Binding site hotspot map of PI3Kα and mTOR in the presence of selective and dual ATP-competitive inhibitors.

The PI3K/Akt/mTOR signaling pathway plays a pivotal role in cellular metabolism, growth and survival. PI3Kα hyperactivation impairs downstream signaling, including mTOR regulation, and are linked to poor prognosis and refractory cancer treatment. To support multi-target drug discovery, we took advantage from existing PI3Kα and mTOR crystallographic structures to map similarities and differences in their ATP-binding pockets in the presence of selective or dual inhibitors. Molecular dynamics and MM/PBSA calculations were employed to study the binding profile and identify the relative contribution of binding site residues. Our analysis showed that while varying parameters of solute and solvent dielectric constant interfered in the absolute binding free energy, it had no effect in the relative per residue contribution. In all complexes, the most important interactions were observed within 3-3.5 Å from inhibitors, responding for ∼75-100% of the total calculated interaction energy. While closest residues are essential for the strength of the binding of all ligands, more distant residues seem to have a larger impact on the binding of the dual inhibitor, as observed for PI3Kα residues Phe934, Lys802 and Asp805 and, mTOR residues Leu2192, Phe2358, Leu2354, Lys2187 and Tyr2225. A detailed description of individual residue contribution in the presence of selective or dual inhibitors is provided as an effort to improve the understanding of molecular mechanisms controlling multi-target inhibition. This work provides key information to support further studies seeking the rational design of potent PI3K/mTOR dual inhibitors for cancer treatment.Communicated by Ramaswamy H. Sarma.

Modulating mTOR Signaling as a Promising Therapeutic Strategy for Atherosclerosis.

For more than a decade, atherosclerosis has been one of the leading causes of death in developed countries. The issue of treatment and prevention of the disease is especially acute. Despite the huge amount of basic and clinical research, a significant number of gaps remain in our understanding of the pathogenesis of atherosclerosis, and only their closure will bring us closer to understanding the causes of the disease at the cellular and molecular levels and, accordingly, to the development of an effective treatment. One of the seemingly well-studied elements of atherogenesis is the mTOR signaling pathway. However, more and more new details are still being clarified. Therapeutic strategies associated with rapamycin have worked well in a number of different diseases, and there is every reason to believe that targeting components of the mTOR pathway may pay off in atherosclerosis as well.

Bioinformatic Analysis of Two TOR (Target of Rapamycin)-Like Proteins Encoded by Entamoeba histolytica Revealed Structural Similarities with Functional Homologs.

The target of rapamycin (TOR), also known as FKBP-rapamycin associated protein (FRAP), is a protein kinase belonging to the PIKK (phosphatidylinositol 3-kinase (PI3K)-related kinases) family. TOR kinases are involved in several signaling pathways that control cell growth and proliferation. Entamoeba histolytica, the protozoan parasite that causes human amoebiasis, contains two genes encoding TOR-like proteins: EhFRAP and EhTOR2. To assess their potential as drug targets to control the cell proliferation of E. histolytica, we studied the structural features of EhFRAP and EhTOR2 using a biocomputational approach. The overall results confirmed that both TOR amoebic homologs share structural similarities with functional TOR kinases, and show inherent abilities to form TORC complexes and participate in protein-protein interaction networks. To our knowledge, this study represents the first in silico characterization of the structure-function relationships of EhFRAP and EhTOR2.

Structural Basis of DEPTOR to Recognize Phosphatidic Acid Using its Tandem DEP Domains.

DEP domain containing mTOR-interacting protein (DEPTOR) plays pivotal roles in regulating metabolism, growth, autophagy and apoptosis by functions as an endogenous inhibitor of mTOR signaling pathway. Activated by phosphatidic acid, a second messenger in mTOR signaling, DEPTOR dissociates from mTORC1 complex with unknown mechanism. Here, we present a 1.5 Å resolution crystal structure, which shows that the N-terminal two tandem DEP domains of hDEPTOR fold into a dumbbell-shaped structure, protruding the characteristic ß-hairpin arms of DEP domains on each side. An 18 amino acids DDEX motif at the end of DEP2 interacts with DEP1 and stabilizes the structure. Biochemical studies showed that the tandem DEP domains directly interact with phosphatidic acid using two distinct positively charged patches. These results provide insights into mTOR activation upon phosphatidic acid stimulation.

