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

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.

利用广谱激酶抑制剂开展激酶富集，可通过质谱（mass spectrometry）鉴定表达激酶组(kinome)的大部分组分。然而，现有抑制剂仍无法覆盖完整的激酶组。本研究中，我们鉴定出一种新型双苯胺基嘧啶类化合物CTx-0294885，其在体外对多种激酶展现出抑制活性，并将其开发为琼脂糖凝胶(Sepharose)锚定的激酶捕获试剂。借助定量蛋白质组学(quantitative proteomics)方法，我们证实了偶联CTx-0294885的亲和微珠具备优异的激酶富集特异性。基于CTx-0294885的大规模亲和纯化结合液相色谱-串联质谱（LC–MS/MS）分析，我们从MDA-MB-231细胞中鉴定出235种蛋白激酶，其中包括此前未被其他广谱激酶抑制剂检出的AKT家族全部成员。将CTx-0294885加入三种常用于激酶富集的抑制剂混合体系后，可鉴定的激酶数量提升至261种，这是迄今为止单细胞系中报道的最大激酶组覆盖范围。将磷酸肽(phosphopeptide)富集与四种抑制剂介导的亲和纯化相结合，我们在183种激酶上鉴定出799个高置信度磷酸化位点(phosphosites)，其中约10%定位于激活环(activation loop)，且包含此前未被报道的BMP2K、MELK、HIPK2及PRKDC上的磷酸化位点。因此，CTx-0294885是一款用于激酶组信号网络分析的高效新型试剂，有望助力靶向治疗策略的开发。本研究的蛋白质组学数据已通过PRIDE合作数据库提交至蛋白质组交换联盟（ProteomeXchange Consortium，http://proteomecentral.proteomexchange.org），数据集标识符为PXD000239。