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1. Functioning of the dimeric GABAB receptor extracellular domain revealed by glycan wedge scanning

Author

Fanny Malhaire, Jianfeng Liu, Carine Monnier, Jean-Philippe Pin, Philippe Rondard, Bertrand Blanchard, Eric Trinquet, Ying Li, Nadia Oueslati, Gilles Labesse, Siluo Huang, Haijun Tu, Rondard, Philippe, Institut de Génomique Fonctionnelle (IGF), Université Montpellier 1 (UM1)-Université Montpellier 2 - Sciences et Techniques (UM2)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Sino-France Laboratory for Drug Screening, Huazhong University of Science and Technology [Wuhan] (HUST), Cisbio, Research Department, CIS BIOINTERNATIONAL, Centre de Biochimie Structurale [Montpellier] (CBS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Université de Montpellier (UM)-Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
Models, Molecular, [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, Plasma protein binding, Protein Structure, Secondary, 0302 clinical medicine, Chlorocebus aethiops, Fluorescence Resonance Energy Transfer, Receptor, gamma-Aminobutyric Acid, 0303 health sciences, General Neuroscience, Transmembrane domain, Biochemistry, COS Cells, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Neurons and Cognition (q-bio.NC), Dimerization, Protein Binding, Agonist, G protein, medicine.drug_class, Blotting, Western, Allosteric regulation, Enzyme-Linked Immunosorbent Assay, Biology, GABAB receptor, Transfection, Article, General Biochemistry, Genetics and Molecular Biology, Cell Line, Structure-Activity Relationship, 03 medical and health sciences, Allosteric Regulation, Polysaccharides, medicine, Animals, Humans, Immunoprecipitation, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Molecular Biology, 030304 developmental biology, G protein-coupled receptor, Binding Sites, General Immunology and Microbiology, [SDV.NEU.NB] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, Computational Biology, Biomolecules (q-bio.BM), Protein Structure, Tertiary, Receptors, GABA-B, nervous system, Quantitative Biology - Biomolecules, FOS: Biological sciences, Quantitative Biology - Neurons and Cognition, Biophysics, 030217 neurology & neurosurgery
Abstract

International audience; The G-protein-coupled receptor (GPCR) activated by the neurotransmitter GABA is made up of two subunits, GABA(B1) and GABA(B2). GABA(B1) binds agonists, whereas GABA(B2) is required for trafficking GABA(B1) to the cell surface, increasing agonist affinity to GABA(B1), and activating associated G proteins. These subunits each comprise two domains, a Venus flytrap domain (VFT) and a heptahelical transmembrane domain (7TM). How agonist binding to the GABA(B1) VFT leads to GABA(B2) 7TM activation remains unknown. Here, we used a glycan wedge scanning approach to investigate how the GABA(B) VFT dimer controls receptor activity. We first identified the dimerization interface using a bioinformatics approach and then showed that introducing an N-glycan at this interface prevents the association of the two subunits and abolishes all activities of GABA(B2), including agonist activation of the G protein. We also identified a second region in the VFT where insertion of an N-glycan does not prevent dimerization, but blocks agonist activation of the receptor. These data provide new insight into the function of this prototypical GPCR and demonstrate that a change in the dimerization interface is required for receptor activation.

Published
2008
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2. In Vitro Models for the Study of the Intracellular Activity of Antibiotics

Author

Julien M. Buyck, Cristina Seral, Sandrine Lemaire, Paul M. Tulkens, Ahalieyah Anantharajah, Frédéric Peyrusson, Françoise Van Bambeke, Pharmacologie des anti-infectieux (PHAR), Université de Poitiers-Institut National de la Santé et de la Recherche Médicale (INSERM), Groupe de recherche Achac (ACHAC), Aix Marseille Université (AMU)-Université Paris 1 Panthéon-Sorbonne (UP1)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Université de Lausanne (UNIL)-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National des Langues et Civilisations Orientales (Inalco)-Centre National de la Recherche Scientifique (CNRS)-Université de Bordeaux (UB)-Université Sorbonne Nouvelle - Paris 3-Université de Lorraine (UL)-Université libre de Bruxelles (ULB), University of Zaragoza - Universidad de Zaragoza [Zaragoza], Cisbio, Research Department, CIS BIOINTERNATIONAL, Pharmacologie Cellulaire et Moléculaire [Brussels], Louvain Drug Research Institute [Bruxelles, Belgique] (LDRI), Université Catholique de Louvain = Catholic University of Louvain (UCL)-Université Catholique de Louvain = Catholic University of Louvain (UCL), Université Catholique de Louvain = Catholic University of Louvain (UCL), Université Paris 1 Panthéon-Sorbonne (UP1)-Université Sorbonne Nouvelle - Paris 3-Université de Lausanne = University of Lausanne (UNIL)-Université de Rennes (UR)-Université Nice Sophia Antipolis (1965 - 2019) (UNS), and COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Aix Marseille Université (AMU)-Université de Bordeaux (UB)-Institut National des Langues et Civilisations Orientales (Inalco)-Université libre de Bruxelles (ULB)-Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA)

