Screening for Specific Inhibitors of Src Family Kinases

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1. In 1987, we reported that Staurosporine can inhibit several species of protein kinases at very low concentrations (nMorder), we started this project by synthesizing more than 50 derivatives of Staurosporine. Using v-src kinase as well as abl kinase synthesized in E. coli, as the tyrosine kinases, we had screened these derivatives but non of them showed specific inhibition at a low concentration (about 100nM).2. Genistein and quercetin that were previously reported as the inhibitors specific to tyrosine kinases were tested in our system. IR^<50> values we determined were, however, were much higher than reported previously. We have also shown that these two reagents have DNA cleaving activity that is DNA topoisomerase dependent. These results indicate that these regents have clear limitation to be used as specific kinase inhibitor in vivo.3. In this project, we isolated a specific inhibitor of protein kinase C from microbiral extract, which was named as UCN1028. We further purified and found that the specific compound, Calphostin C, in UCN1028 is responsible for the specific inhibition. Calphostin C showed very strong antitumor activity when injected into tumor bearing mouse.4. We recently isolated new fos related gene, fra-2. This gene was found to be one member of immediate early genes and to have transforming activity. Induction of the mRNA of this gene was shown to be mediated by several kinds of tyrosine kinase or serine kinase and its gene product, Fra-2 was further shown to be phosphorylated by serum inducible protein kinase activity. This Fra-2 expression as well as modification is expected to be sensitive and effective indicators for the screening of new kinase inhibitors in vivo.