Discovery and Exploitation of Inhibitor-resistant Aurora and Polo Kinase Mutants for the Analysis of Mitotic Networks
Discovery and Exploitation of Inhibitor-resistant Aurora and Polo Kinase Mutants for the Analysis of Mitotic Networks
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DOI：
10.1074/jbc.m109.005694
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发表时间：
2009-06-05
影响因子：
4.8
通讯作者：
Eyers, Patrick A.
中科院分区：
文献类型：
--
作者：
Scutt, Paul J.;Chu, Matthew L. H.;Eyers, Patrick A.
Aurora和Polo样激酶是有丝分裂信号通路的核心组成部分，最近的证据表明Aurora A和Plk 1之间存在大量的串扰。除了作为新型抗癌药物的验证外，小分子激酶抑制剂是帮助剖析临床相关蛋白磷酸化网络的越来越重要的工具。然而，与激酶抑制剂相关的一个主要问题是它们对激酶组的“脱靶”成员的混杂性，这使得从复杂细胞系统获得的数据的解释具有挑战性。此外，患者中抑制剂耐药性的出现清楚地表明，了解耐药机制对于药物设计至关重要。在这项研究中，我们利用VX-680，极光激酶抑制剂，BI 2536，Polo样激酶抑制剂的结合模式的结构知识，设计和评估耐药激酶突变体。使用可诱导的稳定人细胞系，我们验证了两种化合物的有丝分裂靶点，并证明Aurora A突变体对抑制剂VX-680和MLN 8054表现出不同的细胞敏感性。此外，我们验证了Aurora B作为VX-680在模型人癌细胞中的重要抗增殖靶点。最后，这种化学遗传学方法使我们能够证明Aurora A激活环磷酸化是由人类细胞中Plk 1介导的途径控制的。
The Aurora and Polo-like kinases are central components of mitotic signaling pathways, and recent evidence suggests that substantial cross-talk exists between Aurora A and Plk1. In addition to their validation as novel anticancer agents, small molecule kinase inhibitors are increasingly important tools to help dissect clinically relevant protein phosphorylation networks. However, one major problem associated with kinase inhibitors is their promiscuity toward "off-target" members of the kinome, which makes interpretation of data obtained from complex cellular systems challenging. Additionally, the emergence of inhibitor resistance in patients makes it clear that an understanding of resistance mechanisms is essential to inform drug design. In this study, we exploited structural knowledge of the binding modes of VX-680, an Aurora kinase inhibitor, and BI 2536, a Polo-like kinase inhibitor, to design and evaluate drug-resistant kinase mutants. Using inducible stable human cell lines, we authenticated mitotic targets for both compounds and demonstrated that Aurora A mutants exhibit differential cellular sensitivity toward the inhibitors VX-680 and MLN8054. In addition, we validated Aurora B as an important anti-proliferative target for VX-680 in model human cancer cells. Finally, this chemical genetic approach allowed us to prove that Aurora A activation loop phosphorylation is controlled by a Plk1-mediated pathway in human cells.