16 results on '"Jörg Bomke"'
Search Results
2. Identification of Methionine Aminopeptidase-2 (MetAP-2) Inhibitor M8891: A Clinical Compound for the Treatment of Cancer
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Jeyaprakashnarayanan Seenisamy, Frank Becker, Djordje Musil, Jörg Bomke, Jakub Gunera, Uwe Eckert, Timo Heinrich, Felix Rohdich, Kerrin Hansen, Beatrix Blume, Christian Siegl, Frank Zenke, Jens Pfalzgraf, Dieter Spuck, Ansgar Wegener, Birgitta Leuthner, Melanie Dietz, and Manja Friese-Hamim
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A549 cell ,0303 health sciences ,Chemistry ,Cell growth ,Pharmacology ,01 natural sciences ,METAP2 ,0104 chemical sciences ,010404 medicinal & biomolecular chemistry ,03 medical and health sciences ,chemistry.chemical_compound ,In vivo ,Drug Discovery ,Molecular Medicine ,Structure–activity relationship ,Target protein ,Efflux ,Lead compound ,030304 developmental biology - Abstract
The recently disclosed next generation of reversible, selective, and potent MetAP-2 inhibitors introduced a cyclic tartronic diamide scaffold. However, the lead compound 1a suffered from enterohepatic circulation, preventing further development. Nevertheless, 1a served as a starting point for further optimization. Maintaining potent antiproliferation activity, while improving other compound properties, enabled the generation of an attractive array of new MetAP-2 inhibitors. The most promising derivatives were identified by a multiparameter analysis of the compound properties. Essential for the efficient selection of candidates with in vivo activity was the identification of molecules with a long residence time on the target protein, high permeability, and low efflux ratio not only in Caco-2 but also in the MDR-MDCK cell line. Orally bioavailable, potent, and reversible MetAP-2 inhibitors impede the growth of primary endothelial cells and demonstrated antitumoral activity in mouse models. This assessment led to the nomination of the clinical development compound M8891, which is currently in phase I clinical testing in oncology patients.
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- 2019
3. Identification of Methionine Aminopeptidase-2 (MetAP-2) Inhibitor
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Timo, Heinrich, Jeyaprakashnarayanan, Seenisamy, Frank, Becker, Beatrix, Blume, Jörg, Bomke, Melanie, Dietz, Uwe, Eckert, Manja, Friese-Hamim, Jakub, Gunera, Kerrin, Hansen, Birgitta, Leuthner, Djordje, Musil, Jens, Pfalzgraf, Felix, Rohdich, Christian, Siegl, Dieter, Spuck, Ansgar, Wegener, and Frank T, Zenke
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Models, Molecular ,Indoles ,Endothelial Cells ,Mice, Nude ,Antineoplastic Agents ,Apoptosis ,Glioma ,Xenograft Model Antitumor Assays ,Mice ,Structure-Activity Relationship ,A549 Cells ,Tumor Cells, Cultured ,Animals ,Humans ,Methionyl Aminopeptidases ,Female ,Caco-2 Cells ,Enzyme Inhibitors ,Cell Proliferation - Abstract
The recently disclosed next generation of reversible, selective, and potent MetAP-2 inhibitors introduced a cyclic tartronic diamide scaffold. However, the lead compound
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- 2019
4. Estimation of Protein-Ligand Unbinding Kinetics Using Non-Equilibrium Targeted Molecular Dynamics Simulations
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Marta Amaral, Matthias K. Dreyer, Steffen Wolf, Djordje Musil, Jörn Güldenhaupt, Klaus Gerwert, Jörg Bomke, Matthias Frech, Jürgen Schlitter, Maryse Lowinski, and François Vallée
