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1. From a drug repositioning to a structure-based drug design approach to tackle acute lymphoblastic leukemia

Author

Magali Saez-Ayala, Laurent Hoffer, Sébastien Abel, Khaoula Ben Yaala, Benoit Sicard, Guillaume P. Andrieu, Mehdi Latiri, Emma K. Davison, Marco A. Ciufolini, Paul Brémond, Etienne Rebuffet, Philippe Roche, Carine Derviaux, Edwige Voisset, Camille Montersino, Remy Castellano, Yves Collette, Vahid Asnafi, Stéphane Betzi, Patrice Dubreuil, Sébastien Combes, and Xavier Morelli

Subjects
Science
Abstract

Abstract Cancer cells utilize the main de novo pathway and the alternative salvage pathway for deoxyribonucleotide biosynthesis to achieve adequate nucleotide pools. Deoxycytidine kinase is the rate-limiting enzyme of the salvage pathway and it has recently emerged as a target for anti-proliferative therapies for cancers where it is essential. Here, we present the development of a potent inhibitor applying an iterative multidisciplinary approach, which relies on computational design coupled with experimental evaluations. This strategy allows an acceleration of the hit-to-lead process by gradually implementing key chemical modifications to increase affinity and activity. Our lead compound, OR0642, is more than 1000 times more potent than its initial parent compound, masitinib, previously identified from a drug repositioning approach. OR0642 in combination with a physiological inhibitor of the de novo pathway doubled the survival rate in a human T-cell acute lymphoblastic leukemia patient-derived xenograft mouse model, demonstrating the proof-of-concept of this drug design strategy.

Published
2023
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2. Dual protein kinase and nucleoside kinase modulators for rationally designed polypharmacology

Author

Kahina Hammam, Magali Saez-Ayala, Etienne Rebuffet, Laurent Gros, Sophie Lopez, Berengere Hajem, Martine Humbert, Emilie Baudelet, Stephane Audebert, Stephane Betzi, Adrien Lugari, Sebastien Combes, Sebastien Letard, Nathalie Casteran, Colin Mansfield, Alain Moussy, Paulo De Sepulveda, Xavier Morelli, and Patrice Dubreuil

Subjects
Science
Abstract

Masitinib is a protein kinase inhibitor that sensitises refractory pancreatic adenocarcinoma cells to treatment with the nucleoside analog gemcitabine. Here the authors show that Masitinib activates deoxycytidine kinase to enhance phosphorylation of nucleoside analogue pro-drugs, increasing their potency.

Published
2017
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3. Cell-free production and characterisation of human uncoupling protein 1–3

Author

Etienne Rebuffet, Anna Frick, Michael Järvå, and Susanna Törnroth-Horsefield

Subjects
Cell-free expression, Membrane protein, Mitochondrial carrier, Uncoupling protein, Biology (General), QH301-705.5, Biochemistry, QD415-436
Abstract

The uncoupling proteins (UCPs) leak protons across the inner mitochondrial membrane, thus uncoupling the proton gradient from ATP synthesis. The main known physiological role for this is heat generation by UCP1 in brown adipose tissue. However, UCPs are also believed to be important for protection against reactive oxygen species, fine-tuning of metabolism and have been suggested to be involved in disease states such as obesity, diabetes and cancer. Structural studies of UCPs have long been hampered by difficulties in sample preparation with neither expression in yeast nor refolding from inclusion bodies in E. coli yielding sufficient amounts of pure and stable protein. In this study, we have developed a protocol for cell-free expression of human UCP1, 2 and 3, resulting in 1 mg pure protein per 20 mL of expression media. Lauric acid, a natural UCP ligand, significantly improved protein thermal stability and was therefore added during purification. Secondary structure characterisation using circular dichroism spectroscopy revealed the proteins to consist of mostly α-helices, as expected. All three UCPs were able to bind GDP, a well-known physiological inhibitor, as shown by the Fluorescence Resonance Energy Transfer (FRET) technique, suggesting that the proteins are in a natively folded state.

