Kinome-wide Selectivity Profiling of ATP-competitive Mammalian Target of Rapamycin (mTOR) Inhibitors and Characterization of Their Binding Kinetics
Kinome-wide Selectivity Profiling of ATP-competitive Mammalian Target of Rapamycin (mTOR) Inhibitors and Characterization of Their Binding Kinetics
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DOI：
10.1074/jbc.m111.304485
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发表时间：
2012-03-23
影响因子：
4.8
通讯作者：
Gray, Nathanael S.
中科院分区：
文献类型：
--
作者：
Liu, Qingsong;Kirubakaran, Sivapriya;Gray, Nathanael S.
最近开发ATP竞争性mTOR抑制剂的密集努力已经产生了几种有效的和选择性的分子，如Torin 1，PP 242，KU 63794和WYE 354。这些抑制剂被广泛用作mTOR依赖性生物学的药理学探针。为了确定这些药物的效力和特异性，我们已经进行了系统的激酶组范围的努力，使用化学蛋白质组学和酶活性，蛋白质结合和细胞信号传导中断的测定来描述它们的选择性和效力。酶和细胞测定显示，所有四种化合物都是mTORC 1和mTORC 2的有效抑制剂，其中Torin 1对S6激酶上Thr-389磷酸化的抑制效力（EC 50 = 2 nM）比其他抑制剂高出20倍。在10 μ M的体外生物化学分析显示，PP 242结合到许多激酶，虽然WYE 354和KU 63794只结合到p38激酶和PI 3 K亚型和Torin 1共济失调毛细血管扩张症突变，ATM和Rad 3相关蛋白，和DNA-PK。在细胞试验中对这些蛋白质靶点的分析没有发现Torin 1、WYE 354和KU 63794在低于1 μ M的浓度下有任何脱靶活性，但确实表明PP 242有效抑制RET受体（EC 50，42 nM）和JAK 1/2/3激酶（EC 50，780 nM）。此外，Torin 1对mTORC 1/2复合物的抑制表现出异常缓慢的动力学，这一特性可能有助于该抑制剂的药理学。我们的研究结果表明，除了PP 242之外，当以低于1 μ M的浓度应用于细胞时，可用的ATP竞争性化合物是高度选择性的mTOR抑制剂，并且该化合物可能代表旨在开发其他PI 3 K激酶相关激酶抑制剂的药物化学努力的起点。
An intensive recent effort to develop ATP-competitive mTOR inhibitors has resulted in several potent and selective molecules such as Torin1, PP242, KU63794, and WYE354. These inhibitors are being widely used as pharmacological probes of mTOR-dependent biology. To determine the potency and specificity of these agents, we have undertaken a systematic kinome-wide effort to profile their selectivity and potency using chemical proteomics and assays for enzymatic activity, protein binding, and disruption of cellular signaling. Enzymatic and cellular assays revealed that all four compounds are potent inhibitors of mTORC1 and mTORC2, with Torin1 exhibiting similar to 20-fold greater potency for inhibition of Thr-389 phosphorylation on S6 kinases (EC50 = 2 nM) relative to other inhibitors. In vitro biochemical profiling at 10 mu M revealed binding of PP242 to numerous kinases, although WYE354 and KU63794 bound only to p38 kinases and PI3K isoforms and Torin1 to ataxia telangiectasia mutated, ATM and Rad3-related protein, and DNA-PK. Analysis of these protein targets in cellular assays did not reveal any off-target activities for Torin1, WYE354, and KU63794 at concentrations below 1 mu M but did show that PP242 efficiently inhibited the RET receptor (EC50, 42 nM) and JAK1/2/3 kinases (EC50, 780 nM). In addition, Torin1 displayed unusually slow kinetics for inhibition of the mTORC1/2 complex, a property likely to contribute to the pharmacology of this inhibitor. Our results demonstrated that, with the exception of PP242, available ATP-competitive compounds are highly selective mTOR inhibitors when applied to cells at concentrations below 1 mu M and that the compounds may represent a starting point for medicinal chemistry efforts aimed at developing inhibitors of other PI3K kinase-related kinases.