Department of Cancer Biology, Dana Farber Cancer Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Center for Cell Decision Processes, and Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115.
J Biol Chem. 2012 Mar 23;287(13):9742-9752. doi: 10.1074/jbc.M111.304485. Epub 2012 Jan 5.
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 ∼20-fold greater potency for inhibition of Thr-389 phosphorylation on S6 kinases (EC(50) = 2 nM) relative to other inhibitors. In vitro biochemical profiling at 10 μM 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 μM but did show that PP242 efficiently inhibited the RET receptor (EC(50), 42 nM) and JAK1/2/3 kinases (EC(50), 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 μM and that the compounds may represent a starting point for medicinal chemistry efforts aimed at developing inhibitors of other PI3K kinase-related kinases.
最近人们进行了一项密集的研究，旨在开发能够与 ATP 竞争的 mTOR 抑制剂，已经得到了几种高效且有选择性的分子，如 Torin1、PP242、KU63794 和 WYE354。这些抑制剂被广泛用作研究 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，我们利用化学蛋白质组学和酶活性测定、蛋白质结合以及细胞信号转导中断等方法，对激酶组进行了全面的筛选，以分析它们的选择性和效力。酶和细胞测定显示，这四种化合物均为 mTORC1 和 mTORC2 的强效抑制剂，Torin1 对 S6 激酶 Thr-389 磷酸化的抑制作用效力约为其他抑制剂的 20 倍（EC50 = 2 nM）。在 10 μM 下的体外生化筛选发现，PP242 与许多激酶结合，尽管 WYE354 和 KU63794 仅与 p38 激酶和 PI3K 同工型结合，而 Torin1 与共济失调毛细血管扩张突变蛋白、ATR 和 Rad3 相关蛋白以及 DNA-PK 结合。在细胞测定中对这些蛋白靶标进行分析，并未发现 Torin1、WYE354 和 KU63794 在低于 1 μM 的浓度下有任何非靶标活性，但确实显示 PP242 能有效地抑制 RET 受体（EC50，42 nM）和 JAK1/2/3 激酶（EC50，780 nM）。此外，Torin1 对 mTORC1/2 复合物的抑制作用表现出异常缓慢的动力学特性，这一特性可能对该抑制剂的药理学特性有贡献。我们的结果表明，除了 PP242 之外，在低于 1 μM 的浓度下应用于细胞时，现有的 ATP 竞争性化合物都是高度选择性的 mTOR 抑制剂，并且这些化合物可能成为开发其他 PI3K 激酶相关激酶抑制剂的药物化学努力的起点。