Published
2017
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4. Ten years of CAZypedia: a living encyclopedia of carbohydrate-active enzymes

Author

Štefan Janeček, Rohan J. Williams, Geoff Pincher, Darrell Cockburn, Gurvan Michel, Wataru Saburi, David R. Rose, Brian P. Rempel, Glyn R. Hemsworth, Wim Van den Ende, Jerry Ståhlberg, Leila LoLeggio, Tom Wennekes, Kiyohiko Igarashi, Cedric Montanier, Etienne Rebuffet, Naotake Konno, Harry J. Gilbert, Markus Linder, Ed Bayer, Tirso Pons, Jan-Hendrik Hehemann, Tomomi Sumida, Thierry Fontaine, Takane Katayama, Elizabeth Ficko-Blean, Florence Vincent, Zui Fujimoto, Masafumi Hidaka, Kyle Robinson, Ana R. Luís, Yuichi Sakamoto, Bernard Henrissat, Gustav Vaaje-Kolstad, Jens M. Eklöf, Ian R. Greig, Harry Brumer, Ryuichiro Suzuki, Mats Sandgren, Takashi Tonozuka, Ryszard Brzezinski, Brian L. Mark, Bareket Dassa, Haruhide Mori, Junho Lee, Vivian L. Y. Yip, Birte Svensson, Wade Abbott, Alfons K. G. Felice, Juha Rouvinen, Takayuki Ohnuma, Satoshi Kaneko, Franz J. St John, Ramon Hurtado-Guerrero, Pedro M. Coutinho, Sine Larsen, Gideon J. Davies, Yuval Shoham, Kiyotaka Fujita, Warren W. Wakarchuk, Fathima Aidha Shaikh, Alisdair B. Boraston, Breeanna R. Urbanowicz, Vincent G. H. Eijsink, Daniel Kracher, Beatrice Cobucci-Ponzano, Ethan D. Goddard-Borger, Anthony J. Clarke, David J. Vocadlo, Katsuro Yaoi, Seino A. K. Jongkees, Anna A. Kulminskaya, Roland Ludwig, Mirko M. Maksimainen, Magali Remaud-Simeon, Edward J. Taylor, Motomitsu Kitaoka, Spencer J. Williams, Shinya Fushinobu, Marco Moracci, David Wilson, Richard McLean, Toki Taira, Jean-Guy Berrin, Ran Zhang, Hiroyuki Nakai, Tracey M. Gloster, Peter J. Reilly, Wim Nerinckx, Takuya Ishida, Alicia Lammerts van Bueren, Orly Alber, Mirjam Czjzek, Kathleen Piens, Annabelle Varrot, Stephen G. Withers, Nathalie Juge, Maxime Versluys, Gerlind Sulzenbacher, Richard W. Pickersgill, Michael D. L. Suits, Agriculture and Agri-Food [Ottawa] (AAFC), Weizmann Institute of Science [Rehovot, Israël], Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), University of Victoria [Canada] (UVIC), University of British Columbia, Université de Sherbrooke (UdeS), University of Guelph, National Research Council (CNR), Pennsylvania State University (Penn State), Penn State System, Aix Marseille Université (AMU), Laboratoire de Biologie Intégrative des Modèles Marins (LBI2M), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Station biologique de Roscoff (SBR), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Sorbonne Université (SU), University of York, Norwegian University of Life Sciences (NMBU), University of Vienna [Vienna], University of Adelaide, Institut Pasteur [Paris], National Agriculture and Food Research Organization (NARO), Kagoshima University, University of Tokyo, Newcastle University, University of St Andrews [Scotland], The Walter and Eliza Hall Institute of Medical Research (WEHI), Simon Fraser University (SFU.ca), Max Planck Institute for Marine Microbiology, Max-Planck-Gesellschaft, University of Leeds, University of Zaragoza - Universidad de Zaragoza [Zaragoza], Slovak Academy of Sciences (SAS), Quadram Institute, University of the Ryukyus [Okinawa], Ishikawa Prefectural University, Utsunomiya University [Utsunomiya], St Petersburg Nuclear Physics Institute, University of Groningen, Aalto University, University of Copenhagen = Københavns Universitet (KU), University of Lisbon, University of Oulu, University of Manitoba [Winnipeg], University of Lethbridge, Hokkaido University [Sapporo, Japan], Niigata University, Ghent University, Kinki University, Queen Mary University of London (QMUL), Sveriges lantbruksuniversitet, National Center for Biotechnology, Centre de Recherche en Cancérologie de Marseille (CRCM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Aix Marseille Université (AMU), Iowa State University (ISU), Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA), University of Waterloo [Waterloo], University of Eastern Finland, Iwate Biotechnology Research Center (IBRC), Technion - Israel Institute of Technology [Haifa], United States Department of Agriculture, Wilfrid Laurier University (WLU), Architecture et fonction des macromolécules biologiques (AFMB), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Institut National de la Recherche Agronomique (INRA), RIKEN - Institute of Physical and Chemical Research [Japon] (RIKEN), Akita University, Danmarks Tekniske Universitet (DTU), University of Lincoln, Tokyo University of Agriculture and Technology (TUAT), Univ Georgia, University of Georgia [USA], Université Catholique de Louvain = Catholic University of Louvain (UCL), Centre de Recherches sur les Macromolécules Végétales (CERMAV ), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Ryerson University, Utrecht University [Utrecht], University of Melbourne, Cornell University [New York], National Institute of Advanced Industrial Science and Technology (AIST), Natural Sciences and Engineering Research Council of Canada (NSERC), Agriculture and Agri-Food (AAFC), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP), Biotechnology and Biological Sciences Research Council (BBSRC), University of Copenhagen = Københavns Universitet (UCPH), Universidade de Lisboa = University of Lisbon (ULISBOA), Universiteit Gent = Ghent University (UGENT), Aix Marseille Université (AMU)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Danmarks Tekniske Universitet = Technical University of Denmark (DTU), Weizmann Institute of Science, Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), University of Victoria, Sherbrooke University, Sorbonne Universités, University of Vienna, National Agriculture and Food Research Organization, University of St Andrews, Walter and Eliza Hall Institute of Medical Research (WEHI), Slovak Academy of Sciences, Utsunomiya University, Hokkaido University, Iwate Biotechnol Res Ctr, Technion – Israel Institute of Technology, Akita Prefectural University, Université Catholique de Louvain (UCL), Centre de Recherches sur les Macromolécules Végétales (CERMAV), Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Université Grenoble Alpes (UGA), Cornell University, Laboratoire Départemental Vétérinaire et d'Hygiène Alimentaire des Hautes Alpes, and Moracci, M

