PO.ET01.04 · 实验与分子治疗
通过新型全面磷酸化谱分析平台揭示ALK抑制剂的脱靶效应与其不良反应的关联
Association of off targets of ALK inhibitors with their adverse reactions revealed by a novel comprehensive phosphorylation profiling platform
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摘要 Abstract
中文摘要
引言：目前有五种ALK抑制剂获批临床使用。尽管它们均靶向ALK融合蛋白，但其副作用谱差异显著。这些差异被怀疑源于脱靶激酶相互作用，但分子层面的关联仍不清楚。我们旨在使用一种新型的基于磷酸化的谱分析系统来揭示这一关联，该系统全面绘制激酶-底物关系和通路激活。
材料与方法：使用最近开发的无细胞磷酸化阵列，我们测量了细胞内的整体磷酸化活性。该系统不聚焦于特定磷酸化位点；相反，它从1,492种蛋白（190种激酶的816种底物和构成273条通路的845种蛋白）的库中，鉴定在每种实验条件下发生磷酸化的底物。将经ALK抑制剂处理的细胞裂解物应用于阵列，实现模拟细胞内激酶活性的磷酸化反应。计算分析提取了三个层面的信息：条件特异性磷酸化底物、被激活的通路和激酶，从而能够同时评估靶向和脱靶效应。
结果：在EML4-ALK突变细胞系（NCI-H2228 [H2228]）中测量了五种ALK抑制剂诱导的磷酸化谱，并跨底物、激酶和通路进行分析。每种药物均表现出独特的抑制特征，指示了在ALK之外被选择性影响的特定激酶和通路。若干脱靶激酶和通路与已知的临床不良反应相关，为药物特异性毒性提供了机制层面的见解。
结论：这一全面的磷酸化谱分析平台能够同时评估激酶抑制剂的靶向和脱靶效应。通过将分子信号变化与临床副作用相关联，该系统为理解复杂的激酶网络以及指导更安全的下一代抑制剂的开发提供了一种有力的方法。
查看英文原文 English abstract
Introduction : Five ALK inhibitors are currently approved for clinical use. Although all target ALK fusion proteins, their side-effect profiles differ markedly. These variations are suspected to result from off-target kinase interactions, but the molecular links remain unclear. We aimed to reveal this connection using a novel phosphorylation-based profiling system that comprehensively maps kinase-substrate relationships and pathway activations.
Materials and Methods : Using a recently developed cell-free phosphorylation array, we measured the overall phosphorylation activity within cells. This system does not focus on specific phosphorylation sites; instead, it identifies substrates that become phosphorylated under each experimental condition from a pool of 1,492 proteins (816 substrates for 190 kinases and 845 proteins constituting 273 pathways).Cell lysates treated with ALK inhibitors were applied to the array, enabling phosphorylation reactions that mimic intracellular kinase activity. Computational analyses extracted three layers of information, condition-specifically phosphorylated substrates, activated pathways and kinases, allowing simultaneous assessment of both on-target and off-target effects.
Results : Phosphorylation profiles induced by five ALK inhibitors were measured in EML4-ALK mutant cell line (NCI-H2228 [H2228]), and analyzed across substrates, kinases, and pathways. Each drug exhibited a distinct inhibition signature, indicating specific kinases and pathways selectively affected beyond ALK. Several off-target kinases and pathways associated with known clinical adverse reactions, providing mechanistic insight into drug-specific toxicities.
Conclusion : This comprehensive phosphorylation profiling platform enabled simultaneous evaluation of on-target and off-target effects of kinase inhibitors. By linking molecular signaling changes to clinical side effects, the system provides a powerful approach for understanding complex kinase networks and guiding the development of safer next-generation inhibitors.
利益披露 Disclosure
K. Horimoto,
SOCIUM Inc. Stock.
S. Mikami,
SOCIUM Inc. Employment.
L. Feng, None..
Y. Zhang, None..
D. Chen, None..
F. Han, None..
S. Yamasaki, None..
S. Takei, None..
H. Kagamu, None.