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One Question,Multiple Answers: Biochemical and Biophysical Screening Methods Retrieve Deviating Fragment Hit Lists
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Dr. Johannes Schiebel Nedyalka Radeva Dr. Helene Köster Dr. Alexander Metz Timo Krotzky Dr. Maren Kuhnert Prof. Wibke E. Diederich Prof. Andreas Heine Dr. Lars Neumann Dr. Cedric Atmanene Dominique Roecklin Dr. Valérie Vivat‐Hannah Dr. Jean‐Paul Renaud Dr. Robert Meinecke Dr. Nina Schlinck Dr. Astrid Sitte Franziska Popp Dr. Markus Zeeb Prof. Gerhard Klebe 《ChemMedChem》2015,10(9):1511-1521
Fragment‐based lead discovery is gaining momentum in drug development. Typically, a hierarchical cascade of several screening techniques is consulted to identify fragment hits which are then analyzed by crystallography. Because crystal structures with bound fragments are essential for the subsequent hit‐to‐lead‐to‐drug optimization, the screening process should distinguish reliably between binders and non‐binders. We therefore investigated whether different screening methods would reveal similar collections of putative binders. First we used a biochemical assay to identify fragments that bind to endothiapepsin, a surrogate for disease‐relevant aspartic proteases. In a comprehensive screening approach, we then evaluated our 361‐entry library by using a reporter‐displacement assay, saturation‐transfer difference NMR, native mass spectrometry, thermophoresis, and a thermal shift assay. While the combined results of these screening methods retrieve 10 of the 11 crystal structures originally predicted by the biochemical assay, the mutual overlap of individual hit lists is surprisingly low, highlighting that each technique operates on different biophysical principles and conditions. 相似文献
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The harlequin ladybird Harmonia axyridis (Coleoptera: Coccinellidae) is a polyphagous predatory beetle native to Central and Eastern Asia. Since 2007 it has established all over Central Europe. In order to elucidate which defense strategy is responsible for its high resistance to diseases, we tested hemolymph as well as eleven main components of the headspace of H. axyridis for antimicrobial activity against Gram-positive (Bacillus subtilis, B. thuringiensis ssp. tenebrionis, Micrococcus luteus) and Gram-negative bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae). While three of the volatile compounds weakly reduced the growth of microorganisms, hemolymph of adults and larvae of H. axyridis strongly inhibited the growth of Gram-positive and Gram-negative bacteria as well as yeast. Furthermore, we compared the antimicrobial activity in the hemolymph of H. axyridis and Coccinella septempunctata. Antimicrobial activity in H. axyridis was about a thousand times higher compared to hemolymph from C. septempunctata. In contrast to C. septempunctata, the antimicrobial activity in H. axyridis was present without prior challenge. Minimal inhibitory concentration (MIC) of the hemolymph of H. axyridis was lowest against E. coli and yeast followed by B. subtilis, and was highest against entomopathogenic B. thuringiensis ssp. tenebrionidae. Furthermore, MIC values of the hemolymph obtained from live beetles were significantly lower than from frozen insects. This suggests that the active antimicrobial compound is affected by freezing and subsequent thawing of the beetles. Potential implications of our findings for the competitive advantages of H. axyridis over C. septempunctata are discussed. 相似文献
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Sabine Douxchamps François-Lionel Humbert Rein van der Hoek Martin Mena Stefano M. Bernasconi Axel Schmidt Idupulapati Rao Emmanuel Frossard Astrid Oberson 《Nutrient Cycling in Agroecosystems》2010,88(3):447-462
Canavalia brasiliensis (canavalia), a drought tolerant legume, was introduced into the smallholder traditional crop-livestock production system of the Nicaraguan hillsides as green manure to improve soil fertility or as forage during the dry season for improving milk production. Since nitrogen (N) is considered the most limiting nutrient for agricultural production in the target area, the objective of this study was to quantify the soil surface N budgets at plot level in farmers fields over two cropping years for the traditional maize/bean rotation and the alternative maize/canavalia rotation. Mineral fertilizer N, seed N and symbiotically fixed N were summed up as N input to the system. Symbiotic N2 fixation was assessed using the 15N natural abundance method. Nitrogen output was quantified as N export via harvested products. Canavalia derived in average 69% of its N from the atmosphere. The amount of N fixed per hectare varied highly according to the biomass production, which ranged from 0 to 5,700 kg ha?1. When used as green manure, canavalia increased the N balance of the maize/canavalia rotation but had no effect on the N uptake of the following maize crop. When used as forage, it bears the risk of a soil N depletion up to 41 kg N ha?1 unless N would be recycled to the plot by animal manure. Without N mineral fertilizer application, the N budget remains negative even if canavalia was used as green manure. Therefore, the replenishment of soil N stocks by using canavalia may need a few years, during which the application of mineral N fertilizer needs to be maintained to sustain agricultural production. 相似文献
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Spreitler F Brock C Pelzmann A Meyer O Köhler J 《Chembiochem : a European journal of chemical biology》2010,11(17):2419-2423
We have investigated the interaction of carbon monoxide dehydrogenase (CODH), an enzyme that catalyses the oxidation of CO in the aerobic eubacterium Oligotropha carboxidovorans, with the cytoplasmic membrane by using fluorescence correlation spectroscopy (FCS). Our results reveal that in vitro this interaction of CODH is specific for cytoplasmic membranes from CO-grown bacteria. 相似文献
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Johannes Macher Andreas Hausberger Astrid E. Macher Matthias Morak Bernd Schrittesser 《International Journal of Hydrogen Energy》2021,46(43):22574-22590
To reduce loss of hydrogen in storage vessels with high energy-to-weight-ratio, new materials, especially polymers, have to be developed as barrier materials. Very established methods for characterization of barrier materials with permeation measurements are the time-lag and flow rate method along with the differential pressure method, which resembles the nature of hydrogen vessel systems very well. Long measurement durations are necessary to gain suitable measurement data for these evaluation methods, and often restrictive conditions have to be fullfilled. For these reasons, common models for hydrogen permeation through single-layer and multi-layer membranes, as well as models for hydrogen gas properties were collected and reviewed. Using current computer power together with these models can reduce measurement time for characterization of the barrier properties of materials, while additional information about the quality of the measurement results is obtained. 相似文献
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The present paper deals with the computational simulation of a pile of thin sheets. The sheets are not laminated or glued, but they interact by frictional contact. In general, it is not possible to perform a full-scale finite element contact computation for piles containing thousands of sheets; the problem size becomes too large, and numerical solution methods suffer from severe convergence problems due to the large number of strongly coupled contact conditions. In this paper, a macroscopic material model is presented for the two-dimensional case. The pile of sheets is homogenized by introducing an effective anisotropic constitutive law, which is motivated by formulations of the theory of elasto-plasticity. This macroscopic material law models the behavior of a pile of sheets, allowing for no tensile stresses in the direction normal to the sheets and obeying Coulomb??s law of friction in the tangential contact plane. Applying this macroscopic material model, an equivalent homogeneous body can be treated using much coarser discretizations. Computational results for the problems are provided, and a comparison with simplified contact computations is done. 相似文献
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