Tuning a Three-Component Reaction For Trapping Kinase Substrate Complexes
Tuning a Three-Component Reaction For Trapping Kinase Substrate Complexes
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DOI：
10.1021/ja807066f
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发表时间：
2008-12-24
影响因子：
15
通讯作者：
Shokat, Kevan M.
中科院分区：
文献类型：
--
作者：
Statsuk, Alexander V.;Maly, Dustin J.;Shokat, Kevan M.
The upstream protein kinases responsible for thousands of phosphorylation events in the phosphoproteome remain to be discovered. We developed a three-component chemical reaction which converts the transient noncovalent substrate-kinase complex into a covalently cross-linked product by utilizing a dialdehyde-based cross-linker, 1. Unfortunately, the reaction of 1 with a lysine in the kinase active site and an engineered cysteine on the substrate to form an isoindole cross-linked product could not be performed in the presence of competing cellular proteins due to nonspecific side reactions. In order to more selectively target the cross-linker to protein kinases in cell lysates, we replaced the weak, kinase-binding adenosine moiety of 1 with a potent protein kinase inhibitor scaffold. In addition, we replaced the o-phthaldialdehyde moiety in 1 with a less-reactive thiophene-2,3-dicarboxaldehyde moiety. The combination of these two structural modifications provides for cross-linking of a cysteine-containing substrate to its corresponding kinase in the presence of competing cellular proteins.
