Boonlert Cheewatrakoolpong

Phospholipase C and phospholipase D are activated independently of each other in chemotactic peptide-stimulated human neutrophils.

When cytochalasin B‐treated neutrophils were stimulated with fMet‐Leu‐Phe (fMLP) in the presence of Ca2+, phospholipase C (PLC) activity, as measured by inositol‐1,4,5‐trisphosphate (IP3) formation, preceded phospholipase D (PLD)‐catalyzed breakdown of choline‐ containing phosphoglycerides to form choline and dirady 1‐sn ‐glycero‐3‐phosphate (phosphatidic acid), suggesting a possible link between PLC and PLD. However, in the absence of cytochalasin B or extracellular Ca2+, PLC was fully activated by fMLP with minimal activation of PLD, indicating that PLC activation alone is not sufficient for PLD activation. Full activation of PLD by fMLP required the simultaneous presence of both Ca2+ and cytochalasin B, a condition that caused no further enhancement of PLC. This result suggests that PLD products are not involved in the regulation of PLC activation. Furthermore, under conditions of complete inhibition of PLC by phorbol 12‐myristate 13‐acetate (PMA), there was no inhibition of PLD, showing that fMLP can activate PLD in the absence of PLC. Treatment of intact neutrophils with pertussis toxin inhibited both PLC and PLD, with PLC inhibition occurring at lower concentrations than PLD inhibition. These differential effects of pertussis toxin and the observed lack of inhibition of fMLP‐stimulated PLD by PMA, which is believed to inactivate G‐proteins involved in PLC activation, imply that PLC and PLD are linked to fMLP receptors through distinct G‐proteins. Taken together, these observations suggests that, in fMLP‐stimulated neutrophils, PLC and PLD are activated through independent mechanisms.

Human cord blood-derived mast cells synthesize and release I-309 in response to IgE

Mast cells are the central mediating cells of allergic reactions. Binding of allergen specific IgE to high affinity IgE receptor (Fcepsilon RI) and subsequent binding of allergen by the IgE causes receptor cross-linking and activation. In a study examining the differential gene expression in human cord blood-derived mast cells (CBMCs) mediated by activation of Fcepsilon RI both with IgE and IgE followed by cross-linking with alpha-IgE, the chemokine I-309 was found to be upregulated. I-309 is the ligand for the CCR8 receptor and is responsible for chemoattraction of TH2 type T-cells. Interestingly, I-309 RNA and protein levels were elevated not only in response to IgE/alpha-IgE activation but also by IgE alone. In addition, the I-309 levels were augmented by growth of the CBMCs in the presence of the proinflammatory cytokine IL-4. GM-CSF and MIP-1alpha secretion was also induced by IgE. These results suggest that IgE, through the production and release of cytokines such as I-309, GM-CSF and MIP-1alpha could promote an inflammatory reaction in the absence of antigen stimulation of mast cells.

Discovery of SCH 900271, a Potent Nicotinic Acid Receptor Agonist for the Treatment of Dyslipidemia.

Structure-guided optimization of a series of C-5 alkyl substituents led to the discovery of a potent nicotinic acid receptor agonist SCH 900271 (33) with an EC50 of 2 nM in the hu-GPR109a assay. Compound 33 demonstrated good oral bioavailability in all species. Compound 33 exhibited dose-dependent inhibition of plasma free fatty acid (FFA) with 50% FFA reduction at 1.0 mg/kg in fasted male beagle dogs. Compound 33 had no overt signs of flushing at doses up to 10 mg/kg with an improved therapeutic window to flushing as compared to nicotinic acid. Compound 33 was evaluated in human clinical trials.

The neurokinin-1 and neurokinin-2 receptor binding sites of MDL103,392 differ

Several small molecule non-peptide antagonists of the NK-1 and NK-2 receptors have been developed. Mutational analysis of the receptor protein sequence has led to the conclusion that the binding site for these non-peptide antagonists lies within the bundle created by transmembrane domains IV-VII of the receptor and differs from the binding sites of peptide agonists and antagonists. The current investigation uses site-directed mutagenesis of the NK-1 and NK-2 receptors to elucidate the amino acids that are important for binding and functional activity of the first potent dual NK-1/NK-2 antagonist MDL103,392. The amino acids found to be important for MDL103,392 binding to the NK-1 receptor are Gln-165, His-197, Leu-203, Ile-204, Phe-264, His-265 and Tyr-272. The amino acids found to be important for MDL103,392 binding to the NK-2 receptor are Gln-166, His-198, Tyr-266 and Tyr-289. While residues in transmembrane (TM) domains IV and V are important in both receptors (Gln-165/166 and His-197/198), residues in TM V and VI are more important for the NK-1 receptor and residues in TM VII play a more important role in the NK-2 receptor. These data are the first report of the analysis of the binding site of a dual tachykinin receptor antagonist and indicate that a single compound (MDL103,392) binds to each receptor in a different manner despite there being a high degree of homology in the transmembrane bundles. In addition, this is the first report in which a model for the binding of a non-peptide antagonist to the NK-2 receptor is proposed.

