Functionalized pyrrolidines as alpha-mannosidase inhibitors and growth inhibitors of human glioblastoma and melanoma cells

New substituted pyrrolidine-3,4-diol derivatives were prepared from D-(-)- and L-(+)-phenyl glycinol. The influence of the configuration and the substitution of the lateral side chain of these derivatives on the inhibition of 25 commercial glycosidases were determined. (2R,3R,4S)-2-({[(1R)-2-Hydroxy-1-phenylethyl]amino}methyl)pyrrolidine-3,4-diol ((+)-7a) was a potent and selective inhibitor of jack bean alpha-mannosidase (Ki = 135 nM). However, when evaluated on human tumor cells, 7a, and the reference compound swainsonine, did not efficiently inhibit the growth of glioblastoma cells. Further derivatization of the hydroxyl group with lipophilic groups to increase bioavailability improved their growth inhibitory properties for human glioblastoma and melanoma cells. In particular the 4-bromobenzoyl derivative 26 demonstrated high efficacy for human tumor cells whereas primary human fibroblasts were less sensitive to 26. Therefore functionalized pyrrolidines have the potential to inhibit the growth of tumor cells and display selectivity for tumor cells compared to normal cells

Modèle in vitro d'infection urinaire sur cathéter à Pseudomonas aeruginosa traitée par ciprofloxacine : rôle prépondérant de la persistance sur la résistance

International audienc

Increasing the sensitivity of NMR diffusion measurements by paramagnetic longitudinal relaxation enhancement, with application to ribosome–nascent chain complexes

The translational diffusion of macromolecules can be examined non-invasively by stimulated echo (STE) NMR experiments to accurately determine their molecular sizes. These measurements can be important probes of intermolecular interactions and protein folding and unfolding, and are crucial in monitoring the integrity of large macromolecular assemblies such as ribosome–nascent chain complexes (RNCs). However, NMR studies of these complexes can be severely constrained by their slow tumbling, low solubility (with maximum concentrations of up to 10 μM), and short lifetimes resulting in weak signal, and therefore continuing improvements in experimental sensitivity are essential. Here we explore the use of the paramagnetic longitudinal relaxation enhancement (PLRE) agent NiDO2A on the sensitivity of 15N XSTE and SORDID heteronuclear STE experiments, which can be used to monitor the integrity of these unstable complexes. We exploit the dependence of the PLRE effect on the gyromagnetic ratio and electronic relaxation time to accelerate recovery of 1H magnetization without adversely affecting storage on N z during diffusion delays or introducing significant transverse relaxation line broadening. By applying the longitudinal relaxation-optimized SORDID pulse sequence together with NiDO2A to 70S Escherichia coli ribosomes and RNCs, NMR diffusion sensitivity enhancements of up to 4.5-fold relative to XSTE are achieved, alongside ~1.9-fold improvements in two-dimensional NMR sensitivity, without compromising the sample integrity. We anticipate these results will significantly advance the use of NMR to probe dynamic regions of ribosomes and other large, unstable macromolecular assemblies

An analytic and systematic framework for estimating metabolic flux ratios from 13C tracer experiments

