INHIBITION BY 2 LAVENDUSTINS OF THE TYROSINE KINASE-ACTIVITY OF PP60(F527) IN-VITRO AND IN INTACT-CELLS

The mutant pp60(F527) protein possesses an activate protein-tyrosine kinase (PTK) activity correlated with a transforming activity. We have studied the inhibition of the pp60(F527) PTK activity by two EGF-R tyrosine kinase inhibitors, lavendustin A and one of its derivatives, lavendustin C6. In vitro, both molecules were non-competitive inhibitors for the ATP binding site and uncompetitive inhibitors for the peptide binding site. The determined IC(50)s of the inhibition of pp60(F527) kinase activity were 18 mu M for lavendustin A and 5 mu M for lavendustin C6, as determined on the exogenous substrate enolase, showing that lavendustin C6 was more potent than lavendustin A. Lavendustin C6, but not lavendustin A, inhibited the tyrosine phosphorylation of pp60(F527) cellular substrates (the GAP-associated p190, pp125(FAK) and cortactin) in intact cells. However, this in situ inhibitory effect did not result in a reversion of the morphological changes induced by pp60(F527) in cells. On the other hand, lavendustin C6 and lavendustin A exerted antiproliferative effects on cells, suggesting that inhibition of cellular targets related or not to the kinase was also possible.