8 research outputs found
Correlation between interphase and metaphase chromosome arrangements as studied by laser-uv-microbeam experiments
High-Throughput Screening Platform To Identify Inhibitors of Protein Synthesis with Potential for the Treatment of Malaria
Artemisinin-based combination therapies have been crucial in driving down the global burden of malaria, the world’s largest parasitic killer. However, their efficacy is now threatened by the emergence of resistance in Southeast Asia and sub-Saharan Africa. Thus, there is a pressing need to develop new antimalarials with diverse mechanisms of action. One area of Plasmodium metabolism that has recently proven rich in exploitable antimalarial targets is protein synthesis, with a compound targeting elongation factor 2 now in clinical development and inhibitors of several aminoacyl-tRNA synthetases in lead optimization. Given the promise of these components of translation as viable drug targets, we rationalized that an assay containing all functional components of translation would be a valuable tool for antimalarial screening and drug discovery. Here, we report the development and validation of an assay platform that enables specific inhibitors of Plasmodium falciparum translation (PfIVT) to be identified. The primary assay in this platform monitors the translation of a luciferase reporter in a P. falciparum lysate-based expression system. Hits identified in this primary assay are assessed in a counterscreen assay that enables false positives that directly interfere with the luciferase to be triaged. The remaining hit compounds are then assessed in an equivalent human IVT assay. This platform of assays was used to screen MMV’s Pandemic and Pathogen Box libraries, identifying several selective inhibitors of protein synthesis. We believe this new high-throughput screening platform has the potential to greatly expedite the discovery of antimalarials that act via this highly desirable mechanism of action
Cell viability in the presence of PTX derivatives at different concentrations.
<p>(<b>A</b>) HeLa cells were incubated with PTX and caged PTX derivatives at concentrations between 0.01 and 10 µM. After 48 hours cultivation, cells were stained with PrestoBlue™ reagent. Metabolically active cells reduce the cell permeable dye resazurin into fluorescent resorufin, providing a quantitative measure of viable cells (see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043657#s2" target="_blank">Materials and Methods</a>” section for details). (<b>B</b>) The viability of cells incubated with pre-irradiated solutions of 2′,7-bisNvoc-PTX.</p
Effect of the presence of PTX and caged PTXs on the microtubule cytoskeleton of living cells.
<p>Fluorescence microscopy images of HeLa cells stably expressing GFP-tubulin imaged after 1 h (<b>A</b>) and 24 h (<b>B</b>) incubation in medium containing DMSO, PTX and caged derivatives at the indicated concentrations. White arrows indicate the cell edge deprived of microtubules. Scale bars: 20 µm. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043657#pone.0043657.s009" target="_blank">Fig. S5</a> for images of assays with pre-irradiated compounds.</p
Photolytic studies.
<p>(<b>A</b>) HPLC monitoring of the photoreaction and the changes in the composition of a 1 mM solution of 2′,7-bisNvoc-PTX after irradiation at 360 nm (2.7 mW/cm<sup>2</sup>) with increasing dose. (<b>B</b>) Time course of the quantified molar composition of the solution, as obtained from the HPLC data.</p
Development of a Photo-Cross-Linkable Diaminoquinazoline Inhibitor for Target Identification in <i>Plasmodium falciparum</i>
Diaminoquinazolines
represent a privileged scaffold for antimalarial discovery, including
use as putative <i>Plasmodium</i> histone lysine methyltransferase
inhibitors. Despite this, robust evidence for their molecular targets
is lacking. Here we report the design and development of a small-molecule
photo-cross-linkable probe to investigate the targets of our diaminoquinazoline
series. We demonstrate the effectiveness of our designed probe for
photoaffinity labeling of <i>Plasmodium</i> lysates and
identify similarities between the target profiles of the probe and
the representative diaminoquinazoline BIX-01294.
