Natural products AKTing on signal transduction pathways-molecules and methods

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Natural products play an important role as source of inspiration for drug discovery and development and as tools for basic research. Isolated natural products that had shown diverse biological activity in previously published tests were analysed for their direct effect on molecular signalling processes, militarinone A from the entomogenous fungus Paecilomyces militaris and indolinone from Isatis tinctoria. The fungal alkaloid militarinone A was originally identified in a screening for neurotrophic substances, where it induced neuronal outgrowth in PC12 cells. To uncover the mechanism of this action we studied the cell signalling pathways involved in neuronal spike formation and differentiation in two types of neuronal cells (PC12 and N2a) and the interaction of militarinone A with associated pathways. The increased neuronal outgrowth could not be confirmed as a general activity of militarinone A, as this effect was only transiently seen in PC12 cells and all other cell lines tested underwent apoptosis within 24h. We propose that this difference is due to varying constitutive levels of p53. Furthermore, an alkaloid from the traditional European medicinal plant Isatis tinctoria was analysed. Isatis tinctoria contains several known anti-inflammatory components, namely, tryptanthrin, indirubin, and indolinone. In a previous study, indolinone was shown to inhibit degranulation of mast cells and this anti-allergic effect of indolinone should be further characterised. We confirmed the initially observed stabilising effect on mast cells of a different species and in a different assay set-up and showed that indolinone efficiently blocked PtdInsP3 production due to inhibition of all class I PI3- kinases, therefore preventing activation of Akt and subsequent mast cell degranulation. The concentrations necessary to obtain the observed effect in vitro, however, were too high to consider in vivo testing. Since mast cell degranulation depends on phosphoinositide signalling we studied phosphosinositide levels in cells upon stimulation. For this purpose, we developed a method that allows individual analysis of all phosphoinositides, including all PtdInsPand PtdInsP2-isomers. This novel method, based on ion-pair chromatography and ESI-MS detection, offers substantial perspectives for application in phosphoinositidesignalling research as it allows relative quantification of all the different PIs in cells.