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Oss, Netherlands

Falck D.,VU University Amsterdam | De Vlieger J.S.B.,VU University Amsterdam | Niessen W.M.A.,VU University Amsterdam | Kool J.,VU University Amsterdam | And 3 more authors.
Analytical and Bioanalytical Chemistry

A high-resolution screening method was developed for the p38α mitogen-activated protein kinase to detect and identify small-molecule binders. Its central role in inflammatory diseases makes this enzyme a very important drug target. The setup integrates separation by high-performance liquid chromatography with two parallel detection techniques. High-resolution mass spectrometry gives structural information to identify small molecules while an online enzyme binding detection method provides data on p38α binding. The separation step allows the individual assessment of compounds in a mixture and links affinity and structure information via the retention time. Enzyme binding detection was achieved with a competitive binding assay based on fluorescence enhancement which has a simple principle, is inexpensive, and is easy to interpret. The concentrations of p38α and the fluorescence tracer SK&F86002 were optimized as well as incubation temperature, formic acid content of the LC eluents, and the material of the incubation tubing. The latter notably improved the screening of highly lipophilic compounds. For optimization and validation purposes, the known kinase inhibitors BIRB796, TAK715, and MAPKI1 were used among others. The result is a high-quality assay with Z′ factors around 0.8, which is suitable for semi-quantitative affinity measurements and applicable to various binding modes. Furthermore, the integrated approach gives affinity data on individual compounds instead of averaged ones for mixtures. © 2010 The Author(s). Source

Gallwitz B.,University Hospital of Tuebingen | Kusterer K.,Internistische Praxis | Hildemann S.,University Hospital Freiburg | Hildemann S.,MSD Research Laboratories
Diabetologie und Stoffwechsel

Modern antidiabetic therapies must achieve low HbA1c values and avoid hypoglycaemic complications. The register SIRTA included 1522 patients with type 2 diabetes (T2DM) from 306 German medical practices. The patients had an HbA1c > 6.5 % under the maximum tolerated metformin dose. If required, they received combination therapy with other antidiabetics according to the algorithms of the German Diabetes Society (Deutsche Diabetes Gesellschaft DDG) or usual medical practice. Patients were followed up for six months. The target criteria included the achievement of HbA1c target values and the emergence of severe hypoglycaemic episodes. The HbA1c target was < 6.5 % for 64.0 % of patients and 6.5 - 7.0 % for 32.4 % (main reason: individual risk for hypoglycaemia). These HbA1c targets were achieved by 31.3 % and 44.3 % of patients, respectively. Combination therapies increased from 45 % to 56 % over the six months registry. Four patients had severe hypoglycaemias (0.26 %). The register confirms results from other epidemiologic studies on the therapy of T2DM in everyday practice. The treatment strategies applied effectively reduced blood glucose and avoided severe hypoglycaemias. An early therapy of insufficiently controlled patients with T2DM is important, as lower baseline values facilitated achieving HbA1c targets. © Georg Thieme Verlag KG Stuttgart · New York. Source

Blanco-Ania D.,Radboud University Nijmegen | Valderas-Cortina C.,Radboud University Nijmegen | Hermkens P.H.H.,MSD Research Laboratories | Sliedregt L.A.J.M.,Solvay Group | And 2 more authors.

The synthesis of a small library of dihydrouracils spiro-fused to pyrrolidines is described. These compounds are synthesized from ß-aryl pyrrolidines, providing products with the 2-arylethyl amine moiety, a structural feature often encountered in compounds active in the central nervous system. The ß-aryl pyrrolidines are synthesized through a three-step methodology that includes a Knoevenagel condensation reaction, a 1,3-dipolar cycloaddition reaction, and a nitrile reduction. Copyright © 2010 by the authors. Source

Toonen E.J.M.,MSD Research Laboratories | Fleuren W.W.M.,Radboud University Nijmegen | Fleuren W.W.M.,Netherlands Bioinformatics Center 5 | Nassander U.,MSD Research Laboratories | And 5 more authors.

Background: Prednisolone and other glucocorticoids (GCs) are potent anti-inflammatory and immunosuppressive drugs. However, prolonged use at a medium or high dose is hampered by side effects of which the metabolic side effects are most evident. Relatively little is known about their effect on gene-expression in vivo, the effect on cell subpopulations and the relation to the efficacy and side effects of GCs. Aim: To identify and compare prednisolone-induced gene signatures in CD4 + T lymphocytes and CD14 + monocytes derived from healthy volunteers and to link these signatures to underlying biological pathways involved in metabolic adverse effects. Materials & methods: Whole-genome expression profiling was performed on CD4 + T lymphocytes and CD14 + monocytes derived from healthy volunteers treated with prednisolone. Text-mining analyses was used to link genes to pathways involved in metabolic adverse events. Results: Induction of gene-expression was much stronger in CD4 + T lymphocytes than in CD14 + monocytes with respect to fold changes, but the number of truly cell-specific genes where a strong prednisolone effect in one cell type was accompanied by a total lack of prednisolone effect in the other cell type, was relatively low. Subsequently, a large set of genes was identified with a strong link to metabolic processes, for some of which the association with GCs is novel. Conclusion: The identified gene signatures provide new starting points for further study into GC-induced transcriptional regulation in vivo and the mechanisms underlying GC-mediated metabolic side effects. © 2011 Future Medicine Ltd. Source

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