Subjects
0301 basic medicine, History, Carbohydrate, CAZy, Bioinformatics, [SDV]Life Sciences [q-bio], 030106 microbiology, Polysaccharide-Lyases, Glycobiology, Carbohydrates, Glycosyltransferases/chemistry, Computational biology, Biology, Esterase, Biochemistry, History, 21st Century, Databases, 03 medical and health sciences, glycobiology, Esterases/chemistry, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Carbohydrates/chemistry, Glycoscience, Databases, Protein, ComputingMilieux_MISCELLANEOUS, bioinformatic, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Protein, Polysaccharide-Lyases/chemistry, biocuration, Esterases, glycoscience, Glycosyltransferases, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, bioinformatics, 21st Century, Biocuration, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, 030104 developmental biology, carbohydrate-active enzymes, Encyclopedia, Carbohydrate-active enzymes, carbohydrate-active enzyme, Carbohydrate active enzymes, Glycosyltransferase
Abstract

CAZypedia was initiated in 2007 to create a comprehensive, living encyclopedia of the carbohydrate-active enzymes (CAZymes) and associated carbohydrate-binding modules involved in the synthesis, modification, and degradation of complex carbohydrates. CAZypedia is closely connected with the actively-curated CAZy database, which provides a sequence-based foundation for the biochemical, mechanistic, and structural characterization of these diverse proteins. Now celebrating its 10th anniversary online, CAZypedia is a successful example of dynamic, community-driven, and expert-based biocuration. CAZypedia is an open-access resource available at URL http://www.cazypedia.org.

