Kinome-wide Selectivity Profiling of ATP-competitive Mammalian Target of Rapamycin (mTOR) Inhibitors and Characterization of Their Binding Kinetics

• Published in: The Journal of biological chemistry 2012-03, Vol.287 (13), p.9742-9752
• Main Authors: Liu, Qingsong, Kirubakaran, Sivapriya, Hur, Wooyoung, Niepel, Mario, Westover, Kenneth, Thoreen, Carson C., Wang, Jinhua, Ni, Jing, Patricelli, Matthew P., Vogel, Kurt, Riddle, Steve, Waller, David L., Traynor, Ryan, Sanda, Takaomi, Zhao, Zheng, Kang, Seong A., Zhao, Jean, Look, A. Thomas, Sorger, Peter K., Sabatini, David M., Gray, Nathanael S.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate; Ataxia Telangiectasia Mutated Proteins; Cell Cycle Proteins - antagonists & inhibitors; Cell Cycle Proteins - metabolism; Cell Line, Tumor; DNA-Binding Proteins - antagonists & inhibitors; DNA-Binding Proteins - metabolism; Dose-Response Relationship, Drug; Enzyme Inhibitors - pharmacokinetics; Enzyme Inhibitors - pharmacology; Enzyme Kinetics; Enzymology; Humans; Kinetics; KU63794; Mechanistic Target of Rapamycin Complex 1; mTOR; Multiprotein Complexes; p38 Mitogen-Activated Protein Kinases - antagonists & inhibitors; p38 Mitogen-Activated Protein Kinases - metabolism; Phosphatidylinositol 3-Kinases - metabolism; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation - drug effects; PI3K; PP242; Protein-Serine-Threonine Kinases - antagonists & inhibitors; Protein-Serine-Threonine Kinases - metabolism; Proteins - antagonists & inhibitors; Proteins - metabolism; Proteomics - methods; Serine/Threonine-protein Kinase; Slow Off-rate; TOR Serine-Threonine Kinases - antagonists & inhibitors; TOR Serine-Threonine Kinases - metabolism; Torin1; Transcription Factors - antagonists & inhibitors; Transcription Factors - metabolism; Tumor Suppressor Proteins - antagonists & inhibitors; Tumor Suppressor Proteins - metabolism; Tyrosine-protein Kinase (Tyrosine Kinase); WYE-354
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.304485

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 (EC50 = 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 (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 μm and that the compounds may represent a starting point for medicinal chemistry efforts aimed at developing inhibitors of other PI3K kinase-related kinases. Several new ATP-competitive mTOR inhibitors have been described, but their kinome-wide selectivity profiles have not been disclosed. Four different profiling technologies revealed a different spectrum of targets for four recently described mTOR inhibitors. Diverse heterocyclic mTOR inhibitors have unique pharmacology. Profiling data guide choices of mTOR inhibitors for particular applications and provide new potential targets for medicinal chemistry efforts.