Kinome-wide selectivity profiling of ATP-competitive mammalian target of rapamycin (mTOR) inhibitors and characterization of their binding kinetics.
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.
Department of Cancer Biology, Dana Farber Cancer Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Qingsong
Q
Liu
Department of Cancer Biology, Dana Farber Cancer Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Sivapriya
S
Kirubakaran
Department of Cancer Biology, Dana Farber Cancer Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Wooyoung
W
Hur
Center for Cell Decision Processes, and Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115.
Mario
M
Niepel
Harvard Radiation Oncology Program, Boston, Massachusetts 02115.
Kenneth
K
Westover
Department of Cancer Biology, Dana Farber Cancer Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Carson C
CC
Thoreen
Department of Cancer Biology, Dana Farber Cancer Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Jinhua
J
Wang
Department of Cancer Biology, Dana Farber Cancer Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Jing
J
Ni
ActivX Biosciences, Inc., La Jolla, California 92037.
Matthew P
MP
Patricelli
Invitrogen Corp., Madison, Wisconsin 53719.
Kurt
K
Vogel
Invitrogen Corp., Madison, Wisconsin 53719.
Steve
S
Riddle
Department of Cancer Biology, Dana Farber Cancer Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
David L
DL
Waller
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.
Ryan
R
Traynor
Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215.
Takaomi
T
Sanda
High Magnetic Field Laboratory, Chinese Academy of Science, P. O. Box 1110, Hefei, Anhui, 230031, China.
Zheng
Z
Zhao
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, and; Koch Center for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
Seong A
SA
Kang
Department of Cancer Biology, Dana Farber Cancer Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Jean
J
Zhao
Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215.
A Thomas
AT
Look
Center for Cell Decision Processes, and Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115.
Peter K
PK
Sorger
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.
David M
DM
Sabatini
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.
Nathanael S
NS
Gray
D000255:Adenosine Triphosphate; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000493:pharmacokinetics* / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D000076222:Mechanistic Target of Rapamycin Complex 1; D046912:Multiprotein Complexes; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011506:Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
D000255:Adenosine Triphosphate; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D000076222:Mechanistic Target of Rapamycin Complex 1; D046912:Multiprotein Complexes; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011506:Proteins; D058570:TOR Serine-Threonine Kinases; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D048051:p38 Mitogen-Activated Protein Kinases; C493245:CRTC2 protein, human; C546842:MTOR protein, human; C576324:ATM protein, human