Tuning a Three-Component Reaction For Trapping Kinase Substrate Complexes

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• 作者：Alexander V. Statsuk ; Dustin J. Maly ; Markus A. Seeliger ; Miles A. Fabian ; William H. Biggs ; ; David J. Lockhart ; Patrick P. Zarrinkar ; John Kuriyan ; Kevan M. Shokat
• 刊名：Journal of the American Chemical Society
• 出版年：2008
• 出版时间：December 24, 2008
• 年：2008
• 卷：130
• 期：51
• 页码：17568-17574
• 全文大小：328K
• 年卷期：v.130,no.51(December 24, 2008)
• ISSN：1520-5126

文摘

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.