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Protein Conformation ,General Chemical Engineering ,Kinetics ,Static Electricity ,FOS: Physical sciences ,Library and Information Sciences ,Molecular Dynamics Simulation ,Ligands ,01 natural sciences ,symbols.namesake ,Molecular dynamics ,Physics - Chemical Physics ,0103 physical sciences ,Physics - Biological Physics ,Chemical Physics (physics.chem-ph) ,010304 chemical physics ,biology ,Ligand ,Chemistry ,Proteins ,Biomolecules (q-bio.BM) ,General Chemistry ,Computational Physics (physics.comp-ph) ,Electrostatics ,Small molecule ,0104 chemical sciences ,Computer Science Applications ,010404 medicinal & biomolecular chemistry ,Quantitative Biology - Biomolecules ,Chemical physics ,Biological Physics (physics.bio-ph) ,Chaperone (protein) ,FOS: Biological sciences ,symbols ,biology.protein ,van der Waals force ,Physics - Computational Physics ,Protein ligand ,Protein Binding - Abstract
We here report on non-equilibrium targeted Molecular Dynamics simulations as tool for the estimation of protein-ligand unbinding kinetics. Correlating simulations with experimental data from SPR kinetics measurements and X-ray crystallography on two small molecule compound libraries bound to the N-terminal domain of the chaperone Hsp90, we show that the mean non-equilibrium work computed in an ensemble of trajectories of enforced ligand unbinding is a promising predictor for ligand unbinding rates. We furthermore investigate the molecular basis determining unbinding rates within the compound libraries. We propose ligand conformational changes and protein-ligand nonbonded interactions to impact on unbinding rates. Ligands may remain longer at the protein if they exhibit strong electrostatic and/or van der Waals interactions with the target. In the case of ligands with rigid chemical scaffold that exhibit longer residence times however, transient electrostatic interactions with the protein appear to facilitate unbinding. Our results imply that understanding the unbinding pathway and the protein-ligand interactions along this path is crucial for the prediction of small molecule ligands with defined unbinding, This unedited version of the article may be downloaded for personal use only. Any other use requires prior permission of the author and the American Chemical Society. This article appeared in J. Chem. Inf. Model. (2019), 10.1021/acs.jcim.9b00592 and may be found at https://pubs.acs.org/doi/10.1021/acs.jcim.9b00592
- Published
- 2019
5. Novel reversible methionine aminopeptidase-2 (MetAP-2) inhibitors based on purine and related bicyclic templates
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Felix Rohdich, Thorsten Knöchel, Hans-Peter Buchstaller, Mireille Krier, Birgitta Leuthner, Frank Zenke, Jörg Bomke, Timo Heinrich, Djordje Musil, Manja Friese-Hamim, and Bertram Cezanne
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Models, Molecular ,Purine ,Stereochemistry ,Clinical Biochemistry ,Pharmaceutical Science ,Aminopeptidases ,01 natural sciences ,Biochemistry ,Structure-Activity Relationship ,chemistry.chemical_compound ,Drug Discovery ,Hydrolase ,medicine ,Humans ,Methionyl Aminopeptidases ,Imidazole ,Moiety ,Fumagillin ,Molecular Biology ,Protein maturation ,Glycoproteins ,Dose-Response Relationship, Drug ,Molecular Structure ,Bicyclic molecule ,010405 organic chemistry ,Organic Chemistry ,METAP2 ,0104 chemical sciences ,010404 medicinal & biomolecular chemistry ,Pyrimidines ,chemistry ,Purines ,Molecular Medicine ,medicine.drug - Abstract
The natural product fumagillin 1 and derivatives like TNP-470 2 or beloranib 3 bind to methionine aminopeptidase 2 (MetAP-2) irreversibly. This enzyme is critical for protein maturation and plays a key role in angiogenesis. In this paper we describe the synthesis, MetAP-2 binding affinity and structural analysis of reversible MetAP-2 inhibitors. Optimization of enzymatic activity of screening hit 10 (IC50: 1μM) led to the most potent compound 27 (IC50: 0.038μM), with a concomitant improvement in LLE from 2.1 to 4.2. Structural analysis of these MetAP-2 inhibitors revealed an unprecedented conformation of the His339 side-chain imidazole ring being co-planar sandwiched between the imidazole of His331 and the aryl-ether moiety, which is bound to the purine scaffold. Systematic alteration and reduction of H-bonding capability of this metal binding moiety induced an unexpected 180° flip for the triazolo[1,5-a]pyrimdine bicyclic template.