Initial pull-down proteomics experiments identified 104 proteins from
different classes, many of which are essential, highlighting the suitability
of the developed probe as a valuable tool for target identification
in <i>Plasmodium falciparum</i>
Naturally Occurring Genetic Variants of Human Caspase-1 Differ Considerably in Structure and the Ability to Activate Interleukin-1β
Caspase-1 (Interleukin-1 Converting Enzyme, ICE) is a proinflammatory enzyme that plays pivotal roles in innate immunity and many inflammatory conditions such as periodic fever syndromes and gout. In- flammation is often mediated by enzymatic activation of interleukin (IL)-1β and IL-18. We detected seven naturally occurring human CASP1 variants with different effects on protein structure, expression, and enzymatic activity. Most mutations destabilized the caspase-1 dimer interface as revealed by crystal structure analysis and homology modeling followed by molecular dynamics simulations. All variants demonstrated decreased or absent enzymatic and IL-1β releasing activity in vitro, in a cell transfection model, and as low as 25% of normal ex vivo in a whole blood assay of samples taken from subjects with variant CASP1, a subset of whom suffered from unclassified autoinflammation. We conclude that decreased enzymatic activity of caspase-1 is compatible with normal life and does not prevent moderate and severe autoinflammation.Fil: Luksch, Hella. University Hospital Carl Gustav Carus. Department of Pediatrics. Dresden; AlemaniaFil: Romanowski, Michael J.. Sunesis Pharmaceuticals. Department of Structural Biology; Estados UnidosFil: Chara, Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Física de Líquidos y Sistemas Biológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Física de Líquidos y Sistemas Biológicos; Argentina. Technische Universitat Dresden. Center for Information Services and High-Performance Computing; AlemaniaFil: Tuengler, Victoria. University Hospital Carl Gustav Carus. Department of Pediatrics. Dresden; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Caffarena, Ernesto Raúl. Escola Nacional de Saude Publica Sergio Arouca. Fundación Oswaldo Cruz; BrasilFil: Heymann, Michael C.. University Hospital Carl Gustav Carus. Department of Pediatrics. Dresden; AlemaniaFil: Lohse, Peter. Ludwig-Maximilians-Universit; AlemaniaFil: Aksentijevich, Ivona. Inflammatory Disease Section; Estados UnidosFil: Remmers, Elaine F.. Inflammatory Disease Section; Estados UnidosFil: Flecks, Silvana. University Hospital Carl Gustav Carus. Department of Pediatrics. Dresden; AlemaniaFil: Quoos, Nadine. University Hospital Carl Gustav Carus. Department of Pediatrics. Dresden; AlemaniaFil: Gramatté, Johannes. University Hospital Carl Gustav Carus. Department of Pediatrics. Dresden; AlemaniaFil: Petzold, Cathleen. University Hospital Carl Gustav Carus. Department of Pediatrics. Dresden; AlemaniaFil: Hofmann, Sigrun R.. University Hospital Carl Gustav Carus. Department of Pediatrics. Dresden; AlemaniaFil: Winkler, Stefan. University Hospital Carl Gustav Carus. Department of Pediatrics. Dresden; AlemaniaFil: Pessler, Frank. University Hospital Carl Gustav Carus. Department of Pediatrics. Dresden; Alemania. Helmholtz Centre for Infection Research; AlemaniaFil: Kallinich, Tilmann. Universität zu Berlin; AlemaniaFil: Ganser, Gerd. Sendenhorst. St. Josef-Stift; AlemaniaFil: Nimtz-Talaska, Antje. Kinderrheumatologie; AlemaniaFil: Baumann, Ulrich. Hannover Medical School; AlemaniaFil: Runde, Volker. Wilhelm-Anton-Hospital; AlemaniaFil: Grimbacher, Bodo. University Hospital Freiburg; AlemaniaFil: Birmelin, Jennifer. University Hospital Freiburg; AlemaniaFil: Gahr, Manfred. University Hospital Carl Gustav Carus. Department of Pediatrics. Dresden; AlemaniaFil: Roesler, Joachim. University Hospital Carl Gustav Carus. Department of Pediatrics. Dresden; AlemaniaFil: Rösen-Wolff, Angela. University Hospital Carl Gustav Carus. Department of Pediatrics. Dresden; Alemani