<p>Abstract</p> <p>Background</p> <p>Metabolic fluxes provide invaluable insight on the integrated response of a cell to environmental stimuli or genetic modifications. Current computational methods for estimating the metabolic fluxes from ¹³<it>C </it>isotopomer measurement data rely either on manual derivation of analytic equations constraining the fluxes or on the numerical solution of a highly nonlinear system of isotopomer balance equations. In the first approach, analytic equations have to be tediously derived for each organism, substrate or labelling pattern, while in the second approach, the global nature of an optimum solution is difficult to prove and comprehensive measurements of external fluxes to augment the ¹³<it>C </it>isotopomer data are typically needed.</p> <p>Results</p> <p>We present a novel analytic framework for estimating metabolic flux ratios in the cell from ¹³<it>C </it>isotopomer measurement data. In the presented framework, equation systems constraining the fluxes are derived automatically from the model of the metabolism of an organism. The framework is designed to be applicable with all metabolic network topologies, ¹³<it>C </it>isotopomer measurement techniques, substrates and substrate labelling patterns.</p> <p>By analyzing nuclear magnetic resonance (NMR) and mass spectrometry (MS) measurement data obtained from the experiments on glucose with the model micro-organisms <it>Bacillus subtilis </it>and <it>Saccharomyces cerevisiae </it>we show that our framework is able to automatically produce the flux ratios discovered so far by the domain experts with tedious manual analysis. Furthermore, we show by <it>in silico </it>calculability analysis that our framework can rapidly produce flux ratio equations – as well as predict when the flux ratios are unobtainable by linear means – also for substrates not related to glucose.</p> <p>Conclusion</p> <p>The core of ¹³<it>C </it>metabolic flux analysis framework introduced in this article constitutes of flow and independence analysis of metabolic fragments and techniques for manipulating isotopomer measurements with vector space techniques. These methods facilitate efficient, analytic computation of the ratios between the fluxes of pathways that converge to a common junction metabolite. The framework can been seen as a generalization and formalization of existing tradition for computing metabolic flux ratios where equations constraining flux ratios are manually derived, usually without explicitly showing the formal proofs of the validity of the equations.</p

Oxygen dependence of metabolic fluxes and energy generation of Saccharomyces cerevisiae CEN.PK113-1A

<p>Abstract</p> <p>Background</p> <p>The yeast <it>Saccharomyces cerevisiae </it>is able to adjust to external oxygen availability by utilizing both respirative and fermentative metabolic modes. Adjusting the metabolic mode involves alteration of the intracellular metabolic fluxes that are determined by the cell's multilevel regulatory network. Oxygen is a major determinant of the physiology of <it>S. cerevisiae </it>but understanding of the oxygen dependence of intracellular flux distributions is still scarce.</p> <p>Results</p> <p>Metabolic flux distributions of <it>S. cerevisiae </it>CEN.PK113-1A growing in glucose-limited chemostat cultures at a dilution rate of 0.1 h^-1with 20.9%, 2.8%, 1.0%, 0.5% or 0.0% O₂in the inlet gas were quantified by ¹³C-MFA. Metabolic flux ratios from fractional [U-¹³C]glucose labelling experiments were used to solve the underdetermined MFA system of central carbon metabolism of <it>S. cerevisiae</it>.</p> <p>While ethanol production was observed already in 2.8% oxygen, only minor differences in the flux distribution were observed, compared to fully aerobic conditions. However, in 1.0% and 0.5% oxygen the respiratory rate was severely restricted, resulting in progressively reduced fluxes through the TCA cycle and the direction of major fluxes to the fermentative pathway. A redistribution of fluxes was observed in all branching points of central carbon metabolism. Yet only when oxygen provision was reduced to 0.5%, was the biomass yield exceeded by the yields of ethanol and CO₂. Respirative ATP generation provided 59% of the ATP demand in fully aerobic conditions and still a substantial 25% in 0.5% oxygenation. An extensive redistribution of fluxes was observed in anaerobic conditions compared to all the aerobic conditions. Positive correlation between the transcriptional levels of metabolic enzymes and the corresponding fluxes in the different oxygenation conditions was found only in the respirative pathway.</p> <p>Conclusion</p> <p>¹³C-constrained MFA enabled quantitative determination of intracellular fluxes in conditions of different redox challenges without including redox cofactors in metabolite mass balances. A redistribution of fluxes was observed not only for respirative, respiro-fermentative and fermentative metabolisms, but also for cells grown with 2.8%, 1.0% and 0.5% oxygen. Although the cellular metabolism was respiro-fermentative in each of these low oxygen conditions, the actual amount of oxygen available resulted in different contributions through respirative and fermentative pathways.</p

A retrospective analysis of mortality after hip fracture repair according to the anaesthetic strategy