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- 2017
6. Discovery and Structure-Based Optimization of Next-Generation Reversible Methionine Aminopeptidase-2 (MetAP-2) Inhibitors
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Timo Heinrich, Jeyaprakashnarayanan Seenisamy, Beatrix Blume, Jörg Bomke, Michel Calderini, Uwe Eckert, Manja Friese-Hamim, Rainer Kohl, Martin Lehmann, Birgitta Leuthner, Djordje Musil, Felix Rohdich, and Frank T. Zenke
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Male ,Molecular Conformation ,Mice, Nude ,Antineoplastic Agents ,Structure-Activity Relationship ,Methionine ,Metals ,Cell Line, Tumor ,Drug Discovery ,Human Umbilical Vein Endothelial Cells ,Molecular Medicine ,Animals ,Humans ,Methionyl Aminopeptidases ,Female ,Protease Inhibitors ,Cell Proliferation - Abstract
Co- and post-translational processing are crucial maturation steps to generate functional proteins. MetAP-2 plays an important role in this process, and inhibition of its proteolytic activity has been shown to be important for angiogenesis and tumor growth, suggesting that small-molecule inhibitors of MetAP-2 may be promising options for the treatment of cancer. This work describes the discovery and structure-based hit optimization of a novel MetAP-2 inhibitory scaffold. Of critical importance, a cyclic tartronic diamide coordinates the MetAP-2 metal ion in the active site while additional side chains of the molecule were designed to occupy the lipophilic methionine side chain recognition pocket as well as the shallow cavity at the opening of the active site. The racemic screening hit from HTS campaign 11a was discovered with an enzymatic IC
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- 2019
7. Protein conformational flexibility modulates kinetics and thermodynamics of drug binding
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Pedro M. Matias, Jörg Bomke, Daria B. Kokh, Rebecca C. Wade, Hans-Peter Buchstaller, C. Sirrenberg, Ansgar Wegener, Marta Amaral, Matthias Frech, H.-M. Eggenweiler, Molecular, Structural and Cellular Microbiology (MOSTMICRO), and Instituto de Tecnologia Química e Biológica António Xavier (ITQB)
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Models, Molecular ,0301 basic medicine ,Chemistry(all) ,Protein Conformation ,Science ,Entropy ,Kinetics ,General Physics and Astronomy ,Thermodynamics ,Molecular Dynamics Simulation ,Physics and Astronomy(all) ,Crystallography, X-Ray ,Ligands ,Article ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Molecular dynamics ,Protein structure ,Humans ,HSP90 Heat-Shock Proteins ,Binding site ,lcsh:Science ,Binding Sites ,Multidisciplinary ,biology ,Chemistry ,Drug discovery ,Ligand ,Biochemistry, Genetics and Molecular Biology(all) ,Protein dynamics ,General Chemistry ,Surface Plasmon Resonance ,3. Good health ,030104 developmental biology ,Drug Design ,Chaperone (protein) ,Mutagenesis, Site-Directed ,biology.protein ,lcsh:Q ,Crystallization ,Protein Binding - Abstract
Structure-based drug design has often been restricted by the rather static picture of protein–ligand complexes presented by crystal structures, despite the widely accepted importance of protein flexibility in biomolecular recognition. Here we report a detailed experimental and computational study of the drug target, human heat shock protein 90, to explore the contribution of protein dynamics to the binding thermodynamics and kinetics of drug-like compounds. We observe that their binding properties depend on whether the protein has a loop or a helical conformation in the binding site of the ligand-bound state. Compounds bound to the helical conformation display slow association and dissociation rates, high-affinity and high cellular efficacy, and predominantly entropically driven binding. An important entropic contribution comes from the greater flexibility of the helical relative to the loop conformation in the ligand-bound state. This unusual mechanism suggests increasing target flexibility in the bound state by ligand design as a new strategy for drug discovery., An understanding of the dynamics of drug binding and unbinding processes is important for drug discovery. Here, the authors give insights into the binding mechanism of small drug-like molecules to human Hsp90 by combining thermodynamics and kinetics studies as well as molecular dynamics simulations.