Published
2018
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5. Dual protein kinase and nucleoside kinase modulators for rationally designed polypharmacology

Author

Laurent Gros, Stephane Betzi, Paulo De Sepulveda, Nathalie Casteran, Magali Saez-Ayala, Martine Humbert, Stéphane Audebert, Kahina Hammam, Patrice Dubreuil, Alain Moussy, Sébastien Combes, Etienne Rebuffet, Adrien Lugari, Sébastien Letard, Colin Mansfield, Sophie Lopez, Berengere Hajem, Emilie Baudelet, Xavier Morelli, Centre de Recherche en Cancérologie de Marseille (CRCM), Aix Marseille Université (AMU)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), AB Science SA, AB Science, ARC Grant n° SL220130606659, and MORELLI, XAVIER

Subjects
Models, Molecular, Proteomics, 0301 basic medicine, Polypharmacology, Pyridines, General Physics and Astronomy, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, Crystallography, X-Ray, Deoxycytidine, chemistry.chemical_compound, 0302 clinical medicine, Piperidines, [CHIM.CHEM] Chemical Sciences/Cheminformatics, Phosphorylation, lcsh:Science, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], ComputingMilieux_MISCELLANEOUS, Multidisciplinary, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], [CHIM.ORGA]Chemical Sciences/Organic chemistry, Masitinib, [SDV.BBM.MN]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular Networks [q-bio.MN], Deoxycytidine kinase, Protein kinase inhibitor, 3. Good health, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, [SDV.SP.PHARMA] Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, 030220 oncology & carcinogenesis, Benzamides, Imatinib Mesylate, [CHIM.CHEM]Chemical Sciences/Cheminformatics, medicine.drug, [SDV.SP.MED] Life Sciences [q-bio]/Pharmaceutical sciences/Medication, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], medicine.drug_class, Science, [CHIM.THER] Chemical Sciences/Medicinal Chemistry, [SDV.BBM.BP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Antineoplastic Agents, [SDV.CAN]Life Sciences [q-bio]/Cancer, Models, Biological, Article, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, [SDV.CAN] Life Sciences [q-bio]/Cancer, [SDV.SP.MED]Life Sciences [q-bio]/Pharmaceutical sciences/Medication, Cell Line, Tumor, Deoxycytidine Kinase, medicine, [CHIM.CRIS]Chemical Sciences/Cristallography, Humans, [CHIM.CRIS] Chemical Sciences/Cristallography, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Protein kinase A, Protein Kinase Inhibitors, Nucleoside analogue, General Chemistry, [CHIM.ORGA] Chemical Sciences/Organic chemistry, Gemcitabine, Enzyme Activation, Thiazoles, 030104 developmental biology, Imatinib mesylate, [SDV.BBM.MN] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular Networks [q-bio.MN], chemistry, A549 Cells, Drug Resistance, Neoplasm, Drug Design, Cancer research, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, lcsh:Q
Abstract

Masitinib, a highly selective protein kinase inhibitor, can sensitise gemcitabine-refractory cancer cell lines when used in combination with gemcitabine. Here we report a reverse proteomic approach that identifies the target responsible for this sensitisation: the deoxycytidine kinase (dCK). Masitinib, as well as other protein kinase inhibitors, such as imatinib, interact with dCK and provoke an unforeseen conformational-dependent activation of this nucleoside kinase, modulating phosphorylation of nucleoside analogue drugs. This phenomenon leads to an increase of prodrug phosphorylation of most of the chemotherapeutic drugs activated by this nucleoside kinase. The unforeseen dual activity of protein kinase inhibition/nucleoside kinase activation could be of great therapeutic benefit, through either reducing toxicity of therapeutic agents by maintaining effectiveness at lower doses or by counteracting drug resistance initiated via down modulation of dCK target., Masitinib is a protein kinase inhibitor that sensitises refractory pancreatic adenocarcinoma cells to treatment with the nucleoside analog gemcitabine. Here the authors show that Masitinib activates deoxycytidine kinase to enhance phosphorylation of nucleoside analogue pro-drugs, increasing their potency.

