Active-Site Inhibitors of mTOR Target Rapamycin-Resistant Outputs of mTORC1 and mTORC2

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.     

CSN5/Jab1 controls multiple events in the mammalian cell cycle

The COP9 signalosome (CSN) complex is critical for mammalian cell proliferation and survival, but it is not known how the CSN affects the cell cycle. In this study, MEFs lacking CSN5/Jab1 were generated using a CRE-flox system. MEFs ceased to proliferate upon elimination of CSN5/Jab1. Rescue experiments indicated that the JAMM domain of CSN5/Jab1 was essential. CSN5/Jab1-elimination enhanced the neddylation of cullins 1 and 4 and altered the expression of many factors including cyclin E and p53. CSN5/Jab1-elimination inhibited progression of the cell cycle at multiple points, seemed to initiate p53-independent senescence and increased the ploidy of cells. Thus, CSN5/Jab1 controls different events of the cell cycle, preventing senescence and endocycle as well as the proper progression of the somatic cell cycle.

Structured summary

MINT-8046253: Csn1 (uniprotkb:Q99LD4) physically interacts (MI:0914) with Csn5 (uniprotkb:O35864), Csn8 (uniprotkb:Q8VBV7), Csn3 (uniprotkb:O88543), Csn7b (uniprotkb:Q8BV13) and Csn6 (uniprotkb:O88545) by anti bait coimmunoprecipitation (MI:0006)     

Discovery of a novel series of CXCR3 antagonists

The discovery of a novel series of CXCR3 antagonists is described. Starting from an HTS positive, iterative optimization gave potent compounds (IC₅₀ 15 nM in a chemotaxis assay). The strategy employed to improve the metabolic stability of these derivatives is described.     

Transferrin Receptor 2 Is Frequently and Highly Expressed in Glioblastomas

Under physiological conditions, transferrin receptor 2 (TfR2) is expressed in the liver and its balance is related to the cell cycle rather than to intracellular iron levels. We recently showed that TfR2 is highly expressed in glioblastoma cell lines. Here, we demonstrate that, in these cells, TfR2 appears to localize in lipid rafts, induces extracellular signal-regulated kinase 1/2 phosphorylation after transferrin binding, and contributes to cell proliferation, as shown by RNA silencing experiments. In vitro hypoxic conditions induce a significant TfR2 up-regulation, suggesting a role in tumor angiogenesis. As assessed by immunohistochemistry, the level of TfR2 expression in astrocytic tumors is related to histologic grade, with the highest expression observed in glioblastomas. The level of TfR2 expression represents a favorable prognostic factor, which is associated with the higher sensitivity to temozolomide of TfR2-positive tumor cells in vitro. The endothelial cells of glioblastoma vasculature also stain for TfR2, whereas those of the normal brain vessels do not. Importantly, TfR2 is expressed by the subpopulation of glioblastoma cells with properties of cancer-initiating cells. TfR2-positive glioblastoma cells retain their TfR2 expression on xenografting in immunodeficient mice. In conclusion, our observations demonstrate that TfR2 is a neoantigen for astrocytomas that seems attractive for developing target therapies.     

Human recombinant prolidase from eukaryotic and prokaryotic sources. Expression, purification, characterization and long-term stability studies

Prolidase is a Mn(2+)-dependent dipeptidase that cleaves imidodipeptides containing C-terminal proline or hydroxyproline. In humans, a lack of prolidase activity causes prolidase deficiency, a rare autosomal recessive disease, characterized by a wide range of clinical outcomes, including severe skin lesions, mental retardation, and infections of the respiratory tract. In this study, recombinant prolidase was produced as a fusion protein with an N-terminal histidine tag in eukaryotic and prokaryotic hosts and purified in a single step using immobilized metal affinity chromatography. The enzyme was characterized in terms of activity against different substrates, in the presence of various bivalent ions, in the presence of the strong inhibitor Cbz-Pro, and at different temperatures and pHs. The recombinant enzyme with and without a tag showed properties mainly indistinguishable from those of the native prolidase from fibroblast lysate. The protein yield was higher from the prokaryotic source, and a detailed long-term stability study of this enzyme at 37 degrees C was therefore undertaken. For this analysis, an 'on-column' digestion of the N-terminal His tag by Factor Xa was performed. A positive effect of Mn(2+) and GSH in the incubation mixture and high stability of the untagged enzyme are reported. Poly(ethylene glycol) and glycerol had a stabilizing effect, the latter being the more effective. In addition, no significant degradation was detected after up to 6 days of incubation with cellular lysate. Generation of the prolidase in Escherichia coli, because of its high yield, stability, and similarity to native prolidase, appears to be the best approach for future structural studies and enzyme replacement therapy.     

