Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California, 600 16th Street, MC 2280, San Francisco, CA 94158, USA.
Proc Natl Acad Sci U S A. 2011 Sep 13;108(37):15046-52. doi: 10.1073/pnas.1111239108. Epub 2011 Aug 18.
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.
500 个人类激酶的 ATP 结合位点具有保守性，这使得开发特异性抑制剂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，用于克服特异性挑战，利用了看门残基（保守的疏水性氨基酸）的大到小突变，并使用大体积抑制剂通过形状互补性来实现特异性。然而，在许多情况下，引入甘氨酸或丙氨酸看门残基会导致激酶活性和 ATP 亲和力降低。基于工程化看门半胱氨酸与亲电抑制剂之间的共价互补性的新化学遗传方法被开发出来以应对这些挑战。该策略用于评估原癌基因酪氨酸激酶Src，已知使用形状互补化学遗传策略会丧失一些酶活性。我们发现，带有半胱氨酸看门残基的Src 重现了野生型活性，并且可以在体外和细胞中不可逆地抑制。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被解决，以阐明抑制剂结合模式。对 307 种激酶和 MOK（MAPK/MAK/MRK 重叠激酶）（仅有的两种具有内源性半胱氨酸看门残基的人类激酶之一）进行了一组亲电抑制剂的分析。该分析显示靶标很少，这使得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，通过二次位点突变可以提高抑制剂的效力。这些结果表明，基于共价互补性的化学遗传策略应该广泛适用于蛋白激酶的研究。