Published
2017
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6. The Vanadium Iodoperoxidase from the Marine Flavobacteriaceae Species Zobellia galactanivorans Reveals Novel Molecular and Evolutionary Features of Halide Specificity in the Vanadium Haloperoxidase Enzyme Family

Author

Etienne Rebuffet, Gurvan Michel, Jean-Baptiste Fournier, Romain Grijol, Mirjam Czjzek, Laurence Meslet-Cladiere, Catherine Leblanc, Philippe Potin, Ludovic Delage, and Justyna Rzonca

Subjects
Models, Molecular, Molecular Sequence Data, Sequence alignment, medicine.disease_cause, Iodide Peroxidase, Applied Microbiology and Biotechnology, Streptomyces, Substrate Specificity, Evolution, Molecular, Bacterial Proteins, Phylogenetics, Haloperoxidase, medicine, Seawater, Amino Acid Sequence, Enzymology and Protein Engineering, Escherichia coli, Phylogeny, Peroxidase, Ecology, biology, Active site, Vanadium, biology.organism_classification, Kinetics, Biochemistry, Multigene Family, biology.protein, Iodide oxidation, Flavobacteriaceae, Sequence Alignment, Flavobacterium, Food Science, Biotechnology
Abstract

Vanadium haloperoxidases (VHPO) are key enzymes that oxidize halides and are involved in the biosynthesis of organo-halogens. Until now, only chloroperoxidases (VCPO) and bromoperoxidases (VBPO) have been characterized structurally, mainly from eukaryotic species. Three putative VHPO genes were predicted in the genome of the flavobacterium Zobellia galactanivorans , a marine bacterium associated with macroalgae. In a phylogenetic analysis, these putative bacterial VHPO were closely related to other VHPO from diverse bacterial phyla but clustered independently from eukaryotic algal VBPO and fungal VCPO. Two of these bacterial VHPO, heterogeneously produced in Escherichia coli , were found to be strictly specific for iodide oxidation. The crystal structure of one of these vanadium-dependent iodoperoxidases, Zg-VIPO1, was solved by multiwavelength anomalous diffraction at 1.8 Å, revealing a monomeric structure mainly folded into α-helices. This three-dimensional structure is relatively similar to those of VCPO of the fungus Curvularia inaequalis and of Streptomyces sp. and is superimposable onto the dimeric structure of algal VBPO. Surprisingly, the vanadate binding site of Zg-VIPO1 is strictly conserved with the fungal VCPO active site. Using site-directed mutagenesis, we showed that specific amino acids and the associated hydrogen bonding network around the vanadate center are essential for the catalytic properties and also the iodide specificity of Zg-VIPO1. Altogether, phylogeny and structure-function data support the finding that iodoperoxidase activities evolved independently in bacterial and algal lineages, and this sheds light on the evolution of the VHPO enzyme family.

Published
2014
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7. Protein-protein interaction inhibition (2P2I)-oriented chemical library accelerates hit discovery

Author

Carine Derviaux, Brigitt Raux, Etienne Rebuffet, Thomas Roux, Eric Trinquet, Cécilia Eydoux, Michel Aurrand-Lions, Marie-Jeanne Basse, Philippe Roche, Stefan Knapp, Stephane Betzi, Jean-Claude Guillemot, Jean-Claude Lissitzky, Sébastien Combes, Audrey Restouin, Xavier Morelli, Pascale Zimmermann, Marion Badol, Marie-Edith Gourdel, Sabine Milhas, Véronique Hamon, Susanne Müller, Adrien Lugari, Yves Collette, Catherine Rogers, and Rudra Kashyap

Subjects
0301 basic medicine, Validation study, Computer science, Druggability, Nanotechnology, Computational biology, Crystallography, X-Ray, 01 natural sciences, Biochemistry, Chemical library, Protein–protein interaction, Small Molecule Libraries, 03 medical and health sciences, chemistry.chemical_compound, Drug Discovery, Protein Interaction Mapping, 010405 organic chemistry, Drug discovery, Proteins, General Medicine, 0104 chemical sciences, Bromodomain, 030104 developmental biology, chemistry, Homogeneous, Molecular Medicine
Abstract