Role of a heterogeneous free state in the formation of a specific RNA-theophylline complex

The helical regions of RNA are generally very stable, but the single-stranded and loop regions often exist as an ensemble of conformations in solution. The theophylline-binding RNA aptamer forms a very stable structure when bound to the bronchodilator theophylline, but the theophylline binding site is not stably formed in the absence of ligand. The kinetics for theophylline binding were measured here by stopped-flow fluorescence spectroscopy to probe the mechanism for theophylline binding in this RNA aptamer. The kinetic studies showed that formation of the RNA-theophylline complex is over 1000 times slower than a diffusion-controlled rate, and the high affinity of the RNA-theophylline complex arises primarily from a slow dissociation rate for the complex. A theophylline-independent rate was observed for formation of the theophylline-RNA complex at high theophylline concentration, indicating that a conformational change in the RNA is the rate-limiting step in complex formation under these conditions. The RNA-theophylline complex requires divalent metal ions, such as Mg2+, to form a high-affinity complex, and there is a greater than 10000-fold reduction in affinity for theophylline in the absence of Mg2+. This decrease in binding affinity in the absence of Mg2+ results primarily from an increased dissociation rate for the complex. The implications of an ensemble of conformations in the free state of this theophylline-binding RNA are discussed and compared with mechanisms for formation of protein-ligand complexes.     

The suitability of disintegrating force kinetics for studying the effect of manufacturing parameters on spironolactone tablet properties

The aim of this paper was to study the effect of the granulate properties and tablet compression force on disintegrating force behavior in order to investigate the capability of the disintegrating force to characterize tablets that have the same composition but were manufactured in different conditions. Several tablets containing spironolactone in the external or internal granulated mixture of calcium carbonate and maize starch differing in particle size distribution, were prepared at 3 compression levels. The force developed by tablets during water uptake and disintegration was measured and plotted versus time. The curves obtained were analyzed by the Weibull equation in order to calculate the parameters characterizing the tablet disintegration kinetics. The disintegrating force time parameter, the maximum force developed, and the area under the curve were determined. In general, the reduction of time parameter value and/or the increase in maximum force developed corresponded to an acceleration in tablet disintegration. In addition, the area under the force curve increased in stronger tablets, monitoring in a sensitive way the tablet structural changes introduced by compression force. The results showed that the disintegrating force measurement can detect small changes in the structure of the tablet that cannot be discriminated by pharmacopoeia tests. The effect of manufacturing, in particular compression force, on tablet properties was quantified by the parameters of disintegrating force kinetics.     

The global conformation of the hammerhead ribozyme determined using residual dipolar couplings

The global structure of the hammerhead ribozyme was determined in the absence of Mg(2+) by solution NMR experiments. The hammerhead ribozyme motif forms a branched structure consisting of three helical stems connected to a catalytic core. The (1)H-(15)N and (1)H-(13)C residual dipolar couplings were measured in a set of differentially (15)N/(13)C-labeled ribozymes complexed with an unlabeled noncleavable substrate. The residual dipolar couplings provide orientation information on both the local and the global structure of the molecule. Analysis of the residual dipolar couplings demonstrated that the local structure of the three helical stems in solution is well modeled by an A-form conformation. However, the global structure of the hammerhead in solution in the absence of Mg(2+) is not consistent with the Y-shaped conformation observed in crystal structures of the hammerhead. The residual dipolar couplings for the helical stems were combined with standard NOE and J coupling constant NMR data from the catalytic core. The NOE data show formation of sheared G-A base pairs in domain 2. These NMR data were used to determine the global orientation of the three helical stems in the hammerhead. The hammerhead forms a rather extended structure under these conditions with a large angle between stems I and II ( approximately 153 degrees ), a smaller angle between stems II and III ( approximately 100 degrees ), and the smallest angle between stems I and III ( approximately 77 degrees ). The residual dipolar coupling data also contain information on the dynamics of the molecule and were used here to provide qualitative information on the flexibility of the helical domains in the hammerhead ribozyme-substrate complex.     

Solution structure of the calcium channel antagonist omega-conotoxin GVIA.

The three-dimensional solution structure is reported for omega-conotoxin GVIA, which is a potent inhibitor of presynaptic calcium channels in vertebrate neuromuscular junctions. Structures were generated by a hybrid distance geometry and restrained molecular dynamics approach using interproton distance, torsion angle, and hydrogen-bonding constraints derived from 1H NMR data. Conformations of GVIA with low constraint violations converged to a common peptide fold. The secondary structure in the peptide is an antiparallel triple-stranded beta-sheet containing a beta-hairpin and three tight turns. The NMR data are consistent with the region of the peptide from residues S9 to C16 being more dynamic than the rest of the peptide. The peptide has an amphiphilic structure with a positively charged hydrophilic side and an opposite side that contains a small hydrophobic region. Residues that are thought to be important in binding and function are located on the hydrophilic face of the peptide.