2. Academic Validation
  3. 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

  • J Biol Chem. 2012 Mar 23;287(13):9742-9752. doi: 10.1074/jbc.M111.304485.
Qingsong Liu 1,  Sivapriya Kirubakaran 1,  Wooyoung Hur 1,  Mario Niepel 2,  Kenneth Westover 3,  Carson C Thoreen 1,  Jinhua Wang 1,  Jing Ni 1,  Matthew P Patricelli 4,  Kurt Vogel 5,  Steve Riddle 5,  David L Waller 1,  Ryan Traynor 6,  Takaomi Sanda 7,  Zheng Zhao 8,  Seong A Kang 9,  Jean Zhao 1,  A Thomas Look 7,  Peter K Sorger 2,  David M Sabatini 10,  Nathanael S Gray 11
Affiliations

Affiliations

  • 1 Department of Cancer Biology, Dana Farber Cancer Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
  • 2 Center for Cell Decision Processes, and Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115.
  • 3 Harvard Radiation Oncology Program, Boston, Massachusetts 02115.
  • 4 ActivX Biosciences, Inc., La Jolla, California 92037.
  • 5 Invitrogen Corp., Madison, Wisconsin 53719.
  • 6 National Centre for Protein Kinase Profiling, Dundee Division of Signal Transduction Therapy, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
  • 7 Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215.
  • 8 High Magnetic Field Laboratory, Chinese Academy of Science, P. O. Box 1110, Hefei, Anhui, 230031, China.
  • 9 Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, and; Koch Center for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
  • 10 Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, and; Koch Center for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
  • 11 Department of Cancer Biology, Dana Farber Cancer Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115. Electronic address: Nathanael_Gray@dfci.harvard.edu.
PMID: 22223645 DOI: 10.1074/jbc.M111.304485
Abstract

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.

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