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1.
Chemistry ; 2020 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-32633095

RESUMO

Novel 6-alkyl- and 6-alkenyl-3-fluoro-2-pyridinaldoximes have been synthesised by using a mild and efficient chemoselective hydrogenation of 6-alkynyl-3-fluoro-2-pyridinaldoxime scaffolds, without altering the reducible, unprotected, sensitive oxime functionality and the C-F bond. These novel 6-alkyl-3-fluoro-2-pyridinaldoximes may find medicinal application as antidotes to organophosphate poisoning. Indeed, one low-molecular-weight compound exhibited increased affinity for sarin-inhibited acetylcholinesterase (hAChE) and greater reactivation efficiency or resurrection for sarin-inhibited hAChE, compared with those of 2-pyridinaldoxime (2-PAM) and 1-({[4-(aminocarbonyl)pyridinio]methoxy}methyl)-2-[(hydroxyimino)methyl]pyridinium chloride (HI-6), two pyridinium salts currently used as antidote by several countries. In addition, the uncharged 3-fluorinated bifunctional hybrid showed increased in vitro blood-brain barrier permeability compared with those of 2-PAM, HI-6 and obidoxime. These promising features of novel low-molecular-weight alkylfluoropyridinaldoxime open up a new era for the design, synthesis and discovery of central non-quaternary broad spectrum reactivators for organophosphate-inhibited cholinesterases.

2.
Biomolecules ; 10(6)2020 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-32512884

RESUMO

(1) Background: Human exposure to organophosphorus compounds employed as pesticides or as chemical warfare agents induces deleterious effects due to cholinesterase inhibition. One therapeutic approach is the reactivation of inhibited acetylcholinesterase by oximes. While currently available oximes are unable to reach the central nervous system to reactivate cholinesterases or to display a wide spectrum of action against the variety of organophosphorus compounds, we aim to identify new reactivators without such drawbacks. (2) Methods: This study gathers an exhaustive work to assess in vitro and in vivo efficacy, and toxicity of a hybrid tetrahydroacridine pyridinaldoxime reactivator, KM297, compared to pralidoxime. (3) Results: Blood-brain barrier crossing assay carried out on a human in vitro model established that KM297 has an endothelial permeability coefficient twice that of pralidoxime. It also presents higher cytotoxicity, particularly on bone marrow-derived cells. Its strong cholinesterase inhibition potency seems to be correlated to its low protective efficacy in mice exposed to paraoxon. Ventilatory monitoring of KM297-treated mice by double-chamber plethysmography shows toxic effects at the selected therapeutic dose. This breathing assessment could help define the No Observed Adverse Effect Level (NOAEL) dose of new oximes which would have a maximum therapeutic effect without any toxic side effects.

3.
J Chromatogr A ; 1623: 461209, 2020 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-32505293

RESUMO

In most cases, determination of binding constant for analyte-cyclodextrin complexes in capillary electrophoresis is investigated by affinity capillary electrophoresis using a UV detector (ACE-UV). The limitations induced by the UV-detector include : (i) the difficulty of dealing with poor chromogenic analytes and more generally with any analyte presenting strong affinity towards the cyclodextrin (CD), i.e. for which the prerequisite to work with analyte concentration much smaller than those of the CD is difficult to fulfill (ii) the impossibility of studying non-chromogenic analyte. In this paper, two simple methodologies were developed to overcome these limitations. Regarding the analytes which present poor UV-absorbance and/or very high CD-affinity, a methodology using an algorithmic data treatment and taking into account the real analyte concentration in the capillary at the determined migration times allows to correctly estimate the binding constants, even if the experimental prerequisite ([analyte]<<[CD]) is not complied. Moreover, it is proved that classical linearization treatment by picking the migration time of the infinite diluted analytes (at the start of the peak) also provide satisfactory results. Regarding UV-transparent analyte, a competitive methodology combined with algorithmic data treatment allows the determination of their affinity towards cyclodextrins. Last, the applicability of the described competitive method is extended to the study of interaction between two neutral partners, which is another well-known limitation of ACE.