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- 2017
8. Fragment-based discovery of focal adhesion kinase inhibitors
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Ulrich Grädler, Jörg Bomke, Günter Hölzemann, Martin Lehmann, Christina Esdar, Timo Heinrich, Verena Dresing, Mireille Krier, Djordje Musil, and Hartmut Greiner
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Models, Molecular ,Indoles ,Clinical Biochemistry ,Drug Evaluation, Preclinical ,Pharmaceutical Science ,Crystallography, X-Ray ,Biochemistry ,Small Molecule Libraries ,Focal adhesion ,Inhibitory Concentration 50 ,Drug Discovery ,Enzyme Inhibitors ,Surface plasmon resonance ,Molecular Biology ,Sulfonamides ,Molecular Structure ,Fragment (computer graphics) ,Chemistry ,Organic Chemistry ,Surface Plasmon Resonance ,Peptide Fragments ,Enzyme Activation ,Solubility ,Focal Adhesion Protein-Tyrosine Kinases ,Molecular Medicine - Abstract
Chemically diverse fragment hits of focal adhesion kinase (FAK) were discovered by surface plasmon resonance (SPR) screening of our in-house fragment library. Site specific binding of the primary hits was confirmed in a competition setup using a high-affinity ATP-site inhibitor of FAK. Protein crystallography revealed the binding mode of 41 out of 48 selected fragment hits within the ATP-site. Structural comparison of the fragment binding modes with a DFG-out inhibitor of FAK initiated first synthetic follow-up optimization leading to improved binding affinity.
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- 2013
9. Structural and Biophysical Characterization of the Syk Activation Switch
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Hartmut Greiner, Djordje Musil, Matthias Frech, Ulrich Grädler, Stefan Jäkel, Thomas Rysiok, Jörg Bomke, Ansgar Wegener, Daniel Schwarz, Verena Dresing, and Dirk Müller-Pompalla
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Models, Molecular ,Protein Conformation ,Adenylyl Imidodiphosphate ,Syk ,chemical and pharmacologic phenomena ,Crystallography, X-Ray ,Adenosine Triphosphate ,Protein structure ,Structural Biology ,Humans ,Syk Kinase ,Phosphorylation ,Kinase activity ,Phosphotyrosine ,Protein kinase A ,Molecular Biology ,ZAP-70 Protein-Tyrosine Kinase ,Chemistry ,Autophosphorylation ,Intracellular Signaling Peptides and Proteins ,hemic and immune systems ,Protein-Tyrosine Kinases ,Surface Plasmon Resonance ,Kinetics ,enzymes and coenzymes (carbohydrates) ,Protein kinase domain ,Biochemistry ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Mutation ,Biophysics ,Thermodynamics ,Tyrosine ,Tyrosine kinase ,Protein Binding ,Proto-oncogene tyrosine-protein kinase Src - Abstract
Syk is an essential non-receptor tyrosine kinase in intracellular immunological signaling, and the control of Syk kinase function is considered as a valuable target for pharmacological intervention in autoimmune or inflammation diseases. Upon immune receptor stimulation, the kinase activity of Syk is regulated by binding of phosphorylated immune receptor tyrosine-based activating motifs (pITAMs) to the N-terminal tandem Src homology 2 (tSH2) domain and by autophosphorylation with consequences for the molecular structure of the Syk protein. Here, we present the first crystal structures of full-length Syk (fl-Syk) as wild type and as Y348F,Y352F mutant forms in complex with AMP-PNP revealing an autoinhibited conformation. The comparison with the crystal structure of the truncated Syk kinase domain in complex with AMP-PNP taken together with ligand binding studies by surface plasmon resonance (SPR) suggests conformational differences in the ATP sites of autoinhibited and activated Syk forms. This hypothesis was corroborated by studying the thermodynamic and kinetic interaction of three published Syk inhibitors with isothermal titration calorimetry and SPR, respectively. We further demonstrate the modulation of inhibitor binding affinities in the presence of pITAM and discuss the observed differences of thermodynamic and kinetic signatures. The functional relevance of pITAM binding to fl-Syk was confirmed by a strong stimulation of in vitro autophosphorylation. A structural feedback mechanism on the kinase domain upon pITAM binding to the tSH2 domain is discussed in analogy of the related family kinase ZAP-70 (Zeta-chain-associated protein kinase 70). Surprisingly, we observed distinct conformations of the tSH2 domain and the activation switch including Tyr348 and Tyr352 in the interdomain linker of Syk in comparison to ZAP-70.
