Active-site inhibitors of mTOR target rapamycin-resistant outputs of mTORC1 and mTORC2.
Active-site inhibitors of mTOR target rapamycin-resistant outputs of mTORC1 and mTORC2.
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DOI：
10.1371/journal.pbio.1000038
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发表时间：
2009-02-10
期刊：
影响因子：
9.8
通讯作者：
Shokat KM
中科院分区：
文献类型：
--
作者：
Feldman ME;Apsel B;Uotila A;Loewith R;Knight ZA;Ruggero D;Shokat KM
哺乳动物靶标雷帕霉素(MTOR)通过整合营养和激素信号来调节细胞的生长和存活。这些信号功能分布在至少两个不同的mTOR蛋白复合体中：mTORC1和mTORC2。MTORc1对选择性抑制剂雷帕霉素敏感，并通过典型的磷脂酰肌醇3-激酶(PI3K)→Akt→mTOR通路被生长因子刺激激活。激活的mTORC1激酶通过磷酸化mRNA翻译的关键调节因子来上调蛋白质的合成。相比之下，mTORC2对雷帕霉素具有耐药性。遗传学研究表明，mTORC2可能使Akt在S473处磷酸化，S473是Akt激活所需的两个磷酸化位点之一；这一点一直存在争议，部分原因是RNA干扰和基因敲除产生了不同的Akt磷酸化异构体。MTOR在控制关键的细胞生长和生存途径中的核心作用引起了人们对发现mTOR抑制剂的兴趣，这些抑制剂与ATP位点结合，从而靶向mTORC2和mTORC1。我们用两种新的和特定的mTOR激酶结构域抑制剂(TORKinibs)研究了细胞和动物中的mTOR信号转导。与雷帕霉素不同的是，这些TORKinibs(pp242和pp30)抑制mTORC2，我们用它们来证明mTOR的药物抑制阻止了S473处Akt的磷酸化，并阻止了它的完全激活。此外，我们发现TORKinibs比雷帕霉素更能完全抑制原代细胞的增殖。令人惊讶的是，我们发现mTORC2不是这种增强活性的基础，我们表明TORKinib pp242是一种比雷帕霉素更有效的mTORC1抑制剂。重要的是，在分子水平上，pp242在雷帕霉素没有作用的条件下抑制帽子依赖的翻译。我们的发现确定了mTORC1的新功能特征，这些功能对雷帕霉素具有耐药性，但有效地被TORKinibs靶向。这些有效的新药理药物在研究mTOR及其在正常生理和人类疾病中的作用方面与雷帕霉素相辅相成。生长因子途径是正常发育所必需的，但在许多癌症中经常被不适当地激活。癌症研究人员越来越感兴趣的一种生长因子敏感途径依赖于哺乳动物的雷帕霉素靶点(MTOR)，雷帕霉素是一种(像所有激酶一样)将磷酸基团从ATP传递到下游蛋白质的氨基酸残基的激酶。Tor蛋白最初是作为雷帕霉素的细胞靶点在酵母中被发现的，雷帕霉素是一种来自细菌的自然产生的小分子，被广泛用作免疫抑制剂，最近被用于一些癌症治疗。TOR蛋白的研究在很大程度上依赖于雷帕霉素的使用，但雷帕霉素并不直接抑制TOR蛋白的活性，相反，雷帕霉素通过与远离TOR活性部位的结构域结合来影响TOR的酶活性。一些mTOR功能对雷帕霉素耐药，这是由于一种多蛋白复合体mTORC2(MTORC2)的激酶活性所致，而mTOR的某些功能是由于mTOR复合体1(MTORC1)对雷帕霉素敏感。我们已经开发了新的mTOR抑制剂，它可以结合mTOR的ATP结合部位，并且在不抑制其他激酶的情况下抑制mTORC1和mTORC2的催化活性。出乎意料的是，这些抑制剂对蛋白质合成和细胞增殖产生了深远的影响，因为它们抑制的是mTORC1，而不是mTORC2。我们发现，控制蛋白质合成的蛋白质mTORC1底物4E结合蛋白(4EBP)的磷酸化对雷帕霉素有部分抗药性，但完全被我们的新抑制剂抑制。4EBP磷酸化对雷帕霉素具有抗药性的发现表明，活性部位抑制剂在治疗癌症方面可能比雷帕霉素更有效，这可能解释了为什么雷帕霉素在用于免疫抑制时耐受性如此之好。细胞依赖于哺乳动物靶标雷帕霉素激酶(MTOR)来感知生长因子。使用新开发的mTOR活性部位抑制剂抑制所有形式的mTOR，揭示了两个含有mTOR的蛋白质复合体的功能及其作为治疗靶点的潜力。
The mammalian target of rapamycin (mTOR) regulates cell growth and survival by integrating nutrient and hormonal signals. These signaling functions are distributed between at least two distinct mTOR protein complexes: mTORC1 and mTORC2. mTORC1 is sensitive to the selective inhibitor rapamycin and activated by growth factor stimulation via the canonical phosphoinositide 3-kinase (PI3K)→Akt→mTOR pathway. Activated mTORC1 kinase up-regulates protein synthesis by phosphorylating key regulators of mRNA translation. By contrast, mTORC2 is resistant to rapamycin. Genetic studies have suggested that mTORC2 may phosphorylate Akt at S473, one of two phosphorylation sites required for Akt activation; this has been controversial, in part because RNA interference and gene knockouts produce distinct Akt phospho-isoforms. The central role of mTOR in controlling key cellular growth and survival pathways has sparked interest in discovering mTOR inhibitors that bind to the ATP site and therefore target both mTORC2 and mTORC1. We investigated mTOR signaling in cells and animals with two novel and specific mTOR kinase domain inhibitors (TORKinibs). Unlike