Assuntos
Ciclodextrinas/química , Eletroforese Capilar/métodos , Raios Ultravioleta , Ibuprofeno/análise , Cinética , Modelos Lineares , Termodinâmica
5.
Bioorg Med Chem ; 28(10): 115463, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32241621

RESUMO

1-Phenyl-3-methyl-5-pyrazolone is a reagent, known as PMP, used to derivatize monosaccharides for the study of polysaccharides composition and structure, and for the dosage of carbohydrates in complex media. The same molecule is also known as edaravone, a drug approved for the treatment of stroke and amyotrophic lateral sclerosis. It is also a reactive molecule susceptible to form stable adducts with aromatic aldehydes, such as formylpterin and vanillin. In addition, the molecule serves as a scaffold to design of edaravone analogs and drug conjugates, with various pharmacological properties (antioxidant, anticancer, antiviral). We have analyzed the multiple usages of PMP/edaravone to highlight the reactivity of the molecule and its wide range of applications. This phenyl-pyrazolone compound, considered by many as a biochemical reagent and by other as a clinically useful drug, has not yet revealed the full extent of its capacities and benefits.

6.
J Pharm Biomed Anal ; 181: 113063, 2020 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-31927338

RESUMO

Di(2-ethylhexyl) phthalate (DEHP) is a common plasticizer that is largely used for PVC blood bags. The migration of DEHP from medical devices into labile blood products (LBP) is a well-known situation. While DEHP has beneficial effects on the storage of red blood cells, it can have toxicological impact due to its potential reprotoxic effects (classified group 1B). Since July 1st, 2015, the French law prohibits the use of tubing made in DEHP-plasticized PVC in paediatric, neonatal and maternity wards. This provision, which could extend in several years more widely to medical devices used for drugs infusion, dialysis, feeding and blood bags, has led manufacturers to replace DEHP to alternative plasticizers such as diisononylcyclohexane-1,2-dicarboxylate (DINCH). In this paper, a liquid chromatography-tandem mass spectrometry (LCMS/MS) method has been developed and validated for the determination of DEHP, DINCH and their corresponding monoester metabolites (MEHP and MINCH) in four labile blood products (LBP): whole blood (WB), red cells concentrate (RCC), plasma and platelet concentrate (PC). Due to strong contamination of blank LBP by DEHP because of its ubiquitous presence in working environment and despite the attention paid to avoid contamination of solvents and glassware, a trap chromatographic column was implemented between the solvent mixing chamber and the injector of the LC system. This set-up permitted to discriminate DEHP present in the sample to DEHP brought by the environmental contamination. In the optimized conditions, all compounds were separated in less than 10 min. The analytes were extracted from LBP samples using a liquid-liquid extraction. After optimization, recoveries were ranged from 47 to 96 %, depending on the analytes and the nature of LBP. Except for DEHP which exhibited RSD values of intermediate precision higher than 20 % at a concentration of 25 nM, all the precision results (repeatability and intermediate precision) were lower than 16 % and trueness values ranged from -16.2-19.8%. Using the validated method, the leachability of DEHP and DINCH from corresponding PVC-blood bags was investigated and the concentrations of their corresponding metabolites, MEHP and MINCH, were determined in whole blood, red cells concentrate, plasma and platelet concentrate.

7.
Metabolism ; 103: 154042, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31785259

RESUMO

OBJECTIVE: Bile acids (BAs) are signaling molecules controlling lipid and glucose metabolism. Since BA alterations are associated with obesity and insulin resistance, plasma BAs have been considered candidates to predict type 2 diabetes (T2D) risk. We aimed to determine (1) the association of BAs with glucose homeostasis parameters and (2) their predictive association with the risk of conversion from prediabetes to new-onset diabetes (NOD) in a prospective cohort study. DESIGN: 205 patients with impaired fasting glucose (IFG) were followed each year during 5 years in the IT-DIAB cohort study. Twenty-one BA species and 7α-hydroxy-4-cholesten-3-one (C4), a marker of BA synthesis, were quantified by LC/MS-MS in plasma from fasted patients at baseline. Correlations between plasma BA species and metabolic parameters at baseline were assessed by Spearman's coefficients and the association between BAs and NOD was determined using Cox proportional-hazards models. RESULTS: Among the analyzed BA species, total hyocholic acid (HCA) and the total HCA/total chenodeoxycholic acid (CDCA) ratio, reflecting hepatic BA 6α-hydroxylation activity, negatively correlated with BMI and HOMA-IR. The total HCA/total CDCA ratio also correlated negatively with HbA1C. Conversion from IFG to NOD occurred in 33.7% of the participants during the follow-up. Plasma BA species were not independently associated with the conversion to NOD after adjustment with classical T2D risk factors. CONCLUSIONS: Fasting plasma BAs are not useful clinical biomarkers for predicting NOD in patients with IFG. However, an unexpected association between 6α-hydroxylated BAs and glucose parameters was found, suggesting a role for this specific BA pathway in metabolic homeostasis. IT-DIAB study registry number: NCT01218061.


