The Establishment Of A Screening Method For Protein Tyrosine Kinase Inhibitors And The Screening Of The Inhibitor From Curcumin Derivatives

Purpose: A rapid screening method for protein tyrosine kinase (PTK) inhibitors was established for drug screening in vitro and several experimental conditions had been studied. A series of curcumin derivatives was screened by the method and some of them owing PTK inhibitory activity were obtained. Their effect and machanism on the PTK inhibitory activity of p210Bcr-Abl in Ph+ K562 cells were studied. Method: The MTT method was adopted to evaluate the anti-proliferation effect of curcumin derivatives on human chronic myelogeous leukemia cell line K562. The ELISA technique was set up using poly Glu∶Tyr (4∶1) as the substrate; phosphotyrosine monoclonal antibody as the detection tool to the phosphotyrosine residue and tetra-methylbenzidine (TMB) as the chromogenic sustrate of the horseadish peroxidase. And then curcumin derivatives were screened for candidates of potent protein tyrosine kinase inhibitors. The effects of curcumin derivatives on p210Bcr-Abl content and protein tyrosine phosphorylation and the signaling molecules which are relevant with cell growth and apoptosis were observed by Western blotting. Result: The inhibitory effect of curcumin and its derivatives on K562 cell line proliferation in vitro was determined; The IC50 values of curcumin derivatives were all lower than Cur. The primary screening method for the inhibitors of tyrosine kinase was established, and by this method, a series of curcumin derivatives were screened and 4 of them were picked out (FM0815, FM0817, FM0822 and FM0824). And it showed that curcumin derivative FM0824 down-regulated p210Bcr-Abl and protein tyrosine phosphorylation in a dose-dependent manners. Conclusion: This system has a character of high speed, convenience and high throughput in screening of tyrosine kinase inhibitors in vitro. It was observed that Curcumin and its derivatives could effectively inhibit the activity of PTK, and of which FM0824 could down-regulate the content of p210Bcr-Abl and inhibit phosphorylation of p210Bcr-Abl, Erk and Akt.