Characterization of the Novel Broad-Spectrum Kinase Inhibitor CTx-0294885 As an Affinity Reagent for Mass Spectrometry-Based Kinome Profiling

Abstract

Kinase enrichment utilizing broad-spectrum kinase inhibitors enables the identification of large proportions of the expressed kinome by mass spectrometry. However, the existing inhibitors are still inadequate in covering the entire kinome. Here, we identified a novel bisanilino pyrimidine, CTx-0294885, exhibiting inhibitory activity against a broad range of kinases in vitro, and further developed it into a Sepharose-supported kinase capture reagent. Use of a quantitative proteomics approach confirmed the selectivity of CTx-0294885-bound beads for kinase enrichment. Large-scale CTx-0294885-based affinity purification followed by LC-MS/MS led to the identification of 235 protein kinases from MDA-MB-231 cells, including all members of the AKT family that had not been previously detected by other broad-spectrum kinase inhibitors. Addition of CTx-0294885 to a mixture of three kinase inhibitors commonly used for kinase-enrichment increased the number of kinase identifications to 261, representing the largest kinome coverage from a single cell line reported to date. Coupling phosphopeptide enrichment with affinity purification using the four inhibitors enabled the identification of 799 high-confidence phosphosites on 183 kinases, ∼10% of which were localized to the activation loop, and included previously unreported phosphosites on BMP2K, MELK, HIPK2, and PRKDC. Therefore, CTx-0294885 represents a powerful new reagent for analysis of kinome signaling networks that may facilitate development of targeted therapeutic strategies. Proteomics data have been deposited to the ProteomeXchange Consortium ( http://proteomecentral.proteomexchange.org ) via the PRIDE partner repository with the data set identifier PXD000239.

Kinobead and Single-Shot LC-MS Profiling Identifies Selective PKD Inhibitors

“…1a). 5, 24, 28, 33–35 The inhibitor analogs 1 and 3–5 were described previously and the affinity reagents 2 , 6 and 7 were newly developed in our laboratory. Compounds 1–7 were immobilized on carboxy-derivatized sepharose using amide coupling chemistry to yield the corresponding affinity matrices.…”

Conversion of a Single Polypharmacological Agent into Selective Bivalent Inhibitors of Intracellular Kinase Activity

“…To our knowledge, this represents the largest swath of the kinome to be profiled by a single compound via chemical proteomics; narrowly edging CTx-0294885. 24 For 182 protein kinases, a relative affinity for 2 could be estimated by calculating the concentration at which 50% of a given protein remained bound to the affinity matrix (residual binding (RB 50 ) value). As depicted in Figure 3B, full-length protein kinases from the various branches of the kinome tree exhibit measurable affinity for 2 .…”

Optimized Chemical Proteomics Assay for Kinase Inhibitor Profiling

“…It was described to capture more than half of the expressed kinome in MDA-MB-231 and, therefore, could be a beneficial addition to the affinity probe repertoire. 28 Hence we synthesized CTx-0294885 and compared its kinase binding potential to probe 19 and KBγ (Supplemental File 3). The kinase profiles of CTx-0294885 and probe 19 were comparable with differences in detail ( Figure 5C, 5D).…”

“…[17][18][19] Examples for such ideas are drug-centric profiling, [20][21][22] the KiNativ technology and other covalent acyl nucleotide probes, 23,24 the Kinobeads technology 25,26 or other multiplexed kinase inhibitors beads. 27,28 Today, these experiments are typically analyzed using quantitative mass spectrometry as an assay readout. 29 One attraction of chemical proteomic approaches for kinase inhibitor profiling is that it promotes serendipitous finding as less studied or unexpected kinases are more likely to be identified in an unbiased way.…”