amphotericin-b 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 present study evaluated the importance of the association between Na+-K+-ATPase and the actin cytoskeleton on dopamine-induced inhibition of Na+-K+-ATPase activity. The approach used measures the transepithelial transport of Na+ in monolayers of opossum kidney (OK) cells, when the Na+ delivered to Na+-K+-ATPase was increased at the saturating level by amphotericin B. The maximal amphotericin B (1.0 microg mL-1) induced increase in short-circuit current (Isc) was prevented by ouabain (100 microM) or removal of apical Na+. Dopamine (1 microM) applied from the apical side significantly decreased (29 +/- 5% reduction) the amphotericin B-induced increase in Isc, this being prevented by the D1-like receptor antagonist SKF 83566 (1 microM) and the protein kinase C (PKC) inhibitor chelerythrine (1 microM). Exposure of OK cells to cytochalasin B (1 microM) or cytochalasin D (1 microM), inhibitors of actin polymerization, from both cell sides reduced by 31 +/- 4% and 36 +/- 3% the amphotericin B-induced increase in Isc and abolished the inhibitory effect of apical dopamine (1 microM), but not that of the PKC activator phorbol-12,13-dibutyrate (PDBu; 100 nM). Colchicine (1 microM) failed to alter the inhibitory effects of dopamine. The relationship between Na+-K+-ATPase and the concentration of extracellular Na+ showed a Michaelis-Menten constant (Km) of 44.1 +/- 13.7 mM and a Vmax of 49.6 +/- 4.8 microA cm-2 in control monolayers. In the presence of apical dopamine (1 microM) or cytochalasin B (1 microM) Vmax values were significantly (P < 0.05) reduced without changes in Km values. These results are the first, obtained in live cells, showing that the PKC-dependent inhibition of Na+-K+-ATPase activity by dopamine requires the integrity of the association between actin cytoskeleton and Na+-K+-ATPase.

Topics: Actins; Alkaloids; Amphotericin B; Animals; Benzophenanthridines; Cell Line; Colchicine; Cytochalasins; Cytoskeleton; Dopamine; Electric Conductivity; Ion Transport; Kidney; Kinetics; Logistic Models; Opossums; Phenanthridines; Phorbol 12,13-Dibutyrate; Sodium-Potassium-Exchanging ATPase