[New structures of mTORC1: Focus on Rag GTPases]. / Une moisson de nouvelles structures de mTORC1 - Coup de projecteur sur les GTPases Rag.

mTORC1 is a central player in cell growth, a process that is tightly regulated by the availability of nutrients and that controls various aspects of metabolism in the normal cell and in severe diseases such as cancers. mTORC1 is a large multiprotein complex, composed of the kinase subunit mTOR, of Ragulator, which attaches mTOR to the lysosome membrane, of the atypical Rag GTPases and the small GTPase RheB, whose nucleotide states directly dictate its localization to the lysosome and its kinase activity, and of RAPTOR, an adaptor that assembles the complex. The activity of the Rag GTPases is further controlled by the GATOR1 and folliculin complexes, which regulate their GTP/GDP conversion. Here, we review recent structures of important components of the mTORC1 machinery, determined by cryo-electron microscopy for the most part, which allow to reconstitute the architecture of active mTORC1 at near atomic resolution. Notably, we discuss how these structures shed new light on the roles of Rag GTPases and their regulators in mTORC1 regulation, and the perspectives that they open towards understanding the inner workings of mTORC1 on the lysosomal membrane.

TITLE: Une moisson de nouvelles structures de mTORC1 - Coup de projecteur sur les GTPases Rag. ABSTRACT: mTORC1 est un acteur central de la croissance cellulaire, un processus étroitement régulé par la disponibilité de nutriments et qui contrôle diverses étapes du métabolisme dans la cellule normale et au cours de maladies, comme les cancers. mTORC1 est un complexe multiprotéique de grande taille constitué de nombreuses sous-unités, parmi lesquelles deux types de GTPases, Rag et RheB, contrôlent directement sa localisation membranaire et son activité kinase. Dans cette revue, nous faisons le point sur une moisson de structures récentes, déterminées pour la plupart par cryo-microscopie électronique, qui sont en passe de reconstituer le puzzle de l'architecture de mTORC1. Nous discutons ce que ces structures révèlent sur le rôle des GTPases, et ce que leur connaissance ouvre comme perspectives pour comprendre comment mTORC1 fonctionne à la membrane du lysosome.

The dynamic mechanism of 4E-BP1 recognition and phosphorylation by mTORC1.

The activation of cap-dependent translation in eukaryotes requires multisite, hierarchical phosphorylation of 4E-BP by the 1 MDa kinase mammalian target of rapamycin complex 1 (mTORC1). To resolve the mechanism of this hierarchical phosphorylation at the atomic level, we monitored by NMR spectroscopy the interaction of intrinsically disordered 4E binding protein isoform 1 (4E-BP1) with the mTORC1 subunit regulatory-associated protein of mTOR (Raptor). The N-terminal RAIP motif and the C-terminal TOR signaling (TOS) motif of 4E-BP1 bind separate sites in Raptor, resulting in avidity-based tethering of 4E-BP1. This tethering orients the flexible central region of 4E-BP1 toward the mTORC1 kinase site for phosphorylation. The structural constraints imposed by the two tethering interactions, combined with phosphorylation-induced conformational switching of 4E-BP1, explain the hierarchy of 4E-BP1 phosphorylation by mTORC1. Furthermore, we demonstrate that mTORC1 recognizes both free and eIF4E-bound 4E-BP1, allowing rapid phosphorylation of the entire 4E-BP1 pool and efficient activation of translation. Finally, our findings provide a mechanistic explanation for the differential rapamycin sensitivity of the 4E-BP1 phosphorylation sites.

Signalling transduction of O-GlcNAcylation and PI3K/AKT/mTOR-axis in prostate cancer.