Protein-protein interactions (PPIs) represent an enormous source of opportunity for therapeutic intervention. We and others have recently pinpointed key rules that will help in identifying the next generation of innovative drugs to tackle this challenging class of targets within the next decade. We used these rules to design an oriented chemical library corresponding to a set of diverse 'PPI-like' modulators with cores identified as privileged structures in therapeutics. In this work, we purchased the resulting 1664 structurally diverse compounds and evaluated them on a series of representative protein-protein interfaces with distinct "druggability" potential using Homogeneous Time-Resolved Fluorescence (HTRF®) technology. For certain PPI classes, analysis of the hit rates revealed up to 100 enrichment factors compared with non-oriented chemical libraries. This observation correlates with the predicted "druggability" of the targets. A specific focus on selectivity profiles, the three-dimensional (3D) molecular modes of action resolved by X-ray crystallography, and the biological activities of identified hits targeting the well-defined "druggable" bromodomains of the bromo and extraterminal (BET) family are presented as a proof-of-concept. Overall, our present study illustrates the potency of machine learning-based oriented chemical libraries to accelerate the identification of hits targeting PPIs. A generalization of this method to a larger set of compounds will accelerate the discovery of original and potent probes for this challenging class of targets.

Published
2016

8. Vanadium haloperoxidases: from the discovery 30 years ago to X-Ray crystallographic and V K-edge absorption spectroscopic studies

Author

Gurvan Michel, Mirjam Czjzek, Philippe Potin, Catherine Leblanc, Martin C. Feiters, Jean-Baptiste Fournier, P.L. Solari, Ludovic Delage, Hans Vilter, Etienne Rebuffet, Laboratoire de Biologie Intégrative des Modèles Marins (LBI2M), Station biologique de Roscoff [Roscoff] (SBR), Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Zurmaiener Strasse, Centre de Recherche en Cancérologie de Marseille (CRCM), Aix Marseille Université (AMU)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Synchrotron SOLEIL (SSOLEIL), Centre National de la Recherche Scientifique (CNRS), Department Organic Chemistry, Radboud University [Nijmegen], Administateur, HAL Sorbonne Université, Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Station biologique de Roscoff [Roscoff] (SBR), Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), and Radboud university [Nijmegen]

Subjects
X-ray absorption spectroscopy, biology, Chemistry, Vanadium coordination, Crystallographic structure, Oligomeric state, Active site, Halide, Vanadium, chemistry.chemical_element, Synthetic Organic Chemistry, X-ray absorption spectroscopy on biological vanadium, Catalysis, Inorganic Chemistry, Crystallography, Catalytic cycle, [CHIM] Chemical Sciences, Materials Chemistry, biology.protein, Structural evolution, [CHIM]Chemical Sciences, Vanadate, Physical and Theoretical Chemistry, Vanadium bromoperoxidase, Vanadium-dependent haloperoxidase
Abstract

International audience; In the environment, vanadium-dependent haloperoxidases (VHPO) are likely to play a key role in the production of biogenic organo-halogens. These enzymes contain vanadate as a prosthetic group, and catalyze, in the presence of hydrogen peroxide, the oxidation of halide ions (Cl−, Br− or I−). They are classified according to the most electronegative halide that they can oxidize. Since the first discovery of a vanadium bromoperoxidase in the brown alga Ascophyllum nodosum thirty years ago, structural and mechanistic studies have been mainly conducted on two types of VHPO, chloro- and bromoperoxidases, and more recently on a vanadium-dependent iodoperoxidase. In this review, we highlight the main progress obtained on the structure-function relation of these proteins, based on biochemistry, crystallography and X-ray absorption spectroscopy (XAS). The comparison of 3D protein structures of the different VHPO helped identify the residues that govern the molecular mechanisms of catalysis and specificity of VHPO. Vanadium K-edge XAS gave further important insight to understand the fine changes around the vanadium cofactor during the catalytic cycle. The combination of different structural approaches, at different scales of resolution, shed new light on biological vanadium coordination in the active site, and its importance for the catalytic cycle and halide specificity of vanadium haloperoxidases.