Introduction : La fracture du fémur proximal touche essentiellement une population âgée souffrant de multiples comorbidités. La majorité des fractures sont traitées chirurgicalement. La rachianesthésie diminue les complications postopératoires. L’objectif de cette étude est d’examiner la morbidité et la mortalité après une chirurgie d’une fracture du fémur proximal en fonction de la stratégie anesthésique dans notre institution. Méthode : Après accord de la commission d’éthique, nous avons étudié de manière rétrospective les patients de plus de 50 ans opérés d’une fracture du fémur proximal au CHUV durant l’année 2014. Outre la stratégie anesthésique appliquée (rachianesthésie ou anesthésie générale), les informations suivantes ont, entre autres, été extraites : délai entre l’admission et la chirurgie, taux d’hémoglobine (g/l), créatininémie (μmol/l), présence de comorbidités (fibrillation auriculaire, démence, insuffisance cardiaque), complications postopératoires (syndrome coronarien, fibrillation auriculaire, décompensation cardiaque, embolie pulmonaire, pneumonie, péjoration de la fonction rénale, infection urinaire, état confusionnel). La section d’analyse statistique de l’Etat de Vaud (Statistique Vaud) nous a fourni de manière anonyme la mortalité à 7 jours, 1 mois, 3 mois, 6 mois, et 12 mois. Les analyses statistiques univariées et multivariées ont été réalisées avec le logiciel STATA version 14.1 (StataCorp, College Station, TX, USA). Résultats : L’analyse s’est portée sur un collectif de 355 patients. Nous avons constaté que 25% des patients sont décédés dans les 12 mois qui ont suivi la chirurgie. La mortalité était plus importante dans le groupe rachianesthésie à 3 mois (p=0.03), 6 mois (p=0.03) et 12 mois (p=0.02) en analyse univariée. L’influence de la rachianesthésie sur la mortalité s’est montrée non significative lors de l’analyse multivariée à 3 mois (OR : 1.44, IC 95% 0.6-3.5, p=0.42), 6 mois (OR : 1.27, IC 95% 0.6-2.7, p=0.55) et 12 mois (OR : 1.76, IC 95% 0.9-3.6, p=0.11). Nous avons constaté que l’âge (p=0.04) ou la présence d’une pneumonie postopératoire (p=0.04) étaient liés à une augmentation de la mortalité à 6 mois et que la présence d’une démence (p=0.04) ou d’une péjoration de la fonction rénale postopératoire (p=0.03) étaient liées à une augmentation de la mortalité à 12 mois. Conclusion : La stratégie anesthésique n’a pas montré d’association significative avec la mortalité de 7 jours à 12 mois. La mortalité à 6 mois est augmentée lors de la présence d’une pneumonie postopératoire et avec l’âge. La mortalité à 12 mois est augmentée lors de démence ou de péjoration de la fonction rénale postopératoire

Pyrrolidine derivatives as new inhibitors of alpha-mannosidases and growth inhibitors of human cancer cells

New pyrrolidine-3,4-diol derivatives were prepared from D-(-)- and L(+)-phenylglycinol and tested for their ability to inhibit 25 commercial glycosidases. The influence of the substitution of the lateral side chain and of the pyrrolidine ring on the enzyme inhibition was evaluated. (2R,3R,4S)-2-({[(1R)-2-Hydroxy-1-phenylethyl]amino}methyl)pyrrolidine-3,4-diol was a potent and selective inhibitor of α-mannosidase from jack bean. This compound was derivatized into lipophilic esters in order to allow its internalization by human cancer cells. In particular, the 4-bromobenzoate derivative demonstrated promising inhibition of glioblastoma and melanoma cells whereas it was less effective on healthy human fibroblasts

Synthesis of new pyrrolidine derivatives as inhibitors of alpha-mannosidase and of the growth of human glioblastoma cells

New 2-benzylamino-3,4-dihydroxypyrrolidines bearing aromatic and aliphatic amido side chains have been prepared. The influence of the amido substituents on the inhibitory activity of these diamines toward 24 commercially available glycosidases was determined. The most potent and selective a-mannosidase inhibitor (6d) (N-[(2R)-2-([(2R,3R,4S)-3,4-dihydroxypyrrolidin2-yl]methyl amino)-2-phenylethyl]-3-bromobenzamide) of these series was also the most potent inhibitor of the growth of human glioblastoma cells