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Active-site inhibitors of mTOR target rapamycin-resistant outputs of mTORC1 and mTORC2

PLoS Biol. 2009 Feb 10;7(2):e38. doi: 10.1371/journal.pbio.1000038.

Abstract

The mammalian target of rapamycin (mTOR) regulates cell growth and survival by integrating nutrient and hormonal signals. These signaling functions are distributed between at least two distinct mTOR protein complexes: mTORC1 and mTORC2. mTORC1 is sensitive to the selective inhibitor rapamycin and activated by growth factor stimulation via the canonical phosphoinositide 3-kinase (PI3K)-->Akt-->mTOR pathway. Activated mTORC1 kinase up-regulates protein synthesis by phosphorylating key regulators of mRNA translation. By contrast, mTORC2 is resistant to rapamycin. Genetic studies have suggested that mTORC2 may phosphorylate Akt at S473, one of two phosphorylation sites required for Akt activation; this has been controversial, in part because RNA interference and gene knockouts produce distinct Akt phospho-isoforms. The central role of mTOR in controlling key cellular growth and survival pathways has sparked interest in discovering mTOR inhibitors that bind to the ATP site and therefore target both mTORC2 and mTORC1. We investigated mTOR signaling in cells and animals with two novel and specific mTOR kinase domain inhibitors (TORKinibs). Unlike rapamycin, these TORKinibs (PP242 and PP30) inhibit mTORC2, and we use them to show that pharmacological inhibition of mTOR blocks the phosphorylation of Akt at S473 and prevents its full activation. Furthermore, we show that TORKinibs inhibit proliferation of primary cells more completely than rapamycin. Surprisingly, we find that mTORC2 is not the basis for this enhanced activity, and we show that the TORKinib PP242 is a more effective mTORC1 inhibitor than rapamycin. Importantly, at the molecular level, PP242 inhibits cap-dependent translation under conditions in which rapamycin has no effect. Our findings identify new functional features of mTORC1 that are resistant to rapamycin but are effectively targeted by TORKinibs. These potent new pharmacological agents complement rapamycin in the study of mTOR and its role in normal physiology and human disease.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3T3 Cells
  • Actins
  • Animals
  • Catalytic Domain / drug effects
  • Cell Proliferation / drug effects
  • Fibroblasts
  • Gene Expression Regulation
  • Insulin / metabolism
  • Mechanistic Target of Rapamycin Complex 1
  • Mice
  • Mice, Knockout
  • Multiprotein Complexes
  • Phosphorylation / drug effects
  • Protein Kinase Inhibitors / pharmacology*
  • Protein Kinases / genetics
  • Protein Kinases / metabolism*
  • Proteins
  • Pyrimidines / metabolism
  • Sirolimus / pharmacology*
  • TOR Serine-Threonine Kinases
  • Transcription Factors / antagonists & inhibitors
  • Transcription Factors / metabolism

Substances

  • Actins
  • CRTC2 protein, human
  • Insulin
  • Multiprotein Complexes
  • Protein Kinase Inhibitors
  • Proteins
  • Pyrimidines
  • Transcription Factors
  • Protein Kinases
  • MTOR protein, human
  • mTOR protein, mouse
  • Mechanistic Target of Rapamycin Complex 1
  • TOR Serine-Threonine Kinases
  • Sirolimus