Toward a Functional, ChemoProteomic Interrogation of Kinome and Nucleotide Binding Space

Protein kinases represent the single largest mammalian enzyme family with ~ 518 members in the human proteome and have been implicated in a wide array of complex cellular functions and pathways, ranging from metabolic regulation to tumorigenesis. The physiological functions of the majority these enzymes remain unknown. The central role of protein kinases in signal transduction has generated interest in targeting these enzymes for a wide range of therapeutic indications. We have reported a method for identifying and quantifying protein kinases in any biological sample from any species. The ActivX Activity Based Proteomics technology for kinase relies on acylphosphate nucleotide probes, prepared from biotin and ATP or ADP that react covalently at the ATP binding sites of virtually all known protein kinases. Biotinylated peptide fragments from labeled proteomes are captured, sequenced and identified using a mass‐spectrometry based analysis platform to determine the kinases present and their relative levels. Direct competition between the probes and inhibitors can be measured to determine inhibitor potency and selectivity against native protein kinases, as well as hundreds of other ATPases. The ability to broadly profile kinase activities in native proteomes offers an exciting prospect for both target discovery and inhibitor selectivity profiling. In this presentation, the development of the acylphosphate nucleotide probes and the scope of the reaction with the kinome and other nucleotide binding proteins will be discussed. In addition, other issues that speak to the breadth and versatility of this method will be addressed. The proteome‐specific kinase selectivity by drugs will also be demonstrated. Finally, the potential of the approach in other areas of nucleotide binding space, such as the GTP‐dependent enzymes, will be demonstrated.