Subjects
0301 basic medicine, 0303 health sciences, medicine.drug_class, 030306 microbiology, Intracellular parasite, Phagocytosis, 030106 microbiology, Antibiotics, Biology, medicine.disease_cause, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, In vitro, Microbiology, 3. Good health, 03 medical and health sciences, Staphylococcus aureus, medicine, Gentamicin, Intracellular, Bacteria, ComputingMilieux_MISCELLANEOUS, medicine.drug, 030304 developmental biology
Abstract

Intracellular bacteria are poorly responsive to antibiotic treatment. Pharmacological studies are thus needed to determine which antibiotics are most potent or effective against intracellular bacteria as well as to explore the reasons for poor bacterial responsiveness. An in vitro pharmacodynamic model is described, consisting of (1) phagocytosis of pre-opsonized bacteria by eukaryotic cells; (2) elimination of non-internalized bacteria with gentamicin; (3) incubation of infected cells with antibiotics; and (4) determination of surviving bacteria by viable cell counting and normalization of the counts based on sample protein content.

Published
2016
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3. RX-P873, a Novel Protein Synthesis Inhibitor, Accumulates in Human THP-1 Monocytes and Is Active against Intracellular Infections by Gram-Positive (Staphylococcus aureus) and Gram-Negative (Pseudomonas aeruginosa) Bacteria

Author

Julien M. Buyck, Frédéric Peyrusson, Paul M. Tulkens, Françoise Van Bambeke, Pharmacologie des anti-infectieux (PHAR), Université de Poitiers-Institut National de la Santé et de la Recherche Médicale (INSERM), Cisbio, Research Department, CIS BIOINTERNATIONAL, Pharmacologie Cellulaire et Moléculaire [Brussels], Louvain Drug Research Institute [Bruxelles, Belgique] (LDRI), Université Catholique de Louvain = Catholic University of Louvain (UCL)-Université Catholique de Louvain = Catholic University of Louvain (UCL), and Université Catholique de Louvain = Catholic University of Louvain (UCL)

Subjects
Staphylococcus aureus, medicine.drug_class, Antibiotics, Moxifloxacin, Ceftazidime, Microbial Sensitivity Tests, Pyrimidinones, Staphylococcal infections, medicine.disease_cause, Guanidines, Monocytes, Microbiology, 03 medical and health sciences, Daptomycin, Ciprofloxacin, Vancomycin, Drug Resistance, Multiple, Bacterial, medicine, Humans, Pharmacology (medical), Pseudomonas Infections, Cells, Cultured, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Pharmacology, Protein Synthesis Inhibitors, 0303 health sciences, biology, 030306 microbiology, Pseudomonas aeruginosa, Staphylococcal Infections, medicine.disease, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 3. Good health, Anti-Bacterial Agents, Infectious Diseases, Bacteria, medicine.drug, Fluoroquinolones
Abstract

