Abstract
The mammalian target of rapamycin (mTOR) is centrally involved in cell growth, metabolism, and angiogenesis. While showing clinical efficacy in a subset of tumors, rapamycin and rapalogs are specific and allosteric inhibitors of mTOR complex 1 (mTORC1), but they do not directly inhibit mTOR complex 2 (mTORC2), an emerging player in cancer. Here, we report chemical structure and biological characterization of three pyrazolopyrimidine ATP-competitive mTOR inhibitors, WAY-600, WYE-687, and WYE-354 (IC(50), 5-9 nmol/L), with significant selectivity over phosphatidylinositol 3-kinase (PI3K) isofoms (>100-fold). Unlike the rapalogs, these inhibitors acutely blocked substrate phosphorylation by mTORC1 and mTORC2 in vitro and in cells in response to growth factor, amino acids, and hyperactive PI3K/AKT. Unlike the inhibitors of PI3K or dual-pan PI3K/mTOR, cellular inhibition of P-S6K1(T389) and P-AKT(S473) by the pyrazolopyrimidines occurred at significantly lower inhibitor concentrations than those of P-AKT(T308) (PI3K-PDK1 readout), showing mTOR selectivity in cellular setting. mTOR kinase inhibitors reduced AKT downstream function and inhibited proliferation of diverse cancer cell lines. These effects correlated with a strong G(1) cell cycle arrest in both the rapamycin-sensitive and rapamycin-resistant cells, selective induction of apoptosis, repression of global protein synthesis, and down-regulation of angiogenic factors. When injected into tumor-bearing mice, WYE-354 inhibited mTORC1 and mTORC2 and displayed robust antitumor activity in PTEN-null tumors. Together, our results highlight mechanistic differentiation between rapalogs and mTOR kinase inhibitors in targeting cancer cell growth and survival and provide support for clinical development of mTOR kinase inhibitors as new cancer therapy.
Highlights
The historical discovery of the macrolide rapamycin [1] and subsequent biological elucidation of its mammalian target of rapamycin have highlighted a great example of the modern chemical biology that has illuminated human disease and therapy
The recently identified mTOR complex 2 (mTORC2) phosphorylates the serine/threonine kinase AKT, thereby increasing its activity. mammalian target of rapamycin (mTOR) is a critical mediator of the canonical pathways of phosphatidylinositol 3-kinase (PI3K)/AKT and Ras/extracellular signal-regulated kinase (ERK), the two pathways that are most frequently dysregulated in human malignancy [6,7,8]
To test directly the inhibition of mTORC2 catalytic activity in vitro, we immunoprecipitated mTORC2 and mTOR complex 1 (mTORC1) from HEK293 cells and performed immune-complex kinase assay of the mTORC2specific substrate His6-AKT or the mTORC1 substrate His6-S6K
Summary
The historical discovery of the macrolide rapamycin [1] and subsequent biological elucidation of its mammalian target of rapamycin (mTOR) have highlighted a great example of the modern chemical biology that has illuminated human disease and therapy. MTOR is a critical mediator of the canonical pathways of PI3K/AKT and Ras/extracellular signal-regulated kinase (ERK), the two pathways that are most frequently dysregulated in human malignancy [6,7,8] Evidence from both epidemiologic and experimental studies indicate that mTOR signaling contributes to both the tumorigenic effects by numerous oncogenes such as PI3K, AKT, epidermal growth factor receptor, HER2/neu, and BCR-Abl as well as the effects due to loss of tumor suppressor genes such as PTEN, tuberous sclerosis complex, von Hippel-Lindau, and neurofibromatosis 1 [6,7,8]. The positive regulation of AKT by mTORC2 implicates mTOR as acting both upstream and downstream of AKT and its diverse and complex roles in cancer cell growth, survival, and resistance to chemotherapy
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