Hexosamine biosynthetic (HBP) and PI3K/AKT/mTOR pathways are found to predominate the proliferation and survival of prostate cancer cells. Both these pathways have their own specific intermediates to propagate the secondary signals in down-stream cascades and besides having their own structured network, also have shared interconnecting branches. These interconnections are either competitive or co-operative in nature depending on the microenvironmental conditions. Specifically, in prostate cancer HBP and mTOR pathways increases the expression and protein level of androgen receptor in order to support cancer cell proliferation, advancement and metastasis. Pharmacological inhibition of a single pathway is therefore insufficient to stop disease progression as the cancer cells manage to alter the signalling channel. This is one of the primary reasons for the therapeutic failure in prostate cancer and emergence of chemoresistance. Inhibition of these multiple pathways at their common junctures might prove to be of benefit in men suffering from an advanced disease state. Hence, a thorough understanding of these cellular intersecting points and their significance with respect to signal transduction mechanisms might assist in the rational designing of combinations for effective management of prostate cancer.

mTOR Signaling in Pulmonary Vascular Disease: Pathogenic Role and Therapeutic Target.

Pulmonary arterial hypertension (PAH) is a progressive and fatal disease without a cure. The exact pathogenic mechanisms of PAH are complex and poorly understood, yet a number of abnormally expressed genes and regulatory pathways contribute to sustained vasoconstriction and vascular remodeling of the distal pulmonary arteries. Mammalian target of rapamycin (mTOR) is one of the major signaling pathways implicated in regulating cell proliferation, migration, differentiation, and protein synthesis. Here we will describe the canonical mTOR pathway, structural and functional differences between mTOR complexes 1 and 2, as well as the crosstalk with other important signaling cascades in the development of PAH. The pathogenic role of mTOR in pulmonary vascular remodeling and sustained vasoconstriction due to its contribution to proliferation, migration, phenotypic transition, and gene regulation in pulmonary artery smooth muscle and endothelial cells will be discussed. Despite the progress in our elucidation of the etiology and pathogenesis of PAH over the two last decades, there is a lack of effective therapeutic agents to treat PAH patients representing a significant unmet clinical need. In this review, we will explore the possibility and therapeutic potential to use inhibitors of mTOR signaling cascade to treat PAH.

Erianthridin suppresses non-small-cell lung cancer cell metastasis through inhibition of Akt/mTOR/p70S6K signaling pathway.

Cancer metastasis is a major cause of the high mortality rate in lung cancer patients. The cytoskeletal rearrangement and degradation of extracellular matrix are required to facilitate cell migration and invasion and the suppression of these behaviors is an intriguing approach to minimize cancer metastasis. Even though Erianthridin (ETD), a phenolic compound isolated from the Thai orchid Dendrobium formosum exhibits various biological activities, the molecular mechanism of ETD for anti-cancer activity is unclear. In this study, we found that noncytotoxic concentrations of ETD (≤ 50 µM) were able to significantly inhibit cell migration and invasion via disruption of actin stress fibers and lamellipodia formation. The expression of matrix metalloproteinase-2 (MMP-2) and MMP-9 was markedly downregulated in a dose-dependent manner after ETD treatment. Mechanistic studies revealed that protein kinase B (Akt) and its downstream effectors mammalian target of rapamycin (mTOR) and p70 S6 kinase (p70S6K) were strongly attenuated. An in silico study further demonstrated that ETD binds to the protein kinase domain of Akt with both hydrogen bonding and van der Waals interactions. In addition, an in vivo tail vein injection metastasis study demonstrated a significant effect of ETD on the suppression of lung cancer cell metastasis. This study provides preclinical information regarding ETD, which exhibits promising antimetastatic activity against non-small-cell lung cancer through Akt/mTOR/p70S6K-induced actin reorganization and MMPs expression.

Rational design and implementation of a chemically inducible heterotrimerization system.

Chemically inducible dimerization (CID) uses a small molecule to induce binding of two different proteins. CID tools such as the FK506-binding protein-FKBP-rapamycin-binding- (FKBP-FRB)-rapamycin system have been widely used to probe molecular events inside and outside cells. While various CID tools are available, chemically inducible trimerization (CIT) does not exist, due to inherent challenges in designing a chemical that simultaneously binds three proteins with high affinity and specificity. Here, we developed CIT by rationally splitting FRB and FKBP. Cellular and structural datasets showed efficient trimerization of split pairs of FRB or FKBP with full-length FKBP or FRB, respectively, by rapamycin. CIT rapidly induced tri-organellar junctions and perturbed intended membrane lipids exclusively at select membrane contact sites. By conferring one additional condition to what is achievable with CID, CIT expands the types of manipulation in single live cells to address cell biology questions otherwise intractable and engineer cell functions for future synthetic biology applications.

