Screen for chemical modulators of autophagy reveals novel therapeutic inhibitors of mTORC1 signaling.
Screen for chemical modulators of autophagy reveals novel therapeutic inhibitors of mTORC1 signaling.
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DOI：
10.1371/journal.pone.0007124
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发表时间：
2009-09-22
期刊：
影响因子：
3.7
通讯作者：
Roberge M
中科院分区：
文献类型：
--
作者：
Balgi AD;Fonseca BD;Donohue E;Tsang TC;Lajoie P;Proud CG;Nabi IR;Roberge M
雷帕霉素靶蛋白复合物1（mTORC1）是一种蛋白激酶，它传递营养可用性信号以控制众多细胞功能，包括自噬，自噬是一种由营养耗竭激活的细胞自我吞噬过程。要挖掘调节mTORC1信号传导和自噬在人类疾病中的治疗潜力，就需要具有药理学理想特性的活性化学物质。
利用一种基于细胞的自动化检测方法，我们筛选了3500多种化学物质，确定了三种已批准的药物（沛心达、氯硝柳胺、胺碘酮）和一种药理试剂（鱼藤素）能够快速增加自噬体含量。生化检测表明，这四种化合物在营养丰富的条件下能刺激细胞自噬并抑制mTORC1信号传导。这些化合物不抑制mTORC2（mTORC2也含有mTOR作为催化亚基），这表明它们不抑制mTOR的催化活性，而是抑制向mTORC1的信号传导。沛心达、氯硝柳胺或鱼藤素诱导的mTORC1抑制和自噬体积累在停药后迅速逆转，而胺碘酮对mTORC1的抑制基本上是不可逆的。TSC2是mTORC1的负调节因子，鱼藤素对mTORC1信号传导的抑制需要TSC2，但沛心达、氯硝柳胺和胺碘酮对mTORC1的抑制不需要TSC2。在营养丰富的条件下，将永生化小鼠胚胎成纤维细胞短暂暴露于这些药物没有毒性，但在饥饿条件下会通过凋亡导致细胞快速死亡，其机制在很大程度上由结节性硬化复合物蛋白TSC2（mTORC1的上游调节因子）决定。相比之下，短暂暴露于mTORC1抑制剂雷帕霉素会导致基本上不可逆的mTORC1抑制、细胞生长的持续抑制，并且在饥饿条件下没有选择性的细胞杀伤。
已批准用于人类的药物能够可逆地抑制mTORC1并刺激自噬这一发现，应该会极大地促进mTORC1抑制在结节性硬化症、糖尿病、心血管疾病和癌症等适应症方面的临床前和临床测试。
Mammalian target of rapamycin complex 1 (mTORC1) is a protein kinase that relays nutrient availability signals to control numerous cellular functions including autophagy, a process of cellular self-eating activated by nutrient depletion. Addressing the therapeutic potential of modulating mTORC1 signaling and autophagy in human disease requires active chemicals with pharmacologically desirable properties. Using an automated cell-based assay, we screened a collection of >3,500 chemicals and identified three approved drugs (perhexiline, niclosamide, amiodarone) and one pharmacological reagent (rottlerin) capable of rapidly increasing autophagosome content. Biochemical assays showed that the four compounds stimulate autophagy and inhibit mTORC1 signaling in cells maintained in nutrient-rich conditions. The compounds did not inhibit mTORC2, which also contains mTOR as a catalytic subunit, suggesting that they do not inhibit mTOR catalytic activity but rather inhibit signaling to mTORC1. mTORC1 inhibition and autophagosome accumulation induced by perhexiline, niclosamide or rottlerin were rapidly reversed upon drug withdrawal whereas amiodarone inhibited mTORC1 essentially irreversibly. TSC2, a negative regulator of mTORC1, was required for inhibition of mTORC1 signaling by rottlerin but not for mTORC1 inhibition by perhexiline, niclosamide and amiodarone. Transient exposure of immortalized mouse embryo fibroblasts to these drugs was not toxic in nutrient-rich conditions but led to rapid cell death by apoptosis in starvation conditions, by a mechanism determined in large part by the tuberous sclerosis complex protein TSC2, an upstream regulator of mTORC1. By contrast, transient exposure to the mTORC1 inhibitor rapamycin caused essentially irreversible mTORC1 inhibition, sustained inhibition of cell growth and no selective cell killing in starvation. The observation that drugs already approved for human use can reversibly inhibit mTORC1 and stimulate autophagy should greatly facilitate the preclinical and clinical testing of mTORC1 inhibition for indications such as tuberous sclerosis, diabetes, cardiovascular disease and cancer.