Seong A. Kang

An ATP-competitive Mammalian Target of Rapamycin Inhibitor Reveals Rapamycin-resistant Functions of mTORC1

The mammalian target of rapamycin (mTOR) kinase is the catalytic subunit of two functionally distinct complexes, mTORC1 and mTORC2, that coordinately promote cell growth, proliferation, and survival. Rapamycin is a potent allosteric mTORC1 inhibitor with clinical applications as an immunosuppressant and anti-cancer agent. Here we find that Torin1, a highly potent and selective ATP-competitive mTOR inhibitor that directly inhibits both complexes, impairs cell growth and proliferation to a far greater degree than rapamycin. Surprisingly, these effects are independent of mTORC2 inhibition and are instead because of suppression of rapamycin-resistant functions of mTORC1 that are necessary for cap-dependent translation and suppression of autophagy. These effects are at least partly mediated by mTORC1-dependent and rapamycin-resistant phosphorylation of 4E-BP1. Our findings challenge the assumption that rapamycin completely inhibits mTORC1 and indicate that direct inhibitors of mTORC1 kinase activity may be more successful than rapamycin at inhibiting tumors that depend on mTORC1.

The mTOR-regulated phosphoproteome reveals a mechanism of mTORC1-mediated inhibition of growth factor signaling

A search for substrates of a growth-promoting kinase revealed a regulatory feedback loop involved in tumor suppression. The mammalian target of rapamycin (mTOR) protein kinase is a master growth promoter that nucleates two complexes, mTORC1 and mTORC2. Despite the diverse processes controlled by mTOR, few substrates are known. We defined the mTOR-regulated phosphoproteome by quantitative mass spectrometry and characterized the primary sequence motif specificity of mTOR using positional scanning peptide libraries. We found that the phosphorylation response to insulin is largely mTOR dependent and that mTOR exhibits a unique preference for proline, hydrophobic, and aromatic residues at the +1 position. The adaptor protein Grb10 was identified as an mTORC1 substrate that mediates the inhibition of phosphoinositide 3-kinase typical of cells lacking tuberous sclerosis complex 2 (TSC2), a tumor suppressor and negative regulator of mTORC1. Our work clarifies how mTORC1 inhibits growth factor signaling and opens new areas of investigation in mTOR biology.

mTOR Complex 1 Regulates Lipin 1 Localization to Control the SREBP Pathway

The nutrient- and growth factor-responsive kinase mTOR complex 1 (mTORC1) regulates many processes that control growth, including protein synthesis, autophagy, and lipogenesis. Through unknown mechanisms, mTORC1 promotes the function of SREBP, a master regulator of lipo- and sterolgenic gene transcription. Here, we demonstrate that mTORC1 regulates SREBP by controlling the nuclear entry of lipin 1, a phosphatidic acid phosphatase. Dephosphorylated, nuclear, catalytically active lipin 1 promotes nuclear remodeling and mediates the effects of mTORC1 on SREBP target gene, SREBP promoter activity, and nuclear SREBP protein abundance. Inhibition of mTORC1 in the liver significantly impairs SREBP function and makes mice resistant, in a lipin 1-dependent fashion, to the hepatic steatosis and hypercholesterolemia induced by a high-fat and -cholesterol diet. These findings establish lipin 1 as a key component of the mTORC1-SREBP pathway.

Structure of the human mTOR Complex I and its implications for rapamycin inhibition

The mammalian target of rapamycin complex 1 (mTORC1) regulates cell growth in response to the nutrient and energy status of the cell, and its deregulation is common in human cancers. Little is known about the overall architecture and subunit organization of this essential signaling complex. We have determined the three-dimensional (3D) structure of the fully assembled human mTORC1 by cryo-electron microscopy (cryo-EM). Our analyses reveal that mTORC1 is an obligate dimer with an overall rhomboid shape and a central cavity. The dimeric interfaces are formed by interlocking interactions between the mTOR and raptor subunits. Extended incubation with FKBP12-rapamycin compromises the structural integrity of mTORC1 in a stepwise manner, leading us to propose a model in which rapamycin inhibits mTORC1-mediated phosphorylation of 4E-BP1 and S6K1 through different mechanisms.

mTORC1 Phosphorylation Sites Encode Their Sensitivity to Starvation and Rapamycin

