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

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.

Figure 1. In Vitro IC₅₀ Values for PP242 and PP30 Determined in the Presence of 10 μM ATP

Figure 2. Inhibition of mTORC2 by TORKinibs Affects pS473 and pT308 of Akt

(A) Serum-starved L6 myotubes were pre-treated with kinase inhibitors prior to stimulation with insulin for 3 min. Lysates were analyzed by Western blotting. (B) PP242 inhibits pS473 (red) of Akt more potently than pT308 (gray). Serum-starved L6 myotubes were treated with kinase inhibitors prior to stimulation with insulin for 10 min. Akt phosphorylation was measured by in-cell Western and is shown relative to serum starvation and insulin stimulation (n = 3 for each inhibitor dose). EC₅₀ values from the best fit curves are plotted. ***p < 0.001, F test. EC₅₀ values for PIK-90 on pS473 and pT308 were not significantly different (p = 0.2, F test).

Figure 3. PP242 Does Not Directly Inhibit Phosphorylation of Akt at T308

(A) pT308 is not inhibited by PP242 in cells overexpressing S473A Akt. HEK293 cells were transfected with wild-type Akt, S473A Akt, or not transfected (Mock) and were treated with 2.5 μM PP242 or 625 nM PIK-90 as indicated prior to insulin stimulation. Lysates were analyzed by Western blotting. Quantitation of pT308 relative to insulin treated cells overexpressing wild-type Akt (lane 2) is shown below that blot. Data are representative of two independent experiments. (B) pT308 is not inhibited by PP242 in SIN1^−/− MEFs, which lack pS473. Primary wild-type (WT) and SIN1^−/− MEFs were pre-treated with 625 nM PIK-90, 10 μM BX-795, or 100 nM rapamycin for 24 h, 100 nM rapamycin for 30 min, or the indicated concentrations of PP242 prior to stimulation with insulin. Lysates were analyzed by Western blotting.

Figure 4. Phosphorylation of the Akt Substrates GSK3α/β, TSC2, and FoxO1/O3a Is Not Potently Inhibited by PP242

Lysates from L6 myotubes treated with kinase inhibitors and stimulated with insulin were analyzed by Western blotting. Quantitation of pAkt and pTSC2 relative to the insulin control (lane 2) is show below these blots.

Figure 5. PP242 Inhibits Proliferation without Affecting Actin Stress Fibers

(A) NIH 3T3 cells were stained for actin with Alexa 488-phalloidin (green) and for DNA with DAPI (blue). Images are representative of greater that 100 cells. (B) Differential inhibition of cell proliferation by PP242 and rapamycin does not require mTORC2. Proliferation of primary MEFs cultured for 3 d in the presence of kinase inhibitors was assayed by resazurin fluorescence (RF) and is shown in arbitrary units.

Figure 6. PP242 Inhibits Rapamycin-Resistant mTORC1

(A) Western blots of lysates from L6 myotubes treated with kinase inhibitors and stimulated with insulin for 10 min. (B) Western blots of lysates from Figure 3B. Actin loading control is repeated here for clarity.

Figure 7. PP242 Inhibits Cap-Dependent Translation

(A) Cap-binding proteins in lysates from Figure 6A were purified by 7-methyl GTP (m⁷GTP) affinity and analyzed by Western blotting. (B) Primary MEFs were transfected with a bicistronic reporter vector. The ratio of renilla (cap-dependent) to firefly (IRES-dependent) luciferase activity was measured after incubation overnight in either 10% serum (steady state) or with the indicated inhibitors in the presence of 10% serum (n = 3). *p < 0.05, **p < 0.01, ANOVA with Tukey's post test. (C) Primary MEFs were incubated overnight as in (B) prior to labeling new protein synthesis with ³⁵S. Newly synthesized proteins were separated by SDS-PAGE, transferred to nitrocellulose and visualized by autoradiography. (D) Newly synthesized protein from three experiments as in (C) was quantified. *p < 0.05, ANOVA with Tukey's post test.

Figure 8. PP242 Inhibits mTORC2 and Rapamycin-Resistant mTORC1 In Vivo

Rapamycin (5 mg/kg), PP242 (20 mg/kg), or vehicle were injected into the intraperitoneal (IP) cavity of mice, followed by IP injection of 250 mU insulin or saline. Lysates were prepared from perigenital fat, leg muscle, and liver and analyzed by Western blotting.