Assuntos
Ácidos e Sais Biliares/sangue , Diabetes Mellitus Tipo 2/diagnóstico , Intolerância à Glucose/diagnóstico , Glucose/metabolismo , Estado Pré-Diabético/diagnóstico , Estado Pré-Diabético/patologia , Adulto , Idoso , Biomarcadores/sangue , Estudos de Coortes , Diabetes Mellitus Tipo 2/metabolismo , Progressão da Doença , Feminino , Seguimentos , Intolerância à Glucose/metabolismo , Humanos , Resistência à Insulina/fisiologia , Masculino , Pessoa de Meia-Idade , Estado Pré-Diabético/metabolismo , Valor Preditivo dos Testes , Estudos Prospectivos
8.
Food Chem ; 304: 125448, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31491713

RESUMO

Blood, from slaughterhouses, is an inevitable part of meat production, causing environmental problems due to the large volumes recovered and its low valorization. However, the α137-141 peptide, a natural antimicrobial peptide, can be obtained after hydrolysis of hemoglobin, the main constituent of blood red part. To recover it at a sufficient concentration for antimicrobial applications, a new sustainable technology, called electrodialysis with ultrafiltration membrane (EDUF), was investigated. The α137-141 concentration was increased about 4-fold at a feed peptide concentration of 8% with an enrichment factor above 24-fold. This feed peptide concentration also needed the lowest relative energy consumption. Moreover, this peptide fraction protected meat against microbial growth, as well as rancidity, during 14 days under refrigeration. This peptide fraction was validated as a natural preservative and substitute for synthetic additives against food spoilage. Finally, producing antimicrobial/antioxidant peptide from wastes by EDUF fits perfectly with the concept of circular economy.


Assuntos
Anti-Infecciosos/farmacologia , Sangue , Produtos da Carne/análise , Peptídeos/farmacologia , Matadouros , Animais , Antioxidantes/farmacologia , Conservantes de Alimentos , Refrigeração , Ultrafiltração
9.
Pharmacol Ther ; 203: 107396, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31356908

RESUMO

Ursodeoxycholic acid (UDCA) is a secondary bile acid issued from the transformation of (cheno)deoxycholic acid by intestinal bacteria, acting as a key regulator of the intestinal barrier integrity and essential for lipid metabolism. UDCA is also a long-established drug, largely used for the dissolution of cholesterol gallstones, the treatment of primary biliary cholangitis and other hepatobiliary disorders. The history of UDCA is briefly retraced here as well as its multifactorial mechanism of action, based on its anti-inflammatory, antioxidant and cytoprotective activities. The present review is centred around the anticancer properties of UDCA and synthetic antitumor derivatives designed over the past 20 years. Paradoxically, depending on the conditions, UDCA exhibits both pro- and anti-apoptotic properties toward different cell types. In particular, the UDCA drug can protect epithelial cells from damages and apoptosis while inducing inhibition of proliferation and apoptotic and/or autophagic death of cancer cells. The effects of UDCA on cancer cell migration, cancer stem cells and drug-induced dysbiosis are also evoked. The drug has revealed modest activities against colon and gastric cancers but may be useful to improve treatments of hepatocellular carcinoma, notably in combination with other drugs such as sorafenib. UDCA can also protect from damages induced by cancer chemotherapeutic agents. The potential of UDCA in cancer, as a chemo-protecting or chemotherapeutic agent, is highlighted here as well as the design of tumour-active derivatives, including UDCA-drug conjugates. A repurposing of UDCA in oncology should be further considered.