Design and Synthesis of Novel, Selective GPR40 AgoPAMs

GPR40 is a G-protein-coupled receptor expressed primarily in pancreatic islets and intestinal L-cells that has been a target of significant recent therapeutic interest for type II diabetes. Activation of GPR40 by partial agonists elicits insulin secretion only in the presence of elevated blood glucose levels, minimizing the risk of hypoglycemia. GPR40 agoPAMs have shown superior efficacy to partial agonists as assessed in a glucose tolerability test (GTT). Herein, we report the discovery and optimization of a series of potent, selective GPR40 agoPAMs. Compound 24 demonstrated sustained glucose lowering in a chronic study of Goto Kakizaki rats, showing no signs of tachyphylaxis for this mechanism.

Cloning and characterization of the hamster and guinea pig nicotinic acid receptors

In this study, we present the identification and characterization of hamster and guinea pig nicotinic acid receptors. The hamster receptor shares ∼80–90% identity with the nucleotide and amino acid sequences of human, mouse, and rat receptors. The guinea pig receptor shares 76–80% identity with the nucleotide and amino acid sequences of these other species. [3H]nicotinic acid binding affinity at guinea pig and hamster receptors is similar to that in human (dissociation constant = 121 nM for guinea pig, 72 nM for hamster, and 74 nM for human), as are potencies of nicotinic acid analogs in competition binding studies. Inhibition of forskolin-stimulated cAMP production by nicotinic acid and related analogs is also similar to the activity in the human receptor. Analysis of mRNA tissue distribution for the hamster and guinea pig nicotinic acid receptors shows expression across a number of tissues, with higher expression in adipose, lung, skeletal muscle, spleen, testis, and ovary.

Real-time assay of tryptase release from human umbilical cord blood-derived mast cells.

Mast cells are the central mediating cells of allergic reactions responding via the high-affinity IgE receptor (FcεRI) through binding of IgE and specific antigen (1). This activation leads to the release of preformed mediators stored in secretory granules (such as histamine, α and β tryptases, and chymase) as well as to the de novo synthesis of leukotrienes and prostaglandins and the production and release of various cytokines and chemokines (1). The monitoring of mediator release has been used in the past as a direct measurement of mast cell activation. In particular, assays to detect histamine have long been the standard used for studies of mast cell function (2–6). In addition, although the release of the mast cell protease tryptase has been used to monitor mast cell activation, those assays describe a multi-step technique in which tryptase levels are determined at a given endpoint after removal of cells (7,8). We report the development of a real-time assay for mast cell tryptase release from small numbers of cells (104 cells/assay point). This assay measures the release of tryptase from cells real-time and does not require manipulation of the cells other than incubation with appropriate stimuli. To propagate human cord blood-derived mast cells (CMBCs), human umbilical cord blood mononuclear progenitors (Poeitic Technologies, Gaithersburg, MD, USA) were placed into culture medium (RPMI/10% FBS; Invitrogen, Carlsbad, CA, USA) in the presence of stem cell factor and IL-6 (R&D Systems, Minneapolis, MN, USA), both at 100 ng/mL, at a density of 1 × 106 cells/mL (modification of Reference 9). One half of the culture medium was changed each week. After five weeks, IL-4 (R&D Systems) was introduced into the medium at 100 ng/mL. Cultures were continued for eight weeks and evaluated by staining with Toluidine blue dye. Cells used for functional assays were a minimum of 90% Toluidine blue positive. Eightto ten-week-old CBMCs were lysed by three rounds of freeze/ thaw, and the lysate was diluted in tryptase assay buffer (100 mM TrisHCl, pH 8.0, 100 mM NaCl) in a final volume of 50 μL and placed in the wells of a 96-well plate (Pierce Chemical, Rockford, IL, USA). An additional 50 μL of buffer alone or buffer containing protease inhibitors (all from Sigma St. Louis, MO, USA) was added to each well. A final 50 μL substrate [tosyl-glypro-lys-p-nitroanilide (10) (Sigma), 25 mM stock in 100% DMSO] in buffer was added to a final concentration of 500 μM, and the plate was immediately read in a plate reader (Molecular Devices, Sunnyvale, CA, USA) at 410 nm in the kinetic mode for 15 min with data collection every 30 s. Enzyme activity was determined as the rate of change of A due to cleavage of the substrate (velocity) and reported as mOD/min. The proteolytic activity present in varying numbers of cells against a serine protease substrate (tosyl-gly-pro-lys-p-nitro anilide) is shown in Figure 1 along with the activity of 5 nM rh-β-tryptase (Promega, Madison, WI, USA). Proteolytic activity above background could be detected from as few as 100 cells and was linear with cell number through the range tested (r2 = 0.99). This protease was inhibited by serine protease inhibitors (antipain, BABIM, benzamidine, leupeptin, and TLCK) and lactoferrin (which inhibits tryptase by competing for heparin) (data not shown) (11). Cysteine protease inhibitors (aprotinin, pepstatin, and TPCK) were not effective (data not shown). This pattern of inhibition suggests that the proteolytic activity being measured is indeed that of tryptase. To assay the real-time release of tryptase, CBMCs were sensitized for 24 h or longer in the presence of varying concentrations of human IgE (Biodesign, Saco, ME, USA). Before use, the cells were pelleted for 4 min at 1000× g and resuspended in RPMI at 2 × 105 cells/mL. Fifty microliters of cells (approximately 10 000 cells/well) were added to each well of a 96-well plate. Either 50 μL of buffer or buffer containing compounds to be tested was added to each well, and the plate was Benchmarks

