Illuminating function of the understudied druggable kinome

Abstract

The human kinome, with more than 500 proteins, is crucial for cell signaling and disease. Yet, about one-third of kinases lack in-depth study. The Data and Resource Generating Center for Understudied Kinases has developed multiple resources to address this challenge including creation of a heavy amino acid peptide library for parallel reaction monitoring and quantitation of protein kinase expression, use of understudied kinases tagged with a miniTurbo-biotin ligase to determine interaction networks by proximity-dependent protein biotinylation, NanoBRET probe development for screening chemical tool target specificity in live cells, characterization of small molecule chemical tools inhibiting understudied kinases, and computational tools for defining kinome architecture. These resources are available through the Dark Kinase Knowledgebase, supporting further research into these understudied protein kinases.

Keywords: NanoBRET; SureQuant/PRM; chemical tools; dark kinase knowledgebase; human kinome; illuminating the druggable genome; miniTurbo; understudied kinases.

FIGURE 1

The understudied kinome represented by 162 kinases in all subfamilies of the human kinome. The Data and Resource Generating Center for Understudied Kinases used an integrated research strategy involving proteomics, genomics, chemical biology and computational modeling to provide new tools to illuminate function of the understudied kinases and their integration in signaling networks of the human kinome. Coloring is linked to branch of kinome phylogenetic tree (Tree generated by Coral).

FIGURE 2

Tools for quantifying kinase expression and proximity networks. (a) A comprehensive PRM heavy peptide library to quantitate attomole–femtomole protein expression levels of human kinases via mass spectrometry has been generated. The PRM library currently includes high purity peptides for 102 understudied kinases (green nodes) and low purity peptides for 60 understudied kinases (magenta nodes). Peptides have been identified for all 60 understudied kinases from low purity samples that are suitable for high purity peptide synthesis. All 162 understudied kinases will be represented in the PRM heavy peptide library that will have ~400 illuminated and understudied kinases. The PRM heavy peptide library is also being expanded with high purity peptides for illuminated kinases currently not represented, giving a comprehensive human kinome SureQuant/PRM library. To date, established LLOQ and linearity of concentration for ~700 heavy peptides has been completed. Data for all peptide analyses will be on the darkkinome.org and submitted to the Clinical Proteomic Tumor Analysis Consortium (CPTAC) assay portal. (b) Determining the protein neighborhood of understudied kinases for illuminating function and phenotypic cellular regulation. Distribution of understudied kinases across the human kinome with protein–protein interaction networks completed (blue nodes), in progress (yellow nodes) or without networks (smaller gray nodes). A total of 127 understudied kinases have miniTurbo-based proximity biotinylation analysis completed with an additional 5 kinases in progress (trees generated by Coral).

FIGURE 3

Live cell kinase NanoBRET assay. (a) Kinase-Nluc fusion is expressed in HEK293 cells. Kinase inhibitor linked to bodipy fluorophore is used as a tracer. Furimazine is the optimized NLuc substrate. Interaction of the photon with the fluorescent tracer results in a BRET signal. (b) Titration with a kinase inhibitor is used to determine the optimal tracer concentration for the NanoBRET assay. In this example, titration for CDK18-Nluc/cyclin Y using the fluorescent tracer from panel (a) is used with AT-7519 as the CDK inhibitor.

FIGURE 4

Establishing a broader functional network view of the kinome. (a) A literature generated network for NEK7 via INDRA. Connecting lines represent physical interactions (blue), activating (green) and inhibiting (red) relationships, and modifications (black). (b) Sample subview of the broader set of interactions existing between kinases (large circles) and associated protein substrates (small circles). (c) Example of high-level identification of hierarchies of kinase subnetworks based on application of community detection approaches within interaction and functional data sets (green circles represent understudied kinases/groups).

FIGURE 5

Summary of kinase DRGC resources being integrated for illumination of understudied kinases and functional kinome signaling networks.