10.
Exp Physiol ; 104(2): 254-263, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30561141

RESUMO

NEW FINDINGS: What is the central question of this study? Is there an association of plasma concentration of asymmetric dimethylarginine, which is related to exercise capacity in patients with cardiovascular diseases, with oxygen delivery and subsequently exercise capacity in healthy subjects in the absence of the potentially confounding influence of inflammation and oxidative stress? What is the main finding and its importance? Plasma asymmetric dimethylarginine concentrations are not related to exercise capacity in healthy subjects, while O2 delivery in the working skeletal muscle during the maximal graded-exercise test is not associated with any of the l-arginine analogues. ADMA alone does not play a crucial role in local muscle perfusion and in maintaining exercise capacity. ABSTRACT: Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthesis that could limit oxygen (O2 ) delivery in the working skeletal muscles by altering endothelium-dependent vasodilatation. Exercise capacity is associated with plasma ADMA concentrations in patients with cardiovascular diseases, but this issue has still not been investigated in healthy subjects. We aimed to determine whether plasma ADMA concentrations were negatively associated with exercise capacity in young healthy male subjects. Ten men with maximal oxygen uptake ( V ̇ O 2 max ) > 65 mL kg-1  min-1 were included in the high exercise capacity group (HI-FIT), and 10 men with V ̇ O 2 max  < 45 mL kg-1  min-1 were included in the low exercise capacity group (LO-FIT). Plasma ADMA and other l-arginine analogue concentrations were measured before and after a maximal graded-exercise test by liquid chromatography-tandem mass spectrometry. Microvascular O2 delivery during exercise was estimated through the pattern from the sigmoid model of muscle deoxygenation in the vastus lateralis measured by near infrared spectroscopy. V ̇ O 2 max was 60% higher in the HI-FIT group (median: 70.2 mL kg-1  min-1 ; IQR: 68.0-71.9 mL kg-1  min-1 ) than in the LO-FIT group (median: 43.8 mL kg-1  min-1 ; IQR: 34.8-45.3 mL kg-1  min-1 ). Plasma ADMA concentrations did not differ between the LO-FIT and HI-FIT groups before (0.50 ± 0.06 vs. 0.54 ± 0.07 µmol L-1 , respectively) and after the maximal incremental exercise test (0.49 ± 0.08 vs. 0.55 ± 0.03 µmol L-1 , respectively). There was no significant association of plasma ADMA concentrations with the pattern of local muscle deoxygenation and exercise capacity. Exercise capacity and microvascular O2 delivery are not related to plasma ADMA concentrations in young healthy male subjects. Our findings show that ADMA does not play a crucial role in local muscle perfusion and in maintaining exercise capacity without pathological conditions.


Assuntos
Arginina/análogos & derivados , Exercício Físico/fisiologia , Oxigênio/metabolismo , Resistência Física/fisiologia , Adulto , Arginina/sangue , Arginina/metabolismo , Treino Aeróbico/métodos , Teste de Esforço , Humanos , Masculino , Músculos/metabolismo , Óxido Nítrico/metabolismo
11.
Artigo em Inglês | MEDLINE | ID: mdl-29692758

RESUMO

A qualitative study is presented, where the main question was whether food-derived hemorphins, i.e., originating from digested alimentary hemoglobin, could pass the intestinal barrier and/or the blood-brain barrier (BBB). Once absorbed, hemorphins are opioid receptor (OR) ligands that may interact with peripheral and central OR and have effects on food intake and energy balance regulation. LLVV-YPWT (LLVV-H4), LVV-H4, VV-H4, VV-YPWTQRF (VV-H7), and VV-H7 hemorphins that were previously identified in the 120 min digest resulting from the simulated gastrointestinal digestion of hemoglobin have been synthesized to be tested in in vitro models of passage of IB and BBB. LC-MS/MS analyses yielded that all hemorphins, except the LLVV-H4 sequence, were able to cross intact the human intestinal epithelium model with Caco-2 cells within 5-60 min when applied at 5 mM. Moreover, all hemorphins crossed intact the human BBB model with brain-like endothelial cells (BLEC) within 30 min when applied at 100 µM. Fragments of these hemorphins were also detected, especially the YPWT common tetrapeptide that retains OR-binding capacity. A cAMP assay performed in Caco-2 cells indicates that tested hemorphins behave as OR agonists in these cells by reducing cAMP production. We further provide preliminary results regarding the effects of hemorphins on tight junction proteins, specifically here the claudin-4 that is involved in paracellular permeability. All hemorphins at 100 µM, except the LLVV-H4 peptide, significantly decreased claudin-4 mRNA levels in the Caco-2 intestinal model. This in vitro study is a first step toward demonstrating food-derived hemorphins bioavailability which is in line with the growing body of evidence supporting physiological functions for food-derived peptides.