Target of rapamycin signaling inhibits autophagy in sea cucumber Apostichopus japonicus.

Autophagy mediated by mTOR pathway is a particularly important immune defense mechanism in the pathogens infected mammals. However, the role of TOR in echinoderm autophagy is largely unknown. Here, a cDNA encoding TOR protein was cloned and characterized from sea cucumber Apostichopus japonicus (designated as AjTOR) and its biological functions were also investigated. The AjTOR gene encoded a peptide of 2499 amino acids with the representative domains of DUF3385, FAT, FRB, PI3Kc, and FATC, which exhibited highly conservation with vertebrate orthologs. Phylogenetic analysis supported that AjTOR belonged to a new member of TOR family. Moreover, tissues distribution analysis indicated that AjTOR was ubiquitously expressed in all the tested tissues, with the highest transcription in muscle. Vibrio splendidus infection in vivo and LPS challenge in vitro could both significantly down-regulate the mRNA expression of AjTOR. What's more, transmission electron microscopy observations showed that rapamycin treatment resulted in rapid formation of autophagosomes in coelomocytes both at 3 and 6 h, however, injection with mTOR activator of MHY1485 showed an inhibitory effect on autophagosomes formation compared to the control, suggesting blocking the expression of AjTOR could accelerates autophagy signals. Our findings supported that AjTOR served as a negative regulator in sea cucumber authophay.

Daphnane Diterpenoids from Daphne genkwa Inhibit PI3K/Akt/mTOR Signaling and Induce Cell Cycle Arrest and Apoptosis in Human Colon Cancer Cells.

Seven new daphnane-type diterpenoids, daphgenkins A-G (1-7), and 15 known analogues (8-22) were isolated from the flower buds of Daphne genkwa. Their structures and absolute configurations were elucidated by spectroscopic data and calculated ECD analyses. The cytotoxicities of all daphnane-type diterpenoids (1-22) obtained were evaluated against three human colon cancer cell lines (SW620, RKO, and LoVo). Compounds 1, 12, and 13 exhibited cytotoxic effects against the SW620 and RKO cell lines, with IC50 values in the range of 3.0-9.7 µM. The most active new compound, 1, with an IC50 value of 3.0 µM against SW620 cells, was evaluated further for its underlying molecular mechanism. Compound 1 induced G0/G1 cell cycle arrest, leading to the induction of apoptosis in SW620 cells. Also, it induced cancer cell apoptosis by an increased ratio of Bax/Bcl-2, activated cleaved caspase-3 and caspase-9, and upregulated PARP. Finally, compound 1 significantly inhibited PI3K/Akt/mTOR signaling in SW620 cells. Together, the results suggest that compound 1 may be a suitable lead compound for further biological evaluation.

In Vitro and in Vivo Activity of mTOR Kinase and PI3K Inhibitors Against Leishmania donovani and Trypanosoma brucei.

Kinetoplastid parasites, including Leishmania and Trypanosoma spp., are life threatening pathogens with a worldwide distribution. Next-generation therapeutics for treatment are needed as current treatments have limitations, such as toxicity and drug resistance. In this study, we examined the activities of established mammalian target of rapamycin (mTOR)/phosphoinositide 3-kinase (PI3K) inhibitors against these tropical diseases. High-throughput screening of a library of 1742 bioactive compounds against intracellular L. donovani was performed, and seven mTOR/PI3K inhibitors were identified. Dose-dilution assays revealed that these inhibitors had half maximal effective concentration (EC50) values ranging from 0.14 to 13.44 µM for L. donovani amastigotes and from 0.00005 to 8.16 µM for T. brucei. The results of a visceral leishmaniasis mouse model indicated that treatment with Torin2, dactolisib, or NVP-BGT226 resulted in reductions of 35%, 53%, and 54%, respectively, in the numbers of liver parasites. In an acute T. brucei mouse model using NVP-BGT226 parasite numbers were reduced to under the limits of detection by five consecutive days of treatment. Multiple sequence and structural alignment results indicated high similarities between mTOR and kinetoplastid TORs; the inhibitors are predicted to bind in a similar manner. Taken together, these results indicated that the TOR pathways of parasites have potential for the discovery of novel targets and new potent inhibitors.