Structure–Activity Relationship of Novel and Selective Biaryl-Chroman GPR40 AgoPAMs

A series of biaryl chromans exhibiting potent and selective agonism for the GPR40 receptor with positive allosteric modulation of endogenous ligands (AgoPAM) were discovered as potential therapeutics for the treatment of type II diabetes. Optimization of physicochemical properties through modification of the pendant aryl rings resulted in the identification of compound AP5, which possesses an improved metabolic profile while demonstrating sustained glucose lowering.

GPR40 partial agonists and AgoPAMs: Differentiating effects on glucose and hormonal secretions in the rodent

GPR40 agonists are effective antidiabetic agents believed to lower glucose through direct effects on the beta cell to increase glucose stimulated insulin secretion. However, not all GPR40 agonists are the same. Partial agonists lower glucose through direct effects on the pancreas, whereas GPR40 AgoPAMs may incorporate additional therapeutic effects through increases in insulinotrophic incretins secreted by the gut. Here we describe how GPR40 AgoPAMs stimulate both insulin and incretin secretion in vivo over time in diabetic GK rats. We also describe effects of AgoPAMs in vivo to lower glucose and body weight beyond what is seen with partial GPR40 agonists in both the acute and chronic setting. Further comparisons of the glucose lowering profile of AgoPAMs suggest these compounds may possess greater glucose control even in the presence of elevated glucagon secretion, an unexpected feature observed with both acute and chronic treatment with AgoPAMs. Together these studies highlight the complexity of GPR40 pharmacology and the potential additional benefits AgoPAMs may possess above partial agonists for the diabetic patient.

Effect of K+ Depletion on Glutamate Dehydrogenase

Slices of renal cortices were homogenized in Trisolv (Bioteck Laboratories, Houston,Tex., USA) at an approximate ratio of 1 g tissue/10 ml. RNA was precipitated with isopro-panol and washed twice with 75% ethanol. The electrophoresis of RNA was performed in1% agarose-formaldehyde gel, with 20 lg of RNA sample in each lane. After the electrophor-esis, RNA was transferred to a nylon membrane (maximum strength Nytran; Schleicher &Schuell, Keene, N.H., USA) with vacuum blotting. The membrane was prehybridized for2 h with 4¶standard saline citrate (SSC), 5¶Denhart’s solution, 40% formamide, 2.5% SDS,1¶blocking reagent (Bioteck Laboratories), 5% dextran, 0.05 mg/ml sheared salmon sperm,0.1 mg/ml Poly C, 0.05 mg/ml Torula yeast RNA and 20 m