12.
Gut ; 67(2): 271-283, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28377388

RESUMO

OBJECTIVE: To investigate the beneficial role of prebiotics on endothelial dysfunction, an early key marker of cardiovascular diseases, in an original mouse model linking steatosis and endothelial dysfunction. DESIGN: We examined the contribution of the gut microbiota to vascular dysfunction observed in apolipoprotein E knockout (Apoe-/-) mice fed an n-3 polyunsaturated fatty acid (PUFA)-depleted diet for 12 weeks with or without inulin-type fructans (ITFs) supplementation for the last 15 days. Mesenteric and carotid arteries were isolated to evaluate endothelium-dependent relaxation ex vivo. Caecal microbiota composition (Illumina Sequencing of the 16S rRNA gene) and key pathways/mediators involved in the control of vascular function, including bile acid (BA) profiling, gut and liver key gene expression, nitric oxide and gut hormones production were also assessed. RESULTS: ITF supplementation totally reverses endothelial dysfunction in mesenteric and carotid arteries of n-3 PUFA-depleted Apoe-/- mice via activation of the nitric oxide (NO) synthase/NO pathway. Gut microbiota changes induced by prebiotic treatment consist in increased NO-producing bacteria, replenishment of abundance in Akkermansia and decreased abundance in bacterial taxa involved in secondary BA synthesis. Changes in gut and liver gene expression also occur upon ITFs suggesting increased glucagon-like peptide 1 production and BA turnover as drivers of endothelium function preservation. CONCLUSIONS: We demonstrate for the first time that ITF improve endothelial dysfunction, implicating a short-term adaptation of both gut microbiota and key gut peptides. If confirmed in humans, prebiotics could be proposed as a novel approach in the prevention of metabolic disorders-related cardiovascular diseases.


Assuntos
Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiopatologia , Frutanos/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Prebióticos , Aminopeptidases/genética , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Bactérias/efeitos dos fármacos , Ácidos e Sais Biliares/biossíntese , Ácidos e Sais Biliares/sangue , Artérias Carótidas/fisiologia , Ceco/microbiologia , Suplementos Nutricionais , Modelos Animais de Doenças , Ácidos Graxos Ômega-3/administração & dosagem , Ácidos Graxos Ômega-3/deficiência , Expressão Gênica/efeitos dos fármacos , Peptídeo 1 Semelhante ao Glucagon/biossíntese , Masculino , Artérias Mesentéricas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Neurotensina/genética , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Proglucagon/genética , Simportadores/genética , Vasodilatação
13.
J Anal Toxicol ; 41(8): 670-678, 2017 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-28985322

RESUMO

Electronic cigarette use has raised concern worldwide regarding potential health risks and its position in tobacco cessation strategies. As part of any toxicity assessment, the chemical characterization of e-liquids and their related vapors are among fundamental data to be determined. Considering the lack of available reference methods, we developed and validated several analytical procedures in order to conduct a multicomponent analysis of six e-liquid refills and their resultant vapor emissions (generated by a smoking machine), and compared them with tobacco smoke. We combined several techniques including gas-chromatography, high and ultra-performance liquid chromatography and inductively coupled plasma with mass spectrometry or ultraviolet and flame ionization detection in order to identify the main e-liquid constituents (propylene glycol, glycerol and nicotine), as well as multiple potentially harmful components (trace elements, polycyclic aromatic hydrocarbons (PAHs), pesticides and carbonyl compounds). Regarding propylene glycol, glycerol and nicotine concentrations, the six tested e-liquids comply with the advertised composition and contain only traces of pollutants. Noticeable lower concentrations of trace elements (≤3.4 pg/mL puff), pesticides (

