SEC4, an essential gene required for the final stage of secretion in yeast, have been generated by in vitro mutagenesis. Deletion of the two cysteine residues at the C terminus of the protein results in a soluble non-functional protein, indicating that those two residues are required for normal localization of Sec4p to secretory vesicles and the plasma membrane. A mutant allele of SEC4 generated to mimic an activated, transforming allele of H-ras, as predicted, does not bind GTP. The presence of this allele in cells containing wild-type SEC4 causes a secretory defect and the accumulation of secretory vesicles. The results of genetic studies indicate that this allele behaves as a dominant loss of function mutant and as such prevents wild-type protein from functioning properly. We propose a model in which Sec4p cycles between an active and an inactive state in order to mediate the fusion of vesicles to the plasma membrane.

Using either permeabilized cells or microsomes we have reconstituted the early events of the yeast secretory pathway in vitro. In the first stage of the reaction approximately 50-70% of the prepro-alpha-factor, synthesized in a yeast translation lysate, is translocated into the endoplasmic reticulum (ER) of permeabilized yeast cells or directly into yeast microsomes. In the second stage of the reaction 48-66% of the ER form of alpha-factor (26,000 D) is then converted to the high molecular weight Golgi form in the presence of ATP, soluble factors and an acceptor membrane fraction; GTP gamma S inhibits this transport reaction. Donor, acceptor, and soluble fractions can be separated in this assay. This has enabled us to determine the defective fraction in sec23, a secretory mutant that blocks ER to Golgi transport in vivo. When fractions were prepared from mutant cells grown at the permissive or restrictive temperature and then assayed in vitro, the acceptor Golgi fraction was found to be defective.