Introduction The mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) protein kinase promotes cell growth by controlling major anabolic and catabolic processes in response to a variety of environmental and intracellular stimuli, and is deregulated in aging and human diseases such as cancer and diabetes. Rapamycin, an allosteric inhibitor of mTORC1, is used clinically in organ transplantation and the treatment of certain cancers. Exactly how rapamycin perturbs mTORC1 signaling is poorly understood and it remains unknown why certain mTORC1 phosphorylation sites are sensitive to the drug whereas others are not. Here, we test the hypothesis that the inherent capacity of a phosphorylation site to serve as an mTORC1 substrate (a property we call substrate quality) is a key determinant of its sensitivity to rapamycin as well as nutrient and growth factor starvation. mTORC1 Phosphorylation sites encode their sensitivity to physiological and pharmacological modulators of mTORC1. Substrate quality is an important determinant of how effectively mTORC1 phosphorylates its substrates in the response to both pharmacological and natural regulators ofthe kinase. Methods We measured the in vitro kinase activity of mTORC1 towards short synthetic peptides encompassing single mTORC1 phosphorylation sites and refined the established mTORC1 phosphorylation motif. We introduced subtle mutations into bona fide mTORC1 phosphorylation sites that we found to enhance or reduce their phosphorylation by mTORC1 in vitro and monitored the corresponding changes in the sensitivity of these sites to rapamycin treatment within cells. Finally, we assessed whether the modifications of the mTORC1 phosphorylation sites also altered their sensitivities to nutrient and growth factor starvation. Results The response of an mTORC1 phosphorylation site to rapamycin treatment should depend on the balance between the activity of mTORC1 and of the protein phosphatase(s) that dephosphorylates it. We found that the in vitro kinase activity of mTORC1 toward peptides containing established phosphorylation sites strongly correlates with the resistance of the sites to rapamycin within cells. Moreover, the relative affinities of the mTOR kinase domain for the peptides also correlated with its capacity to phosphorylate them. In addition to a preference for either proline or a nonproline hydrophobic residue in the +1 position, our refinement of the mTORC1 phosphorylation motif revealed preferences for noncharged residues surrounding the phosphoacceptor site and for serine over threonine as the phosphoacceptor. Utilizing this improved understanding of the sequence motif specificity of mTORC1, we were able to manipulate mTORC1 activity toward its phosphorylation sites in vitro and alter their sensitivities to rapamycin treatment within cells. Interestingly, mTORC1 phosphorylation sites also varied in their sensitivities to nutrient and growth factor levels and manipulations in substrate quality were sufficient to alter their responses to nutrient and growth factor starvation. Discussion Our findings suggest that the sequence composition of an mTORC1 phosphorylation site, including the presence of serine or threonine as the phosphoacceptor, is one of the key determinants of whether the site is a good or poor mTORC1 substrate within cells. Even though the phosphorylation of mTORC1 sites is subject to varied regulatory mechanisms, we propose that differences in substrate quality are one mechanism for allowing downstream effectors of mTORC1 to respond differentially to temporal and intensity changes in the levels of nutrients and growth factors as well as pharmacological inhibitors such as rapamycin. Such differential responses are likely important for mTORC1 to coordinate and appropriately time the myriad processes that make up the vast starvation program it controls. Lastly, it is likely that the form of hierarchical regulation we describe for mTORC1 substrates also exists in other kinase-driven signaling pathways. Not mTORCing Inhibition of the protein kinase complex mTORC1 has potentially beneficial therapeutic affects that include inhibition of cancer and extension of life span. However, effects of its inhibition in vivo have sometimes been disappointing. One reason may be that the well-studied inhibitor of mTORC1, rapamycin, inhibits some effects of mTORC1 but not others. In line with this idea, Kang et al. (1236566) show that the effect of rapamycin depends on the substrate. Characteristics of the phosphorylation sites on various substrates caused them to be phosphorylated with different efficiency by mTORC1. The substrates that were most efficiently phosphorylated were resistant to inhibition of mTORC1. The results explain how various sites, sometimes within the same protein, can differ in their sensitivity to rapamycin. Inhibition of a protein kinase differentially affects its targets, depending on phosphorylation site characteristics. The mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) protein kinase promotes growth and is the target of rapamycin, a clinically useful drug that also prolongs life span in model organisms. A persistent mystery is why the phosphorylation of many bona fide mTORC1 substrates is resistant to rapamycin. We find that the in vitro kinase activity of mTORC1 toward peptides encompassing established phosphorylation sites varies widely and correlates strongly with the resistance of the sites to rapamycin, as well as to nutrient and growth factor starvation within cells. Slight modifications of the sites were sufficient to alter mTORC1 activity toward them in vitro and to cause concomitant changes within cells in their sensitivity to rapamycin and starvation. Thus, the intrinsic capacity of a phosphorylation site to serve as an mTORC1 substrate, a property we call substrate quality, is a major determinant of its sensitivity to modulators of the pathway. Our results reveal a mechanism through which mTORC1 effectors can respond differentially to the same signals.

Discovery of 1-(4-(4-Propionylpiperazin-1-yl)-3-(trifluoromethyl)phenyl)-9-(quinolin-3-yl)benzo[h][1,6]naphthyridin-2(1H)-one as a Highly Potent, Selective Mammalian Target of Rapamycin (mTOR) Inhibitor for the Treatment of Cancer

The mTOR protein is a master regulator of cell growth and proliferation, and inhibitors of its kinase activity have the potential to become new class of anticancer drugs. Starting from quinoline 1, which was identified in a biochemical mTOR assay, we developed a tricyclic benzonaphthyridinone inhibitor 37 (Torin1), which inhibited phosphorylation of mTORC1 and mTORC2 substrates in cells at concentrations of 2 and 10 nM, respectively. Moreover, Torin1 exhibits 1000-fold selectivity for mTOR over PI3K (EC(50) = 1800 nM) and exhibits 100-fold binding selectivity relative to 450 other protein kinases. Torin1 was efficacious at a dose of 20 mg/kg in a U87MG xenograft model and demonstrated good pharmacodynamic inhibition of downstream effectors of mTOR in tumor and peripheral tissues. These results demonstrate that Torin1 is a useful probe of mTOR-dependent phenomena and that benzonaphthridinones represent a promising scaffold for the further development of mTOR-specific inhibitors with the potential for clinical utility.