Assuntos
Aerossóis/análise , Sistemas Eletrônicos de Liberação de Nicotina , Cromatografia Líquida , Cromatografia Gasosa-Espectrometria de Massas , Substâncias Perigosas/análise , Hidrocarbonetos Policíclicos Aromáticos/análise , Propilenoglicol/análise
14.
Sci Total Environ ; 609: 982-991, 2017 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-28783915

RESUMO

The relevance of Polar Organic Chemical Integrative Samplers (POCIS) was evaluated for the assessment of concentrations of 46 pesticides and 19 pharmaceuticals in a small, peri-urban river with multi-origin inputs. Throughout the period of POCIS deployment, 24h-average water samples were collected automatically, and showed the rapid temporal evolution of concentrations of several micropollutants, as well as permitting the calculation of average concentrations in the water phase for comparison with those estimated from POCIS passive samplers. In the daily water samples, cyproconazol, epoxyconazol and imidacloprid showed high temporal variations with concentrations ranging from under the limit of detection up to several hundreds of ngL-1. Erythromycin, cyprofloxacin and iopromide also increased rapidly up to tens of ngL-1 within a few days. Conversely, atrazine, caffeine, diclofenac, and to a lesser extent carbamazepine and sucralose, were systematically present in the water samples and showed limited variation in concentrations. For most of the substances studied here, the passive samplers gave reliable average concentrations between the minimal and maximal daily concentrations during the time of deployment. For pesticides, a relatively good correlation was clearly established (R2=0.89) between the concentrations obtained by POCIS and those gained from average water samples. A slight underestimation of the concentration by POCIS can be attributed to inappropriate sampling rates extracted from the literature and for our system, and new values are proposed. Considering the all data set, 75% of the results indicate a relatively good agreement between the POCIS and the average water samples concentration (values of the ratio ranging between 0,33 and 3). Note further that this agreement between these concentrations remains valid considering different sampling rates extracted from the literature.

15.
J Pharm Biomed Anal ; 137: 113-122, 2017 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-28110167

RESUMO

The aim of this study was to develop a method combining chiral separation and biophysical techniques to evaluate the enantioselective affinity of original sulfonamide derivatives towards their therapeutic target, the human carbonic anhydrase II (hACII). The first step consisted in the preparation of the enantiomers by chromatographic separation. The performances of HPLC and Supercritical Fluid Chromatography (SFC) were studied at the analytical scale by optimization of various experimental conditions using adsorbed polysaccharide chiral stationary phases (amylose AD-H and cellulose OD-H). Since SFC allowed obtaining higher enantioresolutions per time unit, it was selected for the semi-preparative scale and successfully used to isolate each enantiomer with a satisfactory enantiomeric purity (>98%). Secondly, microscale thermophoresis (MST) method and surface plasmon resonance (SPR) used as reference method were developed to measure potential enantioselective affinities of these enantiomers towards the hACII. The optimizations of both methods were performed using a reference compound, i.e. acetazolamide, which affinity for hCAII has previously been demonstrated. For all compounds, KD values obtained using MST and SPR were in good agreement, leading to similar affinity scales despite both approaches totally differ (labeling for MST versus immobilization of the protein for SPR). The equilibrium dissociation constants of our original compounds for the hCAII were in the range 100-1000nM and an enantioselectivity was observed using the MST and SPR methods for the diarylpyrazole 2. Finally, by comparing the MST and SPR techniques, MST appears especially adapted for further screening of a series of sulfonamide derivatives due to the lower time required to estimate a binding constant while consuming as little hCAII as SPR.