Published
2015
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9. Biochemical and structural investigation of two paralogous glycoside hydrolases from Zobellia galactanivorans: novel insights into the evolution, dimerization plasticity and catalytic mechanism of the GH117 family

Author

Robert Larocque, Gurvan Michel, Agnès Groisillier, Elizabeth Ficko-Blean, Delphine Duffieux, Etienne Rebuffet, Mirjam Czjzek, Laboratoire de Biologie Intégrative des Modèles Marins (LBI2M), Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Station biologique de Roscoff [Roscoff] (SBR), Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Station biologique de Roscoff [Roscoff] (SBR), and Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, Glycoside Hydrolases, Stereochemistry, Protein Conformation, Molecular Sequence Data, Crystallography, X-Ray, Cell wall, Evolution, Molecular, Residue (chemistry), Hydrolysis, Structural Biology, Hydrolase, Glycoside hydrolase, Amino Acid Sequence, Phylogeny, chemistry.chemical_classification, Binding Sites, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, Galactose, General Medicine, biology.organism_classification, Phylogenetic diversity, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Enzyme, Biochemistry, chemistry, Metals, Protein Multimerization, Flavobacteriaceae, Sequence Alignment, Bacteria
Abstract

The family 117 glycoside hydrolase (GH117) enzymes have exo-α-1,3-(3,6-anhydro)-L-galactosidase activity, removing terminal nonreducing α-1,3-linked 3,6-anhydro-L-galactose residues from their red algal neoagarose substrate. These enzymes have previously been phylogenetically divided into clades, and only the clade A enzymes have been experimentally studied to date. The investigation of two GH117 enzymes, Zg3615 and Zg3597, produced by the marine bacteriumZobellia galactanivoransreveals structural, biochemical and further phylogenetic diversity between clades. A product complex with the unusual β-3,6-anhydro-L-galactose residue sheds light on the inverting catalytic mechanism of the GH117 enzymes as well as the structure of this unique sugar produced by hydrolysis of the agarophyte red algal cell wall.

Published
2015
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10. High resolution protein crystals using an efficient convection-free geometry

Author

Willem J. de Grip, Alaa Adawy, Etienne Rebuffet, Susanna Törnroth-Horsefield, Elias Vlieg, and Willem J. P. van Enckevort

Subjects
Diffraction, Convection, Chemical and physical biology [NCMLS 7], Materials science, Macromolecular crystallography, High resolution, Geometry, Solid State Chemistry, General Chemistry, Condensed Matter Physics, law.invention, Crystallinity, law, General Materials Science, Crystallization, Protein crystallization, Macromolecule
Abstract

Macromolecular crystallography is the most direct and accurate approach to determine the three-dimensional structure of biological macromolecules. The growth of high quality single crystals, yielding diffraction to the highest X-ray resolution, remains a bottleneck in this methodology. Here we show that through a modification of the batch crystallization method, an entirely convection-free crystallization environment is achieved, which enhances the purity and crystallinity of protein crystals. This is accomplished by using an upside-down geometry, where crystals grow at the “ceiling” of a growth-cell completely filled with the crystallization solution. The “ceiling crystals” experience the same diffusion-limited conditions as in space microgravity experiments. The new method was tested on bovine insulin and two hen egg-white lysozyme polymorphs. In all cases, ceiling crystals diffracted X-rays to resolution limits beyond that for other methods using similar crystallization conditions without further optim...

