cytochalasin-d and chelerythrine

The presented project started by screening a library consisting of natural and natural based compounds for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity. Active compounds were chemically clustered into groups and further tested on the human cholinesterases isoforms. The aim of the presented study was to identify compounds that could be used as leads to target two key mechanisms associated with the AD's pathogenesis simultaneously: cholinergic depletion and beta amyloid (Aβ) aggregation. Berberin, palmatine and chelerythrine, chemically clustered in the so-called isoquinoline group, showed promising cholinesterase inhibitory activity and were therefore further investigated. Moreover, the compounds demonstrated moderate to good inhibition of Aβ aggregation as well as the ability to disaggregate already preformed Aβ aggregates in an experimental set-up using HFIP as promotor of Aβ aggregates. Analysis of the kinetic mechanism of the AChE inhibition revealed chelerythrine as a mixed inhibitor. Using molecular docking studies, it was further proven that chelerythrine binds on both the catalytic site and the peripheral anionic site (PAS) of the AChE. In view of this, we went on to investigate its effect on inhibiting Aβ aggregation stimulated by AChE. Chelerythrine showed inhibition of fibril formation in the same range as propidium iodide. This approach enabled for the first time to identify a cholinesterase inhibitor of natural origin-chelerythrine-acting on AChE and BChE with a dual ability to inhibit Aβ aggregation as well as to disaggregate preformed Aβ aggregates. This compound could be an excellent starting point paving the way to develop more successful anti-AD drugs.

Topics: Acetylcholinesterase; Amyloid beta-Peptides; Benzophenanthridines; Binding Sites; Butyrylcholinesterase; Catalytic Domain; Cholinesterase Inhibitors; Humans; Isoquinolines; Kinetics; Molecular Docking Simulation; Structure-Activity Relationship

The mechanisms by which uridine triphosphate (UTP) stimulates ATP release from Schwann cells cultured from the sciatic nerve were investigated using online bioluminescence techniques. UTP, a P2Y(2) and P2Y(4) receptor agonist, stimulated ATP release from Schwann cells in a dose-dependent manner with an ED(50) of 0.24 microm. UTP-stimulated ATP release occurs through P2Y(2) receptors as it was blocked by suramin which inhibits P2Y(2) but not P2Y(4) receptors. Furthermore, positive immunostaining of P2Y(2) receptors on Schwann cells was revealed and GTP, an equipotent agonist with UTP at rat P2Y(4) receptors, did not significantly stimulate ATP release. UTP-stimulated ATP release involved second messenger pathways as it was attenuated by the phospholipase C inhibitor U73122, the protein kinase C inhibitor chelerytherine chloride, the IP(3) formation inhibitor lithium chloride, the cell membrane-permeable Ca(2+) chelator BAPTA-AM and the endoplasmic reticulum Ca(2+)-dependent ATPase inhibitor thapsigargin. Evidence that ATP may be stored in vesicles that must be transported to the cell membrane for exocytosis was found as release was significantly reduced by the Golgi-complex inhibitor brefeldin A, microtubule disruption with nocodazole, F-actin disruption with cytochalasin D and the specific exocytosis inhibitor botulinum toxin A. ATP release from Schwann cells also involves anion transport as it was significantly reduced by cystic fibrosis transmembrane conductance regulator inhibitor glibencamide and anion transporter inhibitor furosemide. We suggest that UTP-stimulated ATP release is mediated by activation of P2Y(2) receptors that initiate an IP(3)-Ca(2+) cascade and protein kinase C which promote exocytosis of ATP from vesicles as well as anion transport of ATP across the cell membrane.

Topics: Adenosine Triphosphate; Alkaloids; Animals; Animals, Newborn; Benzophenanthridines; Botulinum Toxins; Botulinum Toxins, Type A; Brefeldin A; Calcium; Cyclic AMP-Dependent Protein Kinases; Cytochalasin D; Diagnostic Imaging; Dose-Response Relationship, Drug; Drug Interactions; Estrenes; Furosemide; Glyburide; Glycyrrhetinic Acid; Guanosine Triphosphate; Immunohistochemistry; Isoquinolines; Microscopy, Confocal; Nucleic Acid Synthesis Inhibitors; Phenanthridines; Phorbol 12,13-Dibutyrate; Protein Kinase C; Protein Synthesis Inhibitors; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Receptors, Purinergic P2Y2; Schwann Cells; Sciatic Nerve; Sulfonamides; Suramin; Thapsigargin; Time Factors; Type C Phospholipases; Uridine Triphosphate

This study investigates the exocytic responses of invertebrate hemocytes to pathogen-associated antigens. It demonstrates that a homologue of complement component C3, a key defensive protein of the innate immune system, is expressed by phagocytic hemocytes (non-refractile vacuolated cells) of the tunicate, Styela plicata. C3-like molecules are localized in sub-cellular vesicles and are rapidly exocytosed after stimulation with bacterial, fungal or algal cell surface molecules. Signal transduction analysis indicated that the induced secretion of C3-like molecules is mediated by a G-protein dependent signaling pathway, which modulates tubulin microtubules. All of this evidence indicates that hemocytes can contribute to host defense responses by rapidly exocytosing C3-like proteins at sites of infection.

