Liu Q, Kirubakaran S, Hur W, Niepel M, Westover K, Thoreen CC, Wang J, Ni J, Patricelli MP, Vogel K, Riddle S, Waller DL, Traynor R, Sanda T, Zhao Z, Kang SA, Zhao J, Look AT, Sorger PK, Sabatini DM, and Gray NS (2012). Kinome-Wide Selectivity Profiling of ATP-Competitive mTOR (Mammalian Target of Rapamycin) Inhibitors and Characterization of Their Binding Kinetics. J Biol Chem 287(13), 9742-9752. PMC3322972 PMID: 22223645.
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 approximately 20-fold greater potency for inhibition of T389 phosphorylation on S6 kinases (EC50 = 2 nM) relative to other inhibitors. In vitro biochemical profiling at 10 muM revealed binding of PP242 to numerous kinases, while WYE354 and KU63794 bound only to p38 kinases and PI3K isoforms and Torin1 to ATR, ATM and DNA-PK. Analysis of these proteins targets in cellular assays did not reveal any off-target activities for Torin1, WYE354 and KU63794 at concentrations below 1 muM but did show that PP242 efficiently inhibited 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 muM, and that the compounds may represent a starting point for medicinal chemistry efforts aimed at developing inhibitors of other PIKK-family kinases.