rapamycin, these TORKinibs (PP242 and PP30) inhibit mTORC2, and we use them to show that pharmacological inhibition of mTOR blocks the phosphorylation of Akt at S473 and prevents its full activation. Furthermore, we show that TORKinibs inhibit proliferation of primary cells more completely than rapamycin. Surprisingly, we find that mTORC2 is not the basis for this enhanced activity, and we show that the TORKinib PP242 is a more effective mTORC1 inhibitor than rapamycin. Importantly, at the molecular level, PP242 inhibits cap-dependent translation under conditions in which rapamycin has no effect. Our findings identify new functional features of mTORC1 that are resistant to rapamycin but are effectively targeted by TORKinibs. These potent new pharmacological agents complement rapamycin in the study of mTOR and its role in normal physiology and human disease. Growth factor pathways are required for normal development but are often inappropriately activated in many cancers. One growth-factor–sensitive pathway of increasing interest to cancer researchers relies on the mammalian target of rapamycin (mTOR), a kinase that (like all kinases) delivers phosphate groups from ATP to amino acid residues of downstream proteins. TOR proteins were first discovered in yeast as the cellular targets of rapamycin, a small, naturally occurring molecule derived from bacteria that is widely used as an immunosuppressant and more recently in some cancer therapies. The study of TOR proteins has relied heavily on the use of rapamycin, but rapamycin does not directly inhibit TOR kinase activity; rather, rapamycin influences TOR's enzymatic activities by binding to a domain far from the kinase's active site. Some mTOR functions are resistant to rapamycin, as a result of the kinase activity of one kind of multiprotein complex, the mTOR complex 2 (mTORC2), whereas rapamycin-sensitive functions of mTOR are due to the mTOR complex 1 (mTORC1). We have developed new inhibitors of mTOR that bind to the ATP-binding site of mTOR and inhibit the catalytic activity of both mTORC1 and mTORC2 without inhibiting other kinases. Unexpectedly, these inhibitors had profound effects on protein synthesis and cell proliferation due to their inhibition of mTORC1 rather than mTORC2. We found that the phosphorylation of a protein that controls protein synthesis, the mTORC1 substrate 4E binding protein (4EBP) is partially resistant to rapamycin but fully inhibited by our new inhibitors. The finding that 4EBP phosphorylation is resistant to rapamycin suggests that active-site inhibitors may be more effective than rapamycin in the treatment of cancer and may explain why rapamycin is so well tolerated when taken for immunosuppression. Cells rely on the mammalian target of rapamycin kinase (mTOR) to sense growth factors. Inhibition of all forms of mTOR using newly developed inhibitors of its active site reveals new insights into the function of two mTOR-containing protein complexes and their potential as therapeutic targets.
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