A Cell Biologist's Field Guide to Aurora Kinase Inhibitors.
A Cell Biologist's Field Guide to Aurora Kinase Inhibitors.
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DOI：
10.3389/fonc.2015.00285
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发表时间：
2015
影响因子：
4.7
通讯作者：
Shiau AK
中科院分区：
文献类型：
--
作者：
de Groot CO;Hsia JE;Anzola JV;Motamedi A;Yoon M;Wong YL;Jenkins D;Lee HJ;Martinez MB;Davis RL;Gahman TC;Desai A;Shiau AK
极光激酶对于细胞分裂至关重要，并且在人类癌症中经常被错误调节。基于其作为癌症治疗的潜力，人们开发了多种小分子极光激酶抑制剂，其中一部分已被用作细胞生物学的工具。在这里，我们通过使用生化和基于细胞的检测系统地分析了一组 10 种市售化合物，这些化合物对 Aurora A（MLN8054、MLN8237、MK-5108、MK-8745、Genentech Aurora Inhibitor 1）、Aurora B（Hesperadin、ZM447439、 AZD1152-HQPA、GSK1070916) 或 Aurora A/B (VX-680)。我们量化了每种抑制剂对单独的 Aurora A 活性以及分别与其激活剂 TPX2 和 INCENP 片段结合的 Aurora A 和 Aurora B 活性的体外影响。我们还报告了这些化合物的一部分的激酶组分析结果，以突出潜在的脱靶效应。在细胞环境中，我们证明基于免疫荧光的 LATS2 和组蛋白 H3 磷酸表位检测提供了一种简便可靠的方法来评估 Aurora A 与 Aurora B 抑制的效力和特异性，并且在实时成像测定中测量的 G2 持续时间是 Aurora A 活性的特定读数。我们的分析还强调了 HeLa、U2OS 和 hTERT-RPE1 细胞之间的差异，这些差异会影响选择性 Aurora A 抑制。对于 Aurora B，所有四种测试化合物均表现出优异的选择性，并且在有效剂量下不会显着抑制 Aurora A。对于 Aurora A，MK-5108 和 MK-8745 的选择性显着高于常用抑制剂 MLN8054 和 MLN8237。我们确定的 Aurora A/MK-5108 复合物的晶体结构表明了这种更高特异性的化学基础。总而言之，我们的定量生化和基于细胞的分析表明 AZD1152-HQPA 和 MK-8745 分别是目前选择性抑制 Aurora B 和 Aurora A 的最佳工具。然而，MK-8745 并不像 AZD1152-HQPA 那样理想，因为它需要高浓度才能在细胞环境中实现完全抑制，这表明需要更有效的 Aurora A 选择性抑制剂。最后，我们提出了一套在细胞生物学实验中使用 Aurora 抑制剂的“良好实践”指南。
Aurora kinases are essential for cell division and are frequently misregulated in human cancers. Based on their potential as cancer therapeutics, a plethora of small molecule Aurora kinase inhibitors have been developed, with a subset having been adopted as tools in cell biology. Here, we fill a gap in the characterization of Aurora kinase inhibitors by using biochemical and cell-based assays to systematically profile a panel of 10 commercially available compounds with reported selectivity for Aurora A (MLN8054, MLN8237, MK-5108, MK-8745, Genentech Aurora Inhibitor 1), Aurora B (Hesperadin, ZM447439, AZD1152-HQPA, GSK1070916), or Aurora A/B (VX-680). We quantify the in vitro effect of each inhibitor on the activity of Aurora A alone, as well as Aurora A and Aurora B bound to fragments of their activators, TPX2 and INCENP, respectively. We also report kinome profiling results for a subset of these compounds to highlight potential off-target effects. In a cellular context, we demonstrate that immunofluorescence-based detection of LATS2 and histone H3 phospho-epitopes provides a facile and reliable means to assess potency and specificity of Aurora A versus Aurora B inhibition, and that G2 duration measured in a live imaging assay is a specific readout of Aurora A activity. Our analysis also highlights variation between HeLa, U2OS, and hTERT-RPE1 cells that impacts selective Aurora A inhibition. For Aurora B, all four tested compounds exhibit excellent selectivity and do not significantly inhibit Aurora A at effective doses. For Aurora A, MK-5108 and MK-8745 are significantly more selective than the commonly used inhibitors MLN8054 and MLN8237. A crystal structure of an Aurora A/MK-5108 complex that we determined suggests the chemical basis for this higher specificity. Taken together, our quantitative biochemical and cell-based analyses indicate that AZD1152-HQPA and MK-8745 are the best current tools for selectively inhibiting Aurora B and Aurora A, respectively. However, MK-8745 is not nearly as ideal as AZD1152-HQPA in that it requires high concentrations to achieve full inhibition in a cellular context, indicating a need for more potent Aurora A-selective inhibitors. We conclude with a set of “good practice” guidelines for the use of Aurora inhibitors in cell biology experiments.