Assuntos
Anidrase Carbônica II/química , Sulfonamidas/química , Acetazolamida/química , Amilose/química , Celulose/química , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia com Fluido Supercrítico/métodos , Humanos , Polissacarídeos/química , Estereoisomerismo , Ressonância de Plasmônio de Superfície/métodos
16.
Mol Neurobiol ; 54(7): 5361-5374, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-27590138

RESUMO

In amyotrophic lateral sclerosis (ALS), motor neuron degeneration is associated with systemic metabolic impairment. However, the evolution of metabolism alteration is partially unknown and its link with disease progression has never been described. For the first time, we ran a study focused on (1) the evolution of metabolism disturbance during disease progression through omics approaches and (2) the relation between metabolome profile and clinical evolution. SOD1-G93A (mSOD1) transgenic mice (n = 11) and wild-type (WT) littermates (n = 17) were studied during 20 weeks. Metabolomic profile of muscle and cerebral cortex was analysed at week 20, and plasma samples were assessed at four time points over 20 weeks. The relevant metabolic pathways highlighted by metabolomic analysis were explored by a targeted transcriptomic approach in mice. Plasma metabolomics were also performed in 24 ALS patients and 24 gender- and age-matched controls. Metabolomic analysis of muscle and cerebral cortex enabled an excellent discrimination between mSOD1 and WT mice (p < 0.001). These alterations included especially tryptophan, arginine, and proline metabolism pathways (including polyamines) as also revealed by transcriptomic analysis and findings in ALS patients. Multivariate models performed to explain clinical findings in ALS mice, and patients were excellent (p < 0.01) and highlighted three main metabolic pathways: arginine and proline, tryptophan, and branched amino acid metabolism. This work is the first longitudinal study that evaluates metabolism alteration in ALS, including the analysis of different tissues and using a combination of omics methods. We particularly identified arginine and proline metabolism. This pathway is also associated with disease progression and may open new perspectives of therapeutic targets.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Arginina/metabolismo , Neurônios Motores/metabolismo , Prolina/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Humanos , Estudos Longitudinais , Masculino , Metabolômica/métodos , Camundongos Transgênicos , Superóxido Dismutase/genética , Triptofano/metabolismo
17.
Anal Biochem ; 511: 42-51, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-27485269

RESUMO

This work was dedicated to the development of a reliable SPR method allowing the simultaneous and quick determination of the affinity and selectivity of designed sulfonamide derivatives for hCAIX and hCAXII versus hCAII, in order to provide an efficient tool to discover drugs for anticancer therapy of solid tumors. We performed for the first time a comparison of two immobilization approaches of hCA isoforms. First one relies on the use of an amine coupling strategy, using a CM7 chip to obtain higher immobilization levels than with a CM5 chip and consequently the affinity with an higher precision (CV% < 10%). The second corresponds to a capture of proteins on a streptavidin chip, named CAP chip, after optimization of biotinylation conditions (amine versus carboxyl coupling, biotin to protein ratio). Thanks to the amine coupling approach, only hCAII and hCAXII isoforms were efficiently biotinylated to reach relevant immobilization (3000 RU and 2700 RU, respectively) to perform affinity studies. For hCAIX, despite a successful biotinylation, capture on the CAP chip was a failure. Finally, concordance between affinities obtained for the three derivatives to CAs isozymes on both chips has allowed to valid the approaches for a further screening of new derivatives.


Assuntos
Biotina/química , Anidrases Carbônicas/química , Enzimas Imobilizadas/química , Sulfonamidas/química , Biotinilação , Humanos , Isoenzimas/química
18.
Neurotherapeutics ; 13(4): 905-917, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27444617

RESUMO

In amyotrophic lateral sclerosis (ALS), motor neuron degeneration occurs simultaneously with systemic metabolic impairment and neuroinflammation. Playing an important role in the regulation of both phenomena, interleukin (IL)-6, a major cytokine of the inflammatory response has been proposed as a target for management of ALS. Although a pilot clinical trial provided promising results in humans, another recent preclinical study showed that knocking out the IL-6 gene in mice carrying ALS did not improve clinical outcome. In this study, we aimed to determine the relevance of the IL-6 pathway blockade in a mouse model of ALS by using a pharmacological antagonist of IL-6, a murine surrogate of tocilizumab, namely MR16-1. We analyzed the immunological and metabolic effects of IL-6 blockade by cytokine measurement, blood cell immunophenotyping, targeted metabolomics, and transcriptomics. A deleterious clinical effect of MR16-1 was revealed, with a speeding up of weight loss (p = 0.0041) and decreasing body weight (p < 0.05). A significant increase in regulatory T-cell count (p = 0.0268) and a decrease in C-X-C ligand-1 concentrations in plasma (p = 0.0479) were observed. Metabolomic and transcriptomic analyses revealed that MR16-1 mainly affected branched-chain amino acid, lipid, arginine, and proline metabolism. IL-6 blockade negatively affected body weight, despite a moderated anti-inflammatory effect. Metabolic effects of IL-6 were mild compared with metabolic disturbances observed in ALS, but a modification of lipid metabolism by therapy was identified. These results indicate that IL-6 blockade did not improve clinical outcome of a mutant superoxide dismutase 1 mouse model of ALS.