The pyrrolocytosine RX-P873, a new broad-spectrum antibiotic in preclinical development, inhibits protein synthesis at the translation step. The aims of this work were to study RX-P873's ability to accumulate in eukaryotic cells, together with its activity against extracellular and intracellular forms of infection by Staphylococcus aureus and Pseudomonas aeruginosa , using a pharmacodynamic approach allowing the determination of maximal relative efficacies ( E max values) and bacteriostatic concentrations ( C s values) on the basis of Hill equations of the concentration-response curves. RX-P873's apparent concentration in human THP-1 monocytes was about 6-fold higher than the extracellular one. In broth, MICs ranged from 0.125 to 0.5 mg/liter ( S. aureus ) and 2 to 8 mg/liter ( P. aeruginosa ), with no significant shift in these values against strains resistant to currently used antibiotics being noted. In concentration-dependent experiments, the pharmacodynamic profile of RX-P873 was not influenced by the resistance phenotype of the strains. E max values (expressed as the decrease in the number of CFU from that in the initial inoculum) against S. aureus and P. aeruginosa reached more than 4 log units and 5 log units in broth, respectively, and 0.7 log unit and 2.7 log units in infected THP-1 cells, respectively, after 24 h. C s values remained close to the MIC in all cases, making RX-P873 more potent than antibiotics to which the strains were resistant (moxifloxacin, vancomycin, and daptomycin for S. aureus ; ciprofloxacin and ceftazidime for P. aeruginosa ). Kill curves in broth showed that RX-P873 was more rapidly bactericidal against P. aeruginosa than against S. aureus . Taken together, these data suggest that RX-P873 may constitute a useful alternative for infections involving intracellular bacteria, especially Gram-negative species.

Published
2015
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4. HER3 as biomarker and therapeutic target in pancreatic cancer: new insights in pertuzumab therapy in preclinical models

Author

Thomas, Gaëlle, Chardès, Thierry, Gaborit, Nadège, Mollevi, Caroline, Leconet, Wilhem, Robert, Bruno, Kharrat, Abdelhakim, Radosevic-Robin, Nina, Penault-Llorca, Frédérique, Gongora, Céline, Colombo, Pierre-Emmanuel, Lazrek, Yassamine, Bras-Goncalves, Rui, Savina, Ariel, Azria, David, Bazin, Hervé, Pèlegrin, André, Larbouret, Christel, Institut de recherche en cancérologie de Montpellier (IRCM - U896 Inserm - UM1), CRLCC Val d'Aurelle - Paul Lamarque-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Montpellier 1 (UM1), Institut de Recherche et Médecine Translationnelle [Boulogne-Billancourt] (IRRMT), Roche France, Unité de biostatistiques, CRLCC Val d'Aurelle - Paul Lamarque, Centre Jean Perrin, CRLCC Jean Perrin, Equipe de recherche sur les traitements individualisés des cancers (ERTICa), Université d'Auvergne - Clermont-Ferrand I (UdA), Institut de Recherche en Cancérologie de Montpellier (IRCM - U1194 Inserm - UM), CRLCC Val d'Aurelle - Paul Lamarque-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Recherches en Cancérologie de Toulouse (CRCT), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Immunociblage des Tumeurs et Ingenierie des Anticorps, Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM), Cis Bio International, This work was supported by the program 'Investissement d’avenir' grant agreement: Labex MabImprove, ANR-10-LABX-53-01 and INCa-DGOS-Inserm 6045., We thank G. Heintz, S. Bousquié, V. Garambois, I. Aït-Arsa and A. Ho-pun-cheung for excellent technical assistance. Flow cytometry analysis was performed thanks to N. Vié using equipment at the Montpellier IRO imaging facility. We thank F. Bibeau for providing pancreatic tumor samples, and A. Cayre and F. Boissière for pathology technical assistance., Université Montpellier 1 (UM1)-CRLCC Val d'Aurelle - Paul Lamarque-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Centre Jean Perrin [Clermont-Ferrand] (UNICANCER/CJP), UNICANCER, Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut Roche de Recherche et Médecine Translationnelle (IRRMT), Millegen SA, MILEGEN-Immeuble BIOSTEP, ROCHE SAS, Roche SAS, Cisbio, Research Department, CIS BIOINTERNATIONAL, This work was supported by the program 'Investissement d’avenir' grant agreement: Labex MabImprove, ANR-10-LABX-53-01 and INCa- DGOS-Inserm 6045., CRLCC Val d'Aurelle - Paul Lamarque-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), and Radosevic-Robin, Nina

