Discovery of a New Class of mTOR Inhibitors Reveals Rapamycin Resistant Outputs of mTORC1 and mTORC2. Kevan M. Shokat HHMI and Department of Cellular and Molecular Pharmacology, UCSF, N512D Genentech Hall, 600 16th St. San Francisco, CA 94158 [email protected] 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 PI3K\#8594;Akt\#8594;mTOR pathway. Activated mTORC1 kinase upregulates protein synthesis by phosphorylating key regulators of mRNA translation. By contrast, mTORC2 is resistant to rapamycin. Growth factor stimulation of PI3K causes activation of Akt by phosphorylation at two key sites: the activation loop (T308) and the C-terminal hydrophobic motif (S473). Of the two phosphorylation sites on Akt, activation loop phosphorylation at T308, mediated by PDK1, is indispensable for kinase activity, while hydrophobic motif phosphorylation at S473 enhances Akt kinase activity by approximately five-fold. 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 RNAi and gene knockouts produce distinct Akt phospho-isoforms. Active Akt promotes cell survival in many ways including suppressing apoptosis, promoting glucose uptake, and modifying cellular metabolism; consequently, there is significant interest in identifying the kinase(s) responsible for each activating phosphorylation, the relationship between these phosphorylation sites, and the role of differential Akt phosphorylation on Akt substrate phosphorylation. 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. Here we investigate mTOR signaling in cells and animals with two novel and specific mTOR kinase domain inhibitors (TORKinibs). These TORKinibs (PP242 and PP30) are the first specific active-site inhibitors of mTOR and therefore the first specific inhibitors of 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 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 isoform specific PI3K inhibitors its role in normal physiology and human disease.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr SY27-1.

100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO