Su Ying, Pan Sijun, Li Zhengqiu, Li Lin, Wu Xiaoyuan, Hao Piliang, Sze Siu Kwan, Yao Shao Q
Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543.
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551.
Sci Rep. 2015 Jan 12;5:7724. doi: 10.1038/srep07724.
MLN8237 is a highly potent and presumably selective inhibitor of Aurora kinase A (AKA) and has shown promising antitumor activities. Like other kinase inhibitors which target the ATP-binding site of kinases, MLN8237 might be expected to have potential cellular off-targets. Herein, we report the first photoaffinity-based, small molecule AKA probe capable of both live-cell imaging of AKA activities and in situ proteome profiling of potential off-targets of MLN8237 (including AKA-associating proteins). By using two mutually compatible, bioorthogonal reactions (copper-catalyzed azide-alkyne cycloaddition chemistry and TCO-tetrazine ligation), we demostrate small molecule-based multiplex bioimaging for simultaneous in situ monitoring of two important cell-cycle regulating kinases (AKA and CDK1). A broad range of proteins, as potential off-targets of MLN8237 and AKA's-interacting partners, is subsequently identified by affinity-based proteome profiling coupled with large-scale LC-MS/MS analysis. From these studies, we discover novel AKA interactions which were further validated by cell-based immunoprecipitation (IP) experiments.
MLN8237是一种高效且可能具有选择性的极光激酶A（AKA）抑制剂，并已显示出有前景的抗肿瘤活性。与其他靶向激酶ATP结合位点的激酶抑制剂一样，预计MLN8237可能存在潜在的细胞脱靶效应。在此，我们报道了首个基于光亲和性的小分子AKA探针，它既能对AKA活性进行活细胞成像，又能对MLN8237的潜在脱靶蛋白（包括与AKA相关的蛋白）进行原位蛋白质组分析。通过使用两种相互兼容的生物正交反应（铜催化的叠氮化物-炔烃环加成化学和反式环辛烯-四嗪连接反应），我们展示了基于小分子的多重生物成像技术，用于同时原位监测两种重要的细胞周期调节激酶（AKA和细胞周期蛋白依赖性激酶1）。随后，通过基于亲和性的蛋白质组分析结合大规模液相色谱-串联质谱分析，鉴定出了一系列作为MLN8237潜在脱靶蛋白以及AKA相互作用伴侣的蛋白质。通过这些研究，我们发现了新的AKA相互作用，并通过基于细胞的免疫沉淀（IP）实验进一步验证。