Discovery of a Brain-Penetrant ATP-Competitive Inhibitor of the Mechanistic Target of Rapamycin (mTOR) for CNS Disorders.

Recent clinical evaluation of everolimus for seizure reduction in patients with tuberous sclerosis complex (TSC), a disease with overactivated mechanistic target of rapamycin (mTOR) signaling, has demonstrated the therapeutic value of mTOR inhibitors for central nervous system (CNS) indications. Given that everolimus is an incomplete inhibitor of the mTOR function, we sought to develop a new mTOR inhibitor that has improved properties and is suitable for CNS disorders. Starting from an in-house purine-based compound, optimization of the physicochemical properties of a thiazolopyrimidine series led to the discovery of the small molecule 7, a potent and selective brain-penetrant ATP-competitive mTOR inhibitor. In neuronal cell-based models of mTOR hyperactivity, 7 corrected the mTOR pathway activity and the resulting neuronal overgrowth phenotype. The new mTOR inhibitor 7 showed good brain exposure and significantly improved the survival rate of mice with neuronal-specific ablation of the Tsc1 gene. These results demonstrate the potential utility of this tool compound to test therapeutic hypotheses that depend on mTOR hyperactivity in the CNS.

Matrine inhibits the development and progression of ovarian cancer by repressing cancer associated phosphorylation signaling pathways.

Ovarian cancer remains the most lethal gynecologic malignancy with late detection and acquired chemoresistance. Advanced understanding of the pathophysiology and novel treatment strategies are urgently required. A growing body of proteomic investigations suggest that phosphorylation has a pivotal role in the regulation of ovarian cancer associated signaling pathways. Matrine has been extensively studied for its potent anti-tumor activities. However, its effect on ovarian cancer cells and underlying molecular mechanisms remain unclear. Herein we showed that matrine treatment inhibited the development and progression of ovarian cancer cells by regulating proliferation, apoptosis, autophagy, invasion and angiogenesis. Matrine treatment retarded the cancer associated signaling transduction by decreasing the phosphorylation levels of ERK1/2, MEK1/2, PI3K, Akt, mTOR, FAK, RhoA, VEGFR2, and Tie2 in vitro and in vivo. Moreover, matrine showed excellent antitumor effect on chemoresistant ovarian cancer cells. No obvious toxic side effects were observed in matrine-administrated mice. As the natural agent, matrine has the potential to be the targeting drug against ovarian cancer cells with the advantages of overcoming the chemotherapy resistance and decreasing the toxic side effects.

A Conformational Restriction Strategy for the Identification of a Highly Selective Pyrimido-pyrrolo-oxazine mTOR Inhibitor.

The mechanistic target of rapamycin (mTOR) plays a pivotal role in growth and tumor progression and is an attractive target for cancer treatment. ATP-competitive mTOR kinase inhibitors (TORKi) have the potential to overcome limitations of rapamycin derivatives in a wide range of malignancies. Herein, we exploit a conformational restriction approach to explore a novel chemical space for the generation of TORKi. Structure-activity relationship (SAR) studies led to the identification of compound 12b with a ∼450-fold selectivity for mTOR over class I PI3K isoforms. Pharmacokinetic studies in male Sprague Dawley rats highlighted a good exposure after oral dosing and a minimum brain penetration. CYP450 reactive phenotyping pointed out the high metabolic stability of 12b. These results identify the tricyclic pyrimido-pyrrolo-oxazine moiety as a novel scaffold for the development of highly selective mTOR inhibitors for cancer treatment.

Angiotensin-converting enzyme 2 regulates autophagy in acute lung injury through AMPK/mTOR signaling.