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- 2013
10. Kinetic and Structural Insights into the Mechanism of Binding of Sulfonamides to Human Carbonic Anhydrase by Computational and Experimental Studies
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C. Rechlin, Andreas Heine, Hans Dieter Gerber, Giovanni Bottegoni, Gerhard Klebe, Jörg Bomke, Walter Rocchia, Andrea Cavalli, Roberto Gaspari, Daniel Schwarz, Gaspari, Roberto, Rechlin, Chri, Heine, Andrea, Bottegoni, Giovanni, Rocchia, Walter, Schwarz, Daniel, Bomke, Jörg, Gerber, Hans-Dieter, Klebe, Gerhard, and Cavalli, Andrea
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Carbonic anhydrase II ,Plasma protein binding ,010402 general chemistry ,Crystallography, X-Ray ,01 natural sciences ,Carbonic Anhydrase II ,Molecular dynamics ,Carbonic anhydrase ,Drug Discovery ,Organic chemistry ,Molecule ,Humans ,Surface plasmon resonance ,Sulfonamides ,biology ,Molecular Structure ,010405 organic chemistry ,Chemistry ,Drug Discovery3003 Pharmaceutical Science ,Lyase ,Ligand (biochemistry) ,0104 chemical sciences ,Kinetics ,Zinc ,Biophysics ,biology.protein ,Molecular Medicine ,Hydrophobic and Hydrophilic Interactions ,Protein Binding - Abstract
The binding of sulfonamides to human carbonic anhydrase II (hCAII) is a complex and long-debated example of protein-ligand recognition and interaction. In this study, we investigate the para-substituted n-alkyl and hydroxyethylene-benzenesulfonamides, providing a complete reconstruction of their binding pathway to hCAII by means of large-scale molecular dynamics simulations, density functional calculations, surface plasmon resonance (SPR) measurements, and X-ray crystallography experiments. Our analysis shows that the protein-ligand association rate (kon) dramatically increases with the ligand's hydrophobicity, pointing to the existence of a prebinding stage largely stabilized by a favorable packing of the ligand's apolar moieties with the hCAII "hydrophobic wall". The characterization of the binding pathway allows an unprecedented understanding of the structure-kinetic relationship in hCAII/benzenesulfonamide complexes, depicting a paradigmatic scenario for the multistep binding process in protein-ligand systems.
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- 2015
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11. Fragment-based discovery of hydroxy-indazole-carboxamides as novel small molecule inhibitors of Hsp90
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Christian Sirrenberg, Harry Schwartz, Astrid Zimmermann, Ansgar Wegener, Jörg Bomke, Hans-Peter Buchstaller, Hans-Michael Eggenweiler, Ulrich Grädler, Edmund Hoppe, Djordje Musil, Joachim März, and Michael Wolf
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Indazoles ,Stereochemistry ,Cell Survival ,Clinical Biochemistry ,Drug Evaluation, Preclinical ,Pharmaceutical Science ,Crystallography, X-Ray ,Biochemistry ,Small Molecule Libraries ,chemistry.chemical_compound ,Structure-Activity Relationship ,Protein structure ,Cell Line, Tumor ,Drug Discovery ,Structure–activity relationship ,Humans ,Computer Simulation ,HSP90 Heat-Shock Proteins ,Binding site ,Molecular Biology ,Indazole ,Binding Sites ,biology ,Organic Chemistry ,Assay ,Small molecule ,Combinatorial chemistry ,Hsp90 ,Amides ,Protein Structure, Tertiary ,chemistry ,Docking (molecular) ,biology.protein ,Molecular Medicine - Abstract
Inhibitors of the Hsp90 molecular chaperone are showing considerable promise as potential molecular therapeutic agents for the treatment of cancer. Here we describe the identification of novel small molecular weight inhibitors of Hsp90 using a fragment based approach. Fragments were selected by docking, tested in a biochemical assay and the confirmed hits were crystallized. Information gained from X-ray structures of these fragments and other chemotypes was used to drive the fragment evolution process. Optimization of these high μM binders resulted in 3-benzylindazole derivatives with significantly improved affinity and anti-proliferative effects in different human cancer cell lines.
