Competitive Kinase Enrichment Proteomics Reveals that Abemaciclib Inhibits GSK3β and Activates WNT Signaling.
Competitive Kinase Enrichment Proteomics Reveals that Abemaciclib Inhibits GSK3β and Activates WNT Signaling.
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DOI：
10.1158/1541-7786.mcr-17-0468
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发表时间：
2018-03
期刊：
影响因子：
--
通讯作者：
Major MB
中科院分区：
文献类型：
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作者：
Cousins EM;Goldfarb D;Yan F;Roques J;Darr D;Johnson GL;Major MB
对小分子激酶抑制剂的细胞和生物体表型反应由抑制剂的靶标及其功能共同定义。小分子激酶抑制剂的选择性通常是使用纯化的激酶和底物在体外测定的。最近，竞争性化学蛋白质组学作为一种互补的、公正的、基于细胞的方法出现，用于定义激酶抑制剂的靶标景观。在这里，我们使用细胞裂解物、活细胞和治疗小鼠评估和优化了竞争性多重抑制剂珠质谱 (MIB/MS) 平台。对几种临床活性激酶抑制剂进行了分析，包括曲美替尼、BMS-777607、达沙替尼、abemaciclib 和 palbociclib。细胞周期蛋白依赖性激酶 4 和 6 (CDK4/6) 抑制剂 abemaciclib 和 palbociclib 的 MIB/MS 竞争分析揭示了重叠且独特的激酶靶点。 abemaciclib 的竞争性 MIB/MS 分析显示了 83 种靶激酶，剂量反应 MIB/MS 分析显示糖原合酶激酶 3 α 和 β（GSK3α 和 β）以及 Ca2+/钙调蛋白依赖性蛋白激酶 II δ 和 γ（CAMKIIδ 和 γ）受到最有效的抑制。基于细胞和体外激酶测定表明，与 palbociclib 相比，abemaciclib 在低纳摩尔浓度下直接抑制 GSK3α/β 和 CAMKIIγ/δ 激酶活性。 GSK3β 磷酸化 β-连环蛋白以抑制 WNT 信号传导，而 abemaciclib（但不是 palbociclib 或 ribociclib）可有效激活 β-连环蛋白依赖性 WNT 信号传导。这些数据说明了竞争性化学蛋白质组学在定义激酶抑制剂的激酶靶标特异性方面的力量，从而为临床疗效、剂量限制毒性和药物再利用工作提供信息。
The cellular and organismal phenotypic response to a small-molecule kinase inhibitor is defined collectively by the inhibitor’s targets and their functions. The selectivity of small-molecule kinase inhibitors is commonly determined in vitro, using purified kinases and substrates. Recently, competitive chemical proteomics has emerged as a complementary, unbiased, cell-based methodology to define the target landscape of kinase inhibitors. Here, we evaluated and optimized a competitive multiplexed inhibitor bead mass spectrometry (MIB/MS) platform using cell lysates, live cells, and treated mice. Several clinically active kinase inhibitors were profiled, including trametinib, BMS-777607, dasatinib, abemaciclib, and palbociclib. MIB/MS competition analyses of the cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors abemaciclib and palbociclib revealed overlapping and unique kinase targets. Competitive MIB/MS analysis of abemaciclib revealed 83 target kinases, and dose-response MIB/MS profiling revealed glycogen synthase kinase 3 alpha and beta (GSK3α and β) and Ca2+/calmodulin-dependent protein kinase II delta and gamma (CAMKIIδ and γ) as the most potently inhibited. Cell-based and in vitro kinase assays show that in contrast to palbociclib, abemaciclib directly inhibits GSK3α/β and CAMKIIγ/δ kinase activity at low nanomolar concentrations. GSK3β phosphorylates β-catenin to suppress WNT signaling, while abemaciclib (but not palbociclib or ribociclib) potently activates β-catenin-dependent WNT signaling. These data illustrate the power of competitive chemical proteomics to define kinase target specificities for kinase inhibitors, thus informing clinical efficacy, dose-limiting toxicities, and drug-repurposing efforts.