Bishop A C, Shah K, Liu Y, Witucki L, Kung C, Shokat K M
Department of Chemistry Princeton University Princeton, New Jersey 08544, USA.
Curr Biol. 1998 Feb 26;8(5):257-66. doi: 10.1016/s0960-9822(98)70198-8.
Deconvoluting protein kinase signaling pathways using conventional genetic and biochemical approaches has been difficult because of the overwhelming number of closely related kinases. If cell-permeable inhibitors of individual kinases could be designed, the role of each kinase could be systematically assessed.
We have devised an approach combining chemistry and genetics to develop the first highly specific cell-permeable inhibitor of the oncogenic tyrosine kinase v-Src. A functionally silent active-site mutation was made in v-Src to distinguish it from all other cellular kinases. A tight-binding cell-permeable inhibitor of this mutant kinase that does not inhibit wild-type kinases was designed and synthesized. In vitro and whole-cell assays established the unique specificity of the mutant v-Src-inhibitor pair. The inhibitor reversed cell transformation by the engineered but not the 'wild type' v-Src, establishing that changes in cellular signaling can be attributed to specific inhibition of the engineered kinase. The generality of the method was tested by engineering another tyrosine kinase, Fyn, to contain the corresponding active-site mutation to the one in v-Src. The same compound that inhibited mutant v-Src could also potently inhibit the engineered Fyn kinase.
Allele-specific cell-permeable inhibitors of individual Src family kinases can be rapidly developed in an approach that should be applicable to all kinases. This approach will be useful for the deconvolution of kinase-mediated cellular pathways and for validating novel kinases as good targets for drug discovery both in vitro and in vivo.
由于密切相关的激酶数量众多，使用传统的遗传和生化方法对蛋白激酶信号通路进行解卷积一直很困难。如果能够设计出可穿透细胞的单个激酶抑制剂，就可以系统地评估每个激酶的作用。
我们设计了一种结合化学和遗传学的方法，开发出了第一种针对致癌酪氨酸激酶v-Src的高度特异性可穿透细胞的抑制剂。在v-Src中引入一个功能沉默的活性位点突变，使其与所有其他细胞激酶区分开来。设计并合成了一种对该突变激酶具有紧密结合能力且不抑制野生型激酶的可穿透细胞的抑制剂。体外和全细胞实验确定了突变型v-Src-抑制剂对的独特特异性。该抑制剂可逆转由工程改造的而非“野生型”v-Src引起的细胞转化，这表明细胞信号传导的变化可归因于对工程改造激酶的特异性抑制。通过对另一种酪氨酸激酶Fyn进行工程改造，使其含有与v-Src中相应的活性位点突变，来测试该方法的通用性。抑制突变型v-Src的同一种化合物也能有效抑制工程改造的Fyn激酶。
可以通过一种应适用于所有激酶的方法快速开发出针对单个Src家族激酶的等位基因特异性可穿透细胞的抑制剂。这种方法将有助于对激酶介导的细胞通路进行解卷积，并在体外和体内验证新型激酶作为药物发现的良好靶点。
