Screening for Specific Inhibitors of Src Family Kinases

1. 1987年，我们报道了Staurosporine在极低浓度（nMorder）下可以抑制几种蛋白激酶，我们通过合成50多种Staurosporine衍生物开始了这个项目。以大肠杆菌合成的v-src激酶和abl激酶作为酪氨酸激酶，我们对这些衍生物进行了筛选，但在低浓度（约100nM）下均未表现出特异性抑制作用。染料木素和槲皮素是先前报道的酪氨酸激酶特异性抑制剂，在我们的系统中进行了测试。然而，我们确定的IR^<50>值比先前报道的要高得多。我们还表明，这两种试剂具有DNA切割活性，这是DNA拓扑异构酶依赖。这些结果表明，这些试剂在体内作为特异性激酶抑制剂有明显的局限性。在本项目中，我们从微生物提取物中分离出一种特异性的蛋白激酶C抑制剂，命名为UCN1028。我们进一步纯化，发现UCN1028中的特定化合物Calphostin C负责特异性抑制。Calphostin C对荷瘤小鼠有很强的抗肿瘤活性。我们最近分离出新的fos相关基因fra-2。该基因被发现是直接早期基因的成员之一，并具有转化活性。该基因的mRNA可由多种酪氨酸激酶或丝氨酸激酶介导，其基因产物Fra-2可通过血清诱导蛋白激酶活性被磷酸化。这种Fra-2的表达和修饰有望成为在体内筛选新的激酶抑制剂的敏感和有效的指标。

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.