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- 2012
12. Structural Basis for Tail-Anchored Membrane Protein Biogenesis by the Get3-Receptor Complex
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Klemens Wild, Simon Reitz, Irmgard Sinning, Christopher Hein, Fei Wang, Jörg Bomke, Susanne Stefer, Volker Dötsch, Daniel Schwarz, Vladimir Denic, Yin Yuin Pang, Frank Bernhard, and Frank Löhr
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Models, Molecular ,Receptor complex ,Saccharomyces cerevisiae Proteins ,Protein subunit ,Molecular Sequence Data ,Plasma protein binding ,Saccharomyces cerevisiae ,Biology ,Crystallography, X-Ray ,Endoplasmic Reticulum ,Protein Structure, Secondary ,Article ,chemistry.chemical_compound ,Protein structure ,Adenosine Triphosphate ,Cytosol ,Catalytic Domain ,Microsomes ,Guanine Nucleotide Exchange Factors ,Protein Interaction Domains and Motifs ,Amino Acid Sequence ,Adenosine Triphosphatases ,Multidisciplinary ,Binding Sites ,integumentary system ,Endoplasmic reticulum ,Signal transducing adaptor protein ,Membrane Proteins ,Protein Structure, Tertiary ,Adaptor Proteins, Vesicular Transport ,Protein Subunits ,chemistry ,Membrane protein ,Biochemistry ,Biophysics ,Protein Multimerization ,Adenosine triphosphate ,Protein Binding - Abstract
Tail-anchored (TA) proteins are involved in cellular processes including trafficking, degradation, and apoptosis. They contain a C-terminal membrane anchor and are posttranslationally delivered to the endoplasmic reticulum (ER) membrane by the Get3 adenosine triphosphatase interacting with the hetero-oligomeric Get1/2 receptor. We have determined crystal structures of Get3 in complex with the cytosolic domains of Get1 and Get2 in different functional states at 3.0, 3.2, and 4.6 angstrom resolution. The structural data, together with biochemical experiments, show that Get1 and Get2 use adjacent, partially overlapping binding sites and that both can bind simultaneously to Get3. Docking to the Get1/2 complex allows for conformational changes in Get3 that are required for TA protein insertion. These data suggest a molecular mechanism for nucleotide-regulated delivery of TA proteins.
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- 2011
13. Structure of the leech protein saratin and characterization of its binding to collagen
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Matthias Frech, Frank Schumann, Werner Kremer, Thomas Rysiok, Wolfram Gronwald, Fritz Huber, Barbara Domogalla, Jörg Bomke, Till Maurer, Florian Fink, and Hans Robert Kalbitzer
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Models, Molecular ,Magnetic Resonance Spectroscopy ,Molecular Sequence Data ,Static Electricity ,Protein Data Bank (RCSB PDB) ,Leech ,Protein Structure, Secondary ,Structural Biology ,Leeches ,Animals ,Amino Acid Sequence ,Binding site ,Salivary Proteins and Peptides ,Molecular Biology ,chemistry.chemical_classification ,biology ,Chemistry ,biology.organism_classification ,Amino acid ,Protein Structure, Tertiary ,Hirudo medicinalis ,Biochemistry ,Docking (molecular) ,Biophysics ,Collagen ,Peptides ,Two-dimensional nuclear magnetic resonance spectroscopy ,Heteronuclear single quantum coherence spectroscopy ,Protein Binding - Abstract
The leech protein Saratin from Hirudo medicinalis prevents thrombocyte aggregation by interfering with the first binding step of the thrombocytes to collagen by binding to collagen. We solved the three-dimensional structure of the leech protein Saratin in solution and identified its collagen binding site by NMR titration experiments. The NMR structure of Saratin consists of one alpha-helix and a five-stranded beta-sheet arranged in the topology betabetaalphabetabetabeta. The C-terminal region, of about 20 amino acids in length, adopts no regular structure. NMR titration experiments with collagen peptides show that the collagen interaction of Saratin takes place in a kind of notch that is formed by the end of the alpha-helix and the beta-sheet. NMR data-driven docking experiments to collagen model peptides were used to elucidate the putative binding mode of Saratin and collagen. Mainly, parts of the first and the end of the fifth beta-strand, the loop connecting the alpha-helix and the third beta-strand, and a short part of the loop connecting the fourth and fifth beta-strand participate in binding.