Assuntos
Esclerose Amiotrófica Lateral/tratamento farmacológico , Esclerose Amiotrófica Lateral/metabolismo , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Interleucina-6/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Esclerose Amiotrófica Lateral/genética , Animais , Peso Corporal/genética , Citocinas/sangue , Avaliação da Deficiência , Modelos Animais de Doenças , Seguimentos , Redes Reguladoras de Genes , Fatores Imunológicos/farmacologia , Fatores Imunológicos/uso terapêutico , Interleucina-6/genética , Locomoção/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos
19.
J Chromatogr A ; 1455: 163-171, 2016 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-27286645

RESUMO

The performances of three neutral static coatings (hydroxypropyl cellulose, polyethylene oxide and poly(N,N-dimethylacrylamide) have been evaluated in order to determine the binding constants of the complexes formed between four polycationic compounds (piperazine derivatives) and four cyclodextrins of pharmaceutical interest (ß-CD, HP-ß-CD, Me-ß-CD and sulfobutyl ether-ß-CD) by affinity capillary electrophoresis. The physically-adsorbed poly(N,N-dimethylacrylamide) coating proves to be the more efficient to mask the silanol groups of the capillary wall since the lowest electroosmotic flow was measured for this coating. Moreover, it drastically reduces the adsorption of the compounds since it allows a correct repeatability of their migration time, higher efficiencies of the peaks and no baseline shift. Then, it was verified for four complexes that this coating allows a correct determination of the binding constants avoiding the CD adsorption which is responsible of an undervaluation of binding constants. The highest binding constants are obtained using the anionic sulfobutyl ether-ß-CD (SBE-ß-CD). The structure of the complex formed between the tacrine derivative and the SBE-ß-CD was further investigated through 2D ROESY NMR experiments and structure-binding constant relationships. Results suggest that the inclusion in the SBE-ß-CD cavity occurs through the aliphatic ring portion of the tacrine moiety.


Assuntos
Ciclodextrinas/química , Eletroforese Capilar , Piperazinas/química , Acrilamidas/química , Celulose/análogos & derivados , Celulose/química , Espectroscopia de Ressonância Magnética , Piperazinas/isolamento & purificação , Polietilenoglicóis/química , Viscosidade
20.
Electrophoresis ; 37(13): 1814-22, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26990205

RESUMO

Consumers and governments have become aware how the daily diet may affect the human health. All proteins from both plant and animal origins are potential sources of a wide range of bioactive peptides and the large majority of those display health-promoting effects. In the meat production food chain, the slaughterhouse blood is an inevitable co-product and, today, the blood proteins remain underexploited despite their bioactive potentiality. Through a comparative food peptidomics approach we illustrate the impact of resolving power, accuracy, sensitivity, and acquisition speed of low-resolution (LR)- and high-resolution (HR)-LC-ESI-MS/MS on the obtained peptide mappings and discuss the limitations of MS-based peptidomics. From in vitro gastrointestinal digestions of partially purified bovine hemoglobin, we have established the peptide maps of each hemoglobin chain. LR technique (normal bore C18 LC-LR-ESI-MS/MS) allows us to identify without ambiguity 75 unique peptides while the HR approach (nano bore C18 LC-HR-ESI-MS/MS) unambiguously identify more than 950 unique peptides (post-translational modifications included). Herein, the food peptidomics approach using the most performant separation methods and mass spectrometers with high-resolution capabilities appears as a promising source of information to assess the health potentiality of proteins.


Assuntos
Cromatografia Líquida/métodos , Digestão , Análise de Alimentos , Hemoglobinas/metabolismo , Peptídeos/metabolismo , Proteômica , Espectrometria de Massas em Tandem/métodos , Animais , Bovinos , Técnicas In Vitro , Mapeamento de Peptídeos
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