Abstract
The cyclic imine marine toxins are characterised by potent nicotinic acetylcholine receptor antagonism causing fast acute toxicity in mice. One member of the family, portimine A, is not toxic but inhibits growth and induces apoptosis in cancer cells at nanomolar concentrations. When cells were protected against apoptosis, portimine A still inhibited growth, suggesting it may be a mixed mechanism of action antineoplastic lead compound. The mechanism through which portimine A exerts its effect is currently unknown. My aim was to identify its primary targets.
The first goal was to compare the anti-proliferative effect of portimine A with other cyclic imine toxins, including the closely related portimine B . This compound, which differs from portimine A by the simple modification of a hydroxyl to a ketone, was found to have a half-maximal growth inhibitory concentration (GI50) of 27 nM in Jurkat T-cell lymphoma cells, far higher than the 0.7 nM measured for portimine A. Pinnatoxins G and H, as well as gymnodimine A, were found to have no growth inhibitory effect at concentrations up to 100 nM. This demonstrated that portimine A contains very specific features that facilitate its biological activity and could provide valuable clues to it mechanism of action.
Previous studies in the host laboratory have used portimine A conjugated to biotin to capture proteins from both whole cell and cell lysate samples through a traditional streptavidin bead-based pulldown. The pulldown experiments were repeated, but it was observed in parallel studies that biotinylated portimine was considerably less effective than unmodified portimine at inhibiting cancer cell growth. Indeed, much of the activity could be ascribed to the 10% of unbiotinylated portimine in the preparation. It was therefore concluded that affinity pulldown methods using biotinylated portimine are of limited benefit.
To overcome this issue, thermal proteome profiling was used to identify direct targets of unlabelled portimine A. This technique measures the change in protein stability induced by a molecule binding its target by measuring the change in the temperature at which the protein target denatures. These results were ranked for pathway analysis to generate a shortlist of candidate proteins for future confirmation by loss-of-function manipulation.