Chemical genetic strategy for targeting protein kinases based on covalent complementarity
Chemical genetic strategy for targeting protein kinases based on covalent complementarity
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基于共价互补的靶向蛋白激酶化学遗传学策略
DOI：
10.1073/pnas.1111239108
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发表时间：
2011-09-13
影响因子：
11.1
通讯作者：
Shokat, Kevan M.
中科院分区：
文献类型：
--
作者：
Garske, Adam L.;Peters, Ulf;Shokat, Kevan M.
超过 500 种人类激酶的 ATP 结合位点的保守性使得特定抑制剂的开发成为一项具有挑战性的任务。一种广泛使用的化学遗传策略来克服特异性挑战，利用守门残基（一种保守的疏水氨基酸）的大到小突变，并使用大的抑制剂通过形状互补来实现特异性。然而，在许多情况下，引入甘氨酸或丙氨酸看门人会导致激酶活性和 ATP 亲和力降低。开发了一种基于工程守门半胱氨酸和亲电抑制剂之间共价互补性的新化学遗传方法来应对这些挑战。该策略用 Src 进行了评估，Src 是一种原癌酪氨酸激酶，已知使用形状互补化学遗传策略会失去一些酶活性。我们发现带有半胱氨酸看门人的 Src 重现了野生型活性，并且在体外和细胞内都可以被不可逆地抑制。解析了 T338C c-Src 与乙烯基磺酰胺衍生的吡唑并嘧啶抑制剂的共晶结构，以阐明抑制剂的结合模式。针对 307 种激酶和 MOK（MAPK/MAK/MRK 重叠激酶）对一组亲电子抑制剂进行了分析，MOK 是已知具有内源半胱氨酸看门人的仅有的两种人类激酶之一。这项分析显示脱靶现象非常少，使这些化合物成为迄今为止报道的最具选择性的化学遗传抑制剂。蛋白质工程研究表明，通过二级位点突变可以提高抑制剂的效力。这些结果表明基于共价互补的化学遗传策略应该广泛适用于蛋白激酶的研究。
The conserved nature of the ATP-binding site of the >500 human kinases renders the development of specific inhibitors a challenging task. A widely used chemical genetic strategy to overcome the specificity challenge exploits a large-to-small mutation of the gatekeeper residue (a conserved hydrophobic amino acid) and the use of a bulky inhibitor to achieve specificity via shape complementarity. However, in a number of cases, introduction of a glycine or alanine gatekeeper results in diminished kinase activity and ATP affinity. A new chemical genetic approach based on covalent complementarity between an engineered gatekeeper cysteine and an electrophilic inhibitor was developed to address these challenges. This strategy was evaluated with Src, a proto-oncogenic tyrosine kinase known to lose some enzymatic activity using the shape complementarity chemical genetic strategy. We found that Src with a cysteine gatekeeper recapitulates wild type activity and can be irreversibly inhibited both in vitro and in cells. A cocrystal structure of T338C c-Src with a vinylsulfonamide-derivatized pyrazolopyrimidine inhibitor was solved to elucidate the inhibitor binding mode. A panel of electrophilic inhibitors was analyzed against 307 kinases and MOK (MAPK/MAK/MRK overlapping kinase), one of only two human kinases known to have an endogenous cysteine gatekeeper. This analysis revealed remarkably few off-targets, making these compounds the most selective chemical genetic inhibitors reported to date. Protein engineering studies demonstrated that it is possible to increase inhibitor potency through secondary-site mutations. These results suggest that chemical genetic strategies based on covalent complementarity should be widely applicable to the study of protein kinases.