Inhibitors of CLK protein kinases suppress cell growth and induce apoptosis by modulating pre-mRNA splicing.
Inhibitors of CLK protein kinases suppress cell growth and induce apoptosis by modulating pre-mRNA splicing.
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DOI：
10.1371/journal.pone.0116929
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Nakanishi O
中科院分区：
文献类型：
--
作者：
Araki S;Dairiki R;Nakayama Y;Murai A;Miyashita R;Iwatani M;Nomura T;Nakanishi O
越来越多的证据表明，选择性剪接在各种生理过程中的重要性，包括不同疾病的发展。cdc样激酶（clk）和丝氨酸精氨酸蛋白激酶（SRPKs）是剪接机制的组成部分，对外显子选择至关重要。针对这些激酶的小分子抑制剂的发现具有重要的价值，不仅可以描述剪接的分子机制，而且还可以确定潜在的治疗机会。在这里，我们描述了一系列抑制clk和SRPKs的小分子，从而调节mrna前剪接。用这些小分子（Cpd-1、Cpd-2或Cpd-3）处理可显著降低内源性磷酸化SR蛋白的水平，并导致MDA-MB-468细胞的核斑增大。此外，这些化合物导致RPS6KB1 （S6K）剪接改变，随后导致S6K蛋白耗竭。有趣的是，选择性clk的化合物的活性与调节S6K剪接的活性以及癌细胞的生长抑制具有良好的相关性。一项全面的mRNA测序方法显示，这些抑制剂诱导了多个基因的剪接改变和蛋白质消耗，包括那些参与生长和生存途径的基因，如S6K、EGFR、EIF3D和PARP。荧光脉冲追踪标记分析表明，使用CLK抑制剂处理后产生的具有过早终止密码子的同工异构体的降解速度比典型mrna快得多。综上所述，这些结果表明CLK抑制剂通过剪接改变参与生长和存活的基因表现出生长抑制和细胞凋亡诱导。这些小分子抑制剂可能是阐明剪接分子机制和开发新型抗肿瘤药物的有价值的工具。
Accumulating evidence has demonstrated the importance of alternative splicing in various physiological processes, including the development of different diseases. CDC-like kinases (CLKs) and serine-arginine protein kinases (SRPKs) are components of the splicing machinery that are crucial for exon selection. The discovery of small molecule inhibitors against these kinases is of significant value, not only to delineate the molecular mechanisms of splicing, but also to identify potential therapeutic opportunities. Here we describe a series of small molecules that inhibit CLKs and SRPKs and thereby modulate pre-mRNA splicing. Treatment with these small molecules (Cpd-1, Cpd-2, or Cpd-3) significantly reduced the levels of endogenous phosphorylated SR proteins and caused enlargement of nuclear speckles in MDA-MB-468 cells. Additionally, the compounds resulted in splicing alterations of RPS6KB1 (S6K), and subsequent depletion of S6K protein. Interestingly, the activity of compounds selective for CLKs was well correlated with the activity for modulating S6K splicing as well as growth inhibition of cancer cells. A comprehensive mRNA sequencing approach revealed that the inhibitors induced splicing alterations and protein depletion for multiple genes, including those involved in growth and survival pathways such as S6K, EGFR, EIF3D, and PARP. Fluorescence pulse-chase labeling analyses demonstrated that isoforms with premature termination codons generated after treatment with the CLK inhibitors were degraded much faster than canonical mRNAs. Taken together, these results suggest that CLK inhibitors exhibit growth suppression and apoptosis induction through splicing alterations in genes involved in growth and survival. These small molecule inhibitors may be valuable tools for elucidating the molecular machinery of splicing and for the potential development of a novel class of antitumor agents.
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