Discovery of 9-(6-aminopyridin-3-yl)-1-(3-(trifluoromethyl)phenyl)benzo[h][1,6]naphthyridin-2(1H)-one (Torin2) as a potent, selective, and orally available mammalian target of rapamycin (mTOR) inhibitor for treatment of cancer.

The mTOR mediated PI3K/AKT/mTOR signal transduction pathway has been demonstrated to play a key role in a broad spectrum of cancers. Starting from the mTOR selective inhibitor 1 (Torin1), a focused medicinal chemistry effort led to the discovery of an improved mTOR inhibitor 3 (Torin2), which possesses an EC(50) of 0.25 nM for inhibiting cellular mTOR activity. Compound 3 exhibited 800-fold selectivity over PI3K (EC(50): 200 nM) and over 100-fold binding selectivity relative to 440 other protein kinases. Compound 3 has significantly improved bioavailability (54%), metabolic stability, and plasma exposure relative to compound 1.

Kinome-wide Selectivity Profiling of ATP-competitive Mammalian Target of Rapamycin (mTOR) Inhibitors and Characterization of Their Binding Kinetics

Background: Several new ATP-competitive mTOR inhibitors have been described, but their kinome-wide selectivity profiles have not been disclosed. Results: Four different profiling technologies revealed a different spectrum of targets for four recently described mTOR inhibitors. Conclusion: Diverse heterocyclic mTOR inhibitors have unique pharmacology. Significance: Profiling data guide choices of mTOR inhibitors for particular applications and provide new potential targets for medicinal chemistry efforts. 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 ∼20-fold greater potency for inhibition of Thr-389 phosphorylation on S6 kinases (EC50 = 2 nm) relative to other inhibitors. In vitro biochemical profiling at 10 μm revealed binding of PP242 to numerous kinases, although WYE354 and KU63794 bound only to p38 kinases and PI3K isoforms and Torin1 to ataxia telangiectasia mutated, ATM and Rad3-related protein, and DNA-PK. Analysis of these protein targets in cellular assays did not reveal any off-target activities for Torin1, WYE354, and KU63794 at concentrations below 1 μm but did show that PP242 efficiently inhibited the 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 μm and that the compounds may represent a starting point for medicinal chemistry efforts aimed at developing inhibitors of other PI3K kinase-related kinases.

Native capillary isoelectric focusing for the separation of protein complex isoforms and subcomplexes.

Here we report the use of capillary isoelectric focusing under native conditions for the separation of protein complex isoforms and subcomplexes. Using biologically relevant HIS-tag and FLAG-tag purified protein complexes, we demonstrate the separations of protein complex isoforms of the mammalian target of rapamycin complex (mTORC1 and 2) and the subcomplexes and different phosphorylation states of the Dam1 complex. The high efficiency capillary isoelectric focusing separation allowed for resolution of protein complexes and subcomplexes similar in size and biochemical composition. By performing separations with native buffers and reduced temperature (15 degrees C) we were able to maintain the complex integrity of the more thermolabile mTORC2 during isoelectric focusing and detection (<45 min). Increasing the separation temperature allowed us to monitor dissociation of the Dam1 complex into its subcomplexes (25 degrees C) and eventually its individual protein components (30 degrees C). The separation of two different phosphorylation states of the Dam1 complex, generated from an in vitro kinase assay with Mps1 kinase, was straightforward due to the large pI shift upon multiple phosphorylation events. The separation of the protein complex isoforms of mTORC, on the other hand, required the addition of a small pI range (4-6.5) of ampholytes to improve resolution and stability of the complexes. We show that native capillary isoelectric focusing is a powerful method for the difficult separations of large, similar, unstable protein complexes. This method shows potential for differentiation of protein complex isoform and subcomplex compositions, post-translational modifications, architectures, stabilities, equilibria, and relative abundances under biologically relevant conditions.

Development of ATP-competitive mTOR inhibitors.

The mammalian Target of Rapamycin (mTOR)-mediated signaling transduction pathway has been observed to be deregulated in a wide variety of cancer and metabolic diseases. Despite extensive clinical development efforts, the well-known allosteric mTOR inhibitor rapamycin and structurally related rapalogs have failed to show significant single-agent antitumor efficacy in most types of cancer. This limited clinical success may be due to the inability of the rapalogs to maintain a complete blockade mTOR-mediated signaling. Therefore, numerous efforts have been initiated to develop ATP-competitive mTOR inhibitors that would block both mTORC1 and mTORC2 complex activity. Here, we describe our experimental approaches to develop Torin1 using a medium throughput cell-based screening assay and structure-guided drug design.