基于质谱的选择性分析可确定一种高度选择性的激酶MELK抑制剂，该抑制剂可延缓癌细胞中的有丝分裂进入。,Journal of Biological Chemistry

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基于质谱的选择性分析可确定一种高度选择性的激酶MELK抑制剂，该抑制剂可延缓癌细胞中的有丝分裂进入。
Journal of Biological Chemistry ( IF 4.0 ) Pub Date : 2020-01-02 , DOI: 10.1074/jbc.ra119.011083
Ian M McDonald ₁ , Gavin D Grant ₂ , Michael P East ₁ , Thomas S K Gilbert ₃ , Emily M Wilkerson ₃ , Dennis Goldfarb ₄ , Joshua Beri ₃ , Laura E Herring ₃ , Cyrus Vaziri ₅ , Jeanette Gowen Cook ₂ , Michael J Emanuele ₁ , Lee M Graves ₆

Affiliation

Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599; Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599.
Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599; Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599.
Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599; UNC Michael Hooker Proteomics Core Facility, University of North Carolina, Chapel Hill, North Carolina 27599.
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110; Institute for Informatics, Washington University School of Medicine, St. Louis, Missouri 63110.
Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599; Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, North Carolina 27599.
Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599; Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599; UNC Michael Hooker Proteomics Core Facility, University of North Carolina, Chapel Hill, North Carolina 27599.

母体胚胎亮氨酸拉链激酶（MELK）已参与癌细胞增殖的调控。RNAi介导的MELK耗损会损害生长并导致许多癌症中的G2 / M阻滞，但对这些作用的潜在机制了解甚少。此外，最近已证明MELK抑制剂OTSSP167对MELK的选择性差，这使该抑制剂作为研究MELK功能的工具化合物的使用变得复杂。在这里，我们使用基于细胞的蛋白质组学技术（称为多重激酶抑制剂珠/质谱（MIB / MS）），我们分析了另外两种MELK抑制剂NVS-MELK8a（8a）和HTH-01-091的选择性。我们的结果表明8a是高度选择性的MELK抑制剂，我们将其进一步用于功能研究。刃天青和结晶紫测定表明8a降低了三阴性乳腺癌细胞的活力，免疫印迹表明生长受损是由于细胞周期进程的扰动而不是细胞凋亡的诱导。使用双胸苷同步和免疫印迹，我们观察到MELK抑制作用可延迟有丝分裂进入，这与Aurora A，Aurora B和细胞周期蛋白依赖性激酶1（CDK1）的激活延迟有关。延迟之后，细胞进入并完成有丝分裂。使用包含荧光增殖细胞核抗原的细胞的活细胞显微镜检查，我们证实8a显着且剂量依赖性延长了G2期。总的来说，我们的结果为使用8a作为MELK功能研究的工具化合物提供了理论依据，并表明MELK抑制作用可延迟有丝分裂进入，

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Mass spectrometry-based selectivity profiling identifies a highly selective inhibitor of the kinase MELK that delays mitotic entry in cancer cells.

The maternal embryonic leucine zipper kinase (MELK) has been implicated in the regulation of cancer cell proliferation. RNAi-mediated MELK depletion impairs growth and causes G2/M arrest in numerous cancers, but the mechanisms underlying these effects are poorly understood. Furthermore, the MELK inhibitor OTSSP167 has recently been shown to have poor selectivity for MELK, complicating the use of this inhibitor as a tool compound to investigate MELK function. Here, using a cell-based proteomics technique called multiplexed kinase inhibitor beads/mass spectrometry (MIB/MS), we profiled the selectivity of two additional MELK inhibitors, NVS-MELK8a (8a) and HTH-01-091. Our results revealed that 8a is a highly selective MELK inhibitor, which we further used for functional studies. Resazurin and crystal violet assays indicated that 8a decreases triple-negative breast cancer cell viability, and immunoblotting revealed that impaired growth is due to perturbation of cell cycle progression rather than induction of apoptosis. Using double-thymidine synchronization and immunoblotting, we observed that MELK inhibition delays mitotic entry, which was associated with delayed activation of Aurora A, Aurora B, and cyclin-dependent kinase 1 (CDK1). Following this delay, cells entered and completed mitosis. Using live-cell microscopy of cells harboring fluorescent proliferating cell nuclear antigen, we confirmed that 8a significantly and dose-dependently lengthens G2 phase. Collectively, our results provide a rationale for using 8a as a tool compound for functional studies of MELK and indicate that MELK inhibition delays mitotic entry, likely via transient G2/M checkpoint activation.

更新日期：2020-02-21

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