Published
2013

11. MARINE-EXPRESS: taking advantage of high throughput cloning and expression strategies for the post-genomic analysis of marine organisms

Author

Isabel Morgado, Catherine Boyen, Mirjam Czjzek, Deborah M. Power, Claire Geslin, Didier Flament, Christiane Herve, Etienne Rebuffet, Alexandra Jeudy, Gurvan Michel, Yann Chevolot, Agnès Groisillier, Margherita Branno, Hervé Moreau, Pierre Francois Pluchon, Végétaux marins et biomolécules, Station biologique de Roscoff [Roscoff] (SBR), Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-GOEMAR-Centre National de la Recherche Scientifique (CNRS), Laboratoire de microbiologie des environnements extrêmophiles (LM2E), Centre National de la Recherche Scientifique (CNRS)-Université de Brest (UBO)-Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER), INL - Chimie et Nanobiotechnologies (INL - C&N), Institut des Nanotechnologies de Lyon (INL), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-École Centrale de Lyon (ECL), Université de Lyon-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-École supérieure de Chimie Physique Electronique de Lyon (CPE)-Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-École supérieure de Chimie Physique Electronique de Lyon (CPE), Centro de Ciências do Mar [Faro] (CCMAR), Universidade do Algarve (UAlg), Cellular and Developmental Biology, Stazione Zoologica Anton Dohrn (SZN), Modèles en biologie cellulaire et évolutive (MBCE), Observatoire océanologique de Banyuls (OOB), Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER)-Université de Brest (UBO)-Centre National de la Recherche Scientifique (CNRS), École Centrale de Lyon (ECL), Université de Lyon-Université de Lyon-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-École Supérieure de Chimie Physique Électronique de Lyon (CPE)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-École Centrale de Lyon (ECL), and Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Pyrococcus abyssi, MESH: High-Throughput Screening Assays, Archaeal Proteins, Recombinant Fusion Proteins, ved/biology.organism_classification_rank.species, lcsh:QR1-502, MESH: Sea Bream, Bioengineering, Computational biology, MESH: Flavobacteriaceae, Biology, Genome, Applied Microbiology and Biotechnology, lcsh:Microbiology, Microbiology, 03 medical and health sciences, Plasmid, Marine bacteriophage, Bacterial Proteins, MESH: Plasmids, MESH: Recombinant Fusion Proteins, Animals, MESH: Animals, MESH: Cloning, Molecular, Cloning, Molecular, Model organism, Gene, MESH: Pyrococcus abyssi, MESH: Bacterial Proteins, 030304 developmental biology, Cloning, 0303 health sciences, Expression vector, ved/biology, Research, 030302 biochemistry & molecular biology, Computational Biology, Eukaryota, MESH: Archaeal Proteins, Sea Bream, High-Throughput Screening Assays, MESH: Eukaryota, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Flavobacteriaceae, Function (biology), Plasmids, Biotechnology, MESH: Computational Biology
Abstract

Background The production of stable and soluble proteins is one of the most important steps prior to structural and functional studies of biological importance. We investigated the parallel production in a medium throughput strategy of genes coding for proteins from various marine organisms, using protocols that involved recombinatorial cloning, protein expression screening and batch purification. This strategy was applied in order to respond to the need for post-genomic validation of the recent success of a large number of marine genomic projects. Indeed, the upcoming challenge is to go beyond the bioinformatic data, since the bias introduced through the genomes of the so called model organisms leads to numerous proteins of unknown function in the still unexplored world of the oceanic organisms. Results We present here the results of expression tests for 192 targets using a 96-well plate format. Genes were PCR amplified and cloned in parallel into expression vectors pFO4 and pGEX-4T-1, in order to express proteins N-terminally fused to a six-histidine-tag and to a GST-tag, respectively. Small-scale expression and purification permitted isolation of 84 soluble proteins and 34 insoluble proteins, which could also be used in refolding assays. Selected examples of proteins expressed and purified to a larger scale are presented. Conclusions The objective of this program was to get around the bottlenecks of soluble, active protein expression and crystallization for post-genomic validation of a number of proteins that come from various marine organisms. Multiplying the constructions, vectors and targets treated in parallel is important for the success of a medium throughput strategy and considerably increases the chances to get rapid access to pure and soluble protein samples, needed for the subsequent biochemical characterizations. Our set up of a medium throughput strategy applied to genes from marine organisms had a mean success rate of 44% soluble protein expression from marine bacteria, archaea as well as eukaryotic organisms. This success rate compares favorably with other protein screening projects, particularly for eukaryotic proteins. Several purified targets have already formed the base for experiments aimed at post-genomic validation.

Published
2010
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