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- 2008
14. Ubiquitin protein ligase Nedd4 binds to connexin43 by a phosphorylation-modulated process
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Matthias Frech, Kerstin Leykauf, Mojibrahman Salek, Jörg Bomke, Matthias Dürst, Angel Alonso, and Wolf-Dieter Lehmann
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Immunoprecipitation ,media_common.quotation_subject ,Nedd4 Ubiquitin Protein Ligases ,Recombinant Fusion Proteins ,Ubiquitin-Protein Ligases ,Amino Acid Motifs ,Molecular Sequence Data ,Connexin ,NEDD4 ,macromolecular substances ,Cell Line ,Ubiquitin ,Animals ,Protein Isoforms ,Phosphorylation ,RNA, Small Interfering ,Internalization ,media_common ,biology ,Endosomal Sorting Complexes Required for Transport ,Colocalization ,Cell Biology ,Cell biology ,Ubiquitin ligase ,Rats ,Biochemistry ,Connexin 43 ,cardiovascular system ,biology.protein ,Peptides ,Protein Binding - Abstract
Connexin43 is degraded by the proteasomal as well as the lysosomal pathway with ubiquitin playing a role in both degradation pathways. So far, no ubiquitin protein ligase has been identified for any of the connexins. By using pull-down assays, here we show binding of a ubiquitin protein ligase, Nedd4, to the C-terminus of connexin43. This observation was confirmed in vivo by coimmunoprecipitation and immunofluorescence, showing colocalization of Nedd4 and connexin43. Binding of Nedd4 to its interaction partners is generally carried out by its WW domains. Our results indicate that the interaction with connexin43 occurs through all three WW domains of Nedd4. Furthermore, whereas WW1 and WW2 domains mainly interact with the unphosphorylated form of connexin43, WW3 binds phosphorylated and unphosphorylated forms equally. In addition, using the surface plasmon resonance approach we show that only the WW2 domain binds to the PY motif located at the C-terminus of connexin43. Suppression of Nedd4 expression with siRNA resulted in an accumulation of gap junction plaques at the plasma membrane, suggesting an involvement of the ubiquitin protein ligase Nedd4 in gap junction internalization.
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- 2006
15. [Untitled]
- Author
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Hans Robert Kalbitzer, Matthias Frech, Till Maurer, Thomas Rysiok, and Jörg Bomke
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biology ,Chemistry ,CD36 ,Fibrinogen ,Biochemistry ,Collagen receptor ,Cell biology ,Von Willebrand factor ,Platelet adhesiveness ,biology.protein ,medicine ,Platelet aggregation inhibitor ,Platelet ,Platelet activation ,Spectroscopy ,medicine.drug - Abstract
Collagen plays an important structural role in the extra-cellular matrix of tissues. Under normal circumstances it is not exposed to flowing blood. Upon injury of the vessel wall, collagen is exposed to flowing blood and its constituents leading to activation and release of a variety of pro-aggregatory and mitotic factors propagating aggregation and thrombosis. The adhesion of platelets to the injured arterial wall is mediated by von Willebrand factor (vWF), which binds to collagen (for review see Sixma et al., 1997). As a consequence the collagen-bound vWF then gathers platelets, leading in the end to platelet activation (for review see Sadler, 1998). Thus vWF could act as a bridge between collagen and platelets and is a prerequisite for platelet adhesion. This process in itself may only be temporary, however, requiring additional, direct interactions between collagen and other receptors on the platelet surface in order to facilitate permanent platelet adhesion, activation and aggregation (Sixma et al., 1997). Such collagen receptors on platelets are known to include, but may not be limited to GP VI, GP Ia/IIa (alpha2beta1), to a lesser extent GP IV (CD36) (Moroi and Jung, 1997) and perhaps p65 (Chiang et al., 1997). In addition, vWF and fibrinogen facilitate cross-linking and further activation of platelets via GP IIb/IIIa receptor binding (Kulkarni et al., 2000), providing stability and strength for the developing thrombus.
