Tuning a Three-Component Reaction For Trapping Kinase Substrate Complexes

Abstract

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 non-covalent 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 proteinsdue to non-specific 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.

Oncogene Mimicry as a Mechanism of Primary Resistance to BRAF Inhibitors

“…Linkable versions of previously described compounds were synthesized based on prior methods; VI-16832l (Daub et al, 2008), Akti-46 (Blake et al, 2010), PP-hydroxyl (Tanaka et al, 2005), sorafenib (Bankston et al, 2001), and JG-4 (Statsuk et al, 2008); purvalanol B was coupled to ether-linked 1,6-diaminohexane Sepharose and all others to ECH following prior methods, with quenching with acetic acid or ethanolamine, respectively (Duncan et al, 2012). Kinase enrichment was performed as described previously (Cooper et al, 2013; Duncan et al, 2012).…”

The Genesis of Tyrosine Phosphorylation

“…One such method involves cross-linking of a catalytic domain to substrate proteins bound to it in the cell; this is achieved by making a Cys substitution mutation at the substrate phosphorylation site, and then using a cross-linker designed to covalently couple the Cys to the Lys in the ATP-binding site, followed by MS identification of the kinase bound to the tagged substrate (Statsuk et al 2008). In another method, an analog-sensitive PK mutant is used to thiophosphorylate substrates in vitro using g-S-ATP, and then thiophosphorylated residues are modified with an adduct that can be recognized by an antibody, allowing the modified peptides to be immunoaffinity enriched and identified by MS (Allen et al 2007).…”

Chemical Methods for Encoding and Decoding of Posttranslational Modifications

“…Again chemistry contributed to solving this problem, in particular with the development of several small molecule cross-linkers. One class of these compounds relies on a three-component reaction between a substrate protein (or peptide) of interest, the small-molecule, and the conserved active-site lysine residue of a kinase (Maly et al, 2004; Riel-Mehan and Shokat, 2014; Statsuk et al, 2008). All of these cross-linkers rely on the genetic incorporation of a cysteine at the normal site of phosphorylation in the substrate.…”