Subjects
MESH: Signal Transduction, Receptor, ErbB-3, MESH: Random Allocation, pancreatic cancer, MESH: Receptor, ErbB-3/metabolism, medicine.disease_cause, Mice, Random Allocation, MESH: Pancreatic Neoplasms/enzymology, MESH: Cell Proliferation/drug effects, MESH: Animals, skin and connective tissue diseases, 3. Good health, MESH: Antibodies, Monoclonal, Humanized/pharmacology, MESH: Pancreatic Neoplasms/drug therapy, Oncology, Monoclonal, Biomarker (medicine), Female, Pertuzumab, MESH: Biomarkers, Tumor, MESH: Receptor, ErbB-3/antagonists & inhibitors, Research Paper, Signal Transduction, medicine.drug, MESH: Xenograft Model Antitumor Assays, medicine.drug_class, Mice, Nude, Antineoplastic Agents, [SDV.CAN]Life Sciences [q-bio]/Cancer, Biology, Antibodies, Monoclonal, Humanized, Monoclonal antibody, [SDV.CAN] Life Sciences [q-bio]/Cancer, MESH: Antineoplastic Agents/pharmacology, pertuzumab, HER3, Pancreatic cancer, HER2, Biomarkers, Tumor, medicine, MESH: Mice, Nude, Animals, Humans, MESH: Mice, Cell Proliferation, MESH: Humans, Cancer, medicine.disease, Xenograft Model Antitumor Assays, Pancreatic Neoplasms, body regions, Tumor progression, Cancer research, Carcinogenesis, MESH: Female, MESH: Pancreatic Neoplasms/pathology
Abstract

// Gaelle Thomas 1,2 , Thierry Chardes 1 , Nadege Gaborit 1 , Caroline Mollevi 5 , Wilhem Leconet 1 , Bruno Robert 1 , Nina Radosevic-Robin 3 , Frederique Penault-Llorca 3 , Celine Gongora 1 , Pierre-Emmanuel Colombo 1 , Yassamine Lazrek 1,4 , Rui Bras-Goncalves 1 , Ariel Savina 6 , David Azria 1 , Herve Bazin 7 , Andre Pelegrin 1 and Christel Larbouret 1 1 IRCM, Institut de Recherche en Cancerologie de Montpellier, Montpellier, F-34298, France; INSERM, Unit 896, Montpellier, F-34298, France; Universite Montpellier1, Montpellier, F-34298, France; ICM, Montpellier, France 2 Institut Roche de Recherche et Medecine Translationnelle, Boulogne Bilancourt, France 3 Department of Biopathology, The Jean Perrin Comprehensive Cancer Center and ERTICa Research Group, University of Auvergne EA4677, Clermont-Ferrand, France 4 Millegen SA, F-31681, Labege, France 5 Unite de Biostatistiques, ICM Val d’Aurelle, Montpellier, France 6 Roche SAS Scientific Partnerships, Boulogne Billancourt, France 7 CisBio Bioassays, Le Codolet, France Correspondence: Christel Larbouret, email: // Keywords : HER3; HER2; pertuzumab; pancreatic cancer Received : June 30, 2014 Accepted : July 16, 2014 Published : July 17, 2014 Abstract The anti-HER2 antibody pertuzumab inhibits HER2 dimerization and affects HER2/HER3 dimer formation and signaling. As HER3 and its ligand neuregulin are implicated in pancreatic tumorigenesis, we investigated whether HER3 expression could be a predictive biomarker of pertuzumab efficacy in HER2 low -expressing pancreatic cancer. We correlated in vitro and in vivo HER3 expression and neuregulin dependency with the inhibitory effect of pertuzumab on cell viability and tumor progression. HER3 knockdown in BxPC-3 cells led to resistance to pertuzumab therapy. Pertuzumab treatment of HER3-expressing pancreatic cancer cells increased HER3 at the cell membrane, whereas the anti-HER3 monoclonal antibody 9F7-F11 down-regulated it. Both antibodies blocked HER3 and AKT phosphorylation and inhibited HER2/HER3 heterodimerization but affected differently HER2 and HER3 homodimers. The pertuzumab/9F7-F11 combination enhanced tumor inhibition and the median survival time in mice xenografted with HER3-expressing pancreatic cancer cells. Finally, HER2 and HER3 were co-expressed in 11% and HER3 alone in 27% of the 45 pancreatic ductal adenocarcinomas analyzed by immunohistochemