Chemical proteomics reveals target selectivity of clinical Jak inhibitors in human primary cells
Chemical proteomics reveals target selectivity of clinical Jak inhibitors in human primary cells
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DOI：
10.1038/s41598-019-50335-5
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发表时间：
2019-10-02
影响因子：
4.6
通讯作者：
Bantscheff, Marcus
中科院分区：
文献类型：
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作者：
Eberl, H. Christian;Werner, Thilo;Bantscheff, Marcus
Kinobeads是一组固定在琼脂糖凝胶珠上的混杂激酶抑制剂，用于从细胞系和组织中全面富集内源性表达的蛋白激酶。这些微珠能够在剂量依赖性竞争研究中结合定量质谱法对感兴趣的激酶抑制剂进行化学蛋白质组学分析。我们提出了改进的珠基质，捕获超过350个蛋白激酶和15个脂质激酶从人类细胞裂解物，分别。多重策略的建议，使在一个单一的质谱实验中的表观解离常数的测定。该程序的小型化使得能够确定临床BCR-ABL抑制剂达沙替尼在来自个体供体的外周血单核细胞（PBMC）裂解物中的靶选择性。对一组Jak激酶抑制剂的分析揭示了来自几乎所有激酶家族的激酶脱靶，这支持了针对激酶组分析激酶抑制剂的需要。临床抑制剂的有效结合脱靶提示了多药理学，例如通过MRCK α和β，其以纳摩尔亲和力与地赛诺替尼结合。
Kinobeads are a set of promiscuous kinase inhibitors immobilized on sepharose beads for the comprehensive enrichment of endogenously expressed protein kinases from cell lines and tissues. These beads enable chemoproteomics profiling of kinase inhibitors of interest in dose-dependent competition studies in combination with quantitative mass spectrometry. We present improved bead matrices that capture more than 350 protein kinases and 15 lipid kinases from human cell lysates, respectively. A multiplexing strategy is suggested that enables determination of apparent dissociation constants in a single mass spectrometry experiment. Miniaturization of the procedure enabled determining the target selectivity of the clinical BCR-ABL inhibitor dasatinib in peripheral blood mononuclear cell (PBMC) lysates from individual donors. Profiling of a set of Jak kinase inhibitors revealed kinase off-targets from nearly all kinase families underpinning the need to profile kinase inhibitors against the kinome. Potently bound off-targets of clinical inhibitors suggest polypharmacology, e.g. through MRCK alpha and beta, which bind to decernotinib with nanomolar affinity.