- Published
- 2001
16. Abstract 2639: Discovery of novel small molecule inhibitors of Hsp90 for oral treatment
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Astrid Zimmermann, Hans-Peter Buchstaller, Ulrich Grädler, Djordje Musil, Harry Schwartz, Hans-Michael Eggenweiler, Wegener Ansgar, Christiane Amendt, Jörg Bomke, Edmund Hoppe, Johannes Gleitz, Joachim März, Michael Wolf, and Christian Sirrenberg
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Cancer Research ,Kinase ,Angiogenesis ,Cancer ,Biology ,Pharmacology ,medicine.disease ,Hsp90 ,Small molecule ,In vitro ,Oncology ,In vivo ,medicine ,biology.protein ,EGFR inhibitors - Abstract
Hsp90 is a molecular chaperone that plays an important role in the folding and function of client protein substrates in the cell. Many of these client proteins are well known oncogenes that are often over-expressed and mutated in tumor cells and association of Hsp90 with these client proteins maintains their ability to function in the deregulated state. Therefore Hsp90 inhibitors target tumor growth by multiple parallel mechanisms involving several oncogenes, which are critically involved in cell cycle, tumor growth, angiogenesis and apoptosis. Here we will present HTS and structure based design efforts to identify novel chemotypes that show potent binding to HSP90. In particular a 5-methyl-1H-pyrazol-3-yl-benzene-1,3-diol derivative (1) was identified during an HTS as a potent starting point for further optimization. This initial hit already showed an IC50 of 240nM in a HSP90 binding assay. The HSP90 co-crystal structure of this initial HTS hit revealed its binding mode to the N-terminal ATPase pocket and thus guided subsequent optimization of this structural series towards potency and oral bioavailability. Especially the identification of a new binding pocket for the ethyl group in (1) opened the route for the introduction of a broad variety of substituents. In addition to these variations the pyrazole core in (1) was replaced by a triazolone system. Although the SAR with respect to in vitro potency is comparable for both series, this fine tuning led to compounds with improved bioavailability and in vivo potency. The most promising candidates from both series were screened for selectivity against other ATPases, kinases and ion channels where they turned out to be completely HSP90 selective. In vivo tumor xenografts using prostate cancer cell line PC3, colon carcinoma HCT116 (KRAS mutation: mechanism of resistance against EGFR inhibitor in mCRC) and NSCLC tumor H1975 (EGFR-T790M mutation: mechanism of resistance against EGFR TKI) demonstrated excellent anti-tumor activity under different schedules after oral and iv administration. Biomarker analysis demonstrated efficient dose and time-dependent down-regulation of EGFR, phospho-Erk, cyclin D1 and up-regulation of Hsp70 as well as induction of apoptosis as determined by the increase of cleaved PARP and cleaved caspase 3. Favorably PK/PD studies revealed extraordinary long lasting compound levels in tumor tissue and long lasting pharmacodynamic effects (for more than 5 days in the tumors), indicating prolonged Hsp90 inhibition in tumor tissue in contrast to healthy tissue, from which the triazolones were rapidly cleared. The overall profile of the triazolones including synthesis, structure activity relationships, X-ray structures as well as a comprehensive pharmacological in vitro and in vivo characterization, together with potential pharmacodynamic and predictive biomarkers will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2639.
- Published
- 2010
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