Topics: Alkaloids; Animals; Benzophenanthridines; Blotting, Western; Calcimycin; Carrageenan; Cell Survival; Cholera Toxin; Colchicine; Colforsin; Complement C3; Cytochalasin D; Cytoplasmic Vesicles; Enzyme-Linked Immunosorbent Assay; Exocytosis; Hemocytes; Immunohistochemistry; Kinetics; Lipopolysaccharides; Mannans; Microscopy, Electron; Phenanthridines; Staurosporine; Tetradecanoylphorbol Acetate; Thapsigargin; Urochordata

Chemoattractant-stimulated pseudopod growth in human neutrophils was used as a model system to study the rate-limiting mechanism of cytoskeleton rearrangement induced by activated G-protein-coupled receptors. Cells were activated with N-formyl-Met-Leu-Phe, and the temperature dependence of the rate of pseudopod extension was measured in the presence of pharmacological inhibitors with known mechanisms of action. Three groups of inhibitors were used: (i) inhibitors sequestering substrates involved in F-actin polymerization (latrunculin A for G-actin and cytochalasin D for actin filament-free barbed ends) or sequestering secondary messengers (PIP-binding peptide for phosphoinositide lipids); (ii) competitively binding inhibitors (Akt-inhibitor for Akt/protein kinase B); and (iii) inhibitors that reduce enzyme activity (wortmannin for phosphoinositide 3-kinase and chelerythrine for protein kinase C). The experimental data are consistent with a model in which the relative involvement of a given pathway of F-actin polymerization to the measured rate of pseudopod extension is limited by a slowest (bottleneck) reaction in the cascade of reactions involved in the overall signaling pathway. The approach we developed was used to demonstrate that chemoattractant-induced pseudopod growth and mechanically stimulated cytoskeleton rearrangement are controlled by distinct pathways of F-actin polymerization.

Topics: Actins; Alkaloids; Androstadienes; Benzophenanthridines; Bridged Bicyclo Compounds, Heterocyclic; Cytochalasin D; Cytoskeleton; Enzyme Inhibitors; Enzymes; Humans; Kinetics; Lipids; Models, Chemical; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Peptides; Phenanthridines; Phosphatidylinositol 3-Kinases; Protein Binding; Protein Kinase C; Receptors, G-Protein-Coupled; Temperature; Thiazoles; Thiazolidines; Time Factors; Wortmannin

The activated leukocyte cell adhesion molecule (ALCAM) is dynamically regulated by the actin cytoskeleton. In this study we explored the molecular mechanisms and signaling pathways underlying the cytoskeletal restraints of this homotypic adhesion molecule. We observed that ALCAM-mediated adhesion induced by cytoskeleton-disrupting agents is accompanied by activation of the small GTPases RhoA, Rac1 and Cdc42. Interestingly, unlike adhesion mediated by integrins or cadherins, ALCAM-mediated adhesion appears to be independent of Rho-like GTPase activity. By contrast, we demonstrated that protein kinase C (PKC) plays a major role in ALCAM-mediated adhesion. PKC inhibition by chelerythrine chloride and myristoylated PKC pseudosubstrate, as well as PKC downregulation by PMA strongly reduce cytoskeleton-dependent ALCAM-mediated adhesion. Since serine and threonine residues are dispensable for ALCAM-mediated adhesion and ALCAM is not phosphorylated, we can rule out that ALCAM itself is a direct PKC substrate. We conclude that PKCalpha plays a dominant role in cytoskeleton-dependent avidity modulation of ALCAM.

Topics: Activated-Leukocyte Cell Adhesion Molecule; Alkaloids; Antibodies, Monoclonal; Benzophenanthridines; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Cell Adhesion; Cytochalasin D; Cytoskeleton; Dose-Response Relationship, Drug; Down-Regulation; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Humans; K562 Cells; Microscopy, Fluorescence; Phenanthridines; Phosphorus Radioisotopes; Protein Kinase C; Protein Kinase C-alpha; Retroviridae; rho GTP-Binding Proteins; Substrate Specificity; Tetradecanoylphorbol Acetate; Thiazoles; Thiazolidines