Natural products AKTing on signal transduction pathways-molecules and methods

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.