Autophagy exerts a dual role in promoting cell death or survival. Recent studies have shown that it may play an important role in lipopolysaccharide (LPS)-induced acute lung injury (ALI). It was also suggested that angiotensin converting enzyme 2 (ACE2) may participate in the regulation of autophagy. The present study aims to investigate the role of autophagy in ALI and the involvement of ACE2. The regulation of the APMK/mTOR pathway was explored to clarify the underlying mechanism. The results showed that autophagy played an important role in ALI induced by LPS, as the autophagy inhibitor 3-methyladenine (3-MA) mitigated the severity of ALI. ACE2 activator resorcinolnaphthalein and inhibitor MLN-4760 significantly affected the histological appearance and wet/dry (W/D) ratio of the lung and altered the ACE2 activity of the lung, tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) levels in bronchoalveolar lavage fluid (BALF) and myeloperoxidase (MPO) levels in lung tissue. Furthermore, LPS, resorcinolnaphthalein and MLN-4760 significantly affected the expression of autophagy proteins Beclin-1, LC3-I and LC3-II. To explore the mechanism of ACE2 on lung autophagy, we measured the phosphorylation of AMPK/mTOR after mice were treated with LPS and resorcinolnaphthalein or MLN-4760. The results revealed that resorcinolnaphthalein and MLN-4760 both significantly altered the phosphorylation of AMPK/mTOR. Finally, we found that AMPK inhibitor (8-bAMP) and mTOR activator (propranolol) both abolished the effects of ACE2 activator (resorcinolnaphthalein) on the expression of lung autophagy proteins Beclin-1, LC3-I and LC3-II. In conclusion, these findings suggest that ACE2 could alleviate the severity of ALI, inflammation and autophagy in lung tissue through the AMPK/mTOR pathway.

A Binding Site Hotspot Map of the FKBP12-Rapamycin-FRB Ternary Complex by Photoaffinity Labeling and Mass Spectrometry-Based Proteomics.

Structural characterization of small molecule binding site hotspots within the global proteome is uniquely enabled by photoaffinity labeling (PAL) coupled with chemical enrichment and unbiased analysis by mass spectrometry (MS). MS-based binding site maps provide structural resolution of interaction sites in conjunction with identification of target proteins. However, binding site hotspot mapping has been confined to relatively simple small molecules to date; extension to more complex compounds would enable the structural definition of new binding modes in the proteome. Here, we extend PAL and MS methods to derive a binding site hotspot map for the immunosuppressant rapamycin, a complex macrocyclic natural product that forms a ternary complex with the proteins FKBP12 and FRB. Photo-rapamycin was developed as a diazirine-based PAL probe for rapamycin, and the FKBP12-photo-rapamycin-FRB ternary complex formed readily in vitro. Photoirradiation, digestion, and MS analysis of the ternary complex revealed a McLafferty rearrangement product of photo-rapamycin conjugated to specific surfaces on FKBP12 and FRB. Molecular modeling based on the binding site map revealed two distinct conformations of complex-bound photo-rapamycin, providing a 5.0 Å distance constraint between the conjugated residues and the diazirine carbon and a 9.0 Å labeling radius for the diazirine upon photoactivation. These measurements may be broadly useful in the interpretation of binding site measurements from PAL. Thus, in characterizing the ternary complex of photo-rapamycin by MS, we applied binding site hotspot mapping to a macrocyclic natural product and extracted precise structural measurements for interpretation of PAL products that may enable the discovery of new binding sites in the "undruggable" proteome.

Anticancer compound XL765 as PI3K/mTOR dual inhibitor: A structural insight into the inhibitory mechanism using computational approaches.

The PI3K-AKT-mTOR pathway is often a commonly disrupted pathway in human cancer and, therefore, it is widely exploited for cancer therapy. The inhibitors for the important proteins of the pathway including PI3K and mTOR have been increasingly designed. The dual inhibitors targeting PI3K and mTOR both have proven to be more effective than those targeting single protein only. An orally-active compound XL765 is well established as PI3K/mTOR dual inhibitor and have shown in vitro and in vivo anticancer activity against a variety of cancer types and is undergoing clinical trials. The present study explored the exact binding pose and the the interactive forces holding XL765 within the active sites of PI3Kγ and mTOR using molecular docking analyses. The XL765 interacting residues of both the proteins were delineated and the degree of participation in binding was estimated by various methods. In the process, among the interacting residues of PI3Kγ, the Lys-890 and the Met-953 were recognized as the key residues involved in XL765 binding. While, in mTOR case, the Trp-2239 was recognized as the key residue playing role in the XL765 binding. In order to explore the better inhibitors, the study also generated combinatorial chemical library by modifying the scaffold considered from XL765. The virtual screening of the generated compound library led to identification of six novel promising compounds proposed as PI3K/mTOR dual inhibitors. Thus, the present work will through light on the drug inhibitory mechanism of XL765 for PI3K and mTOR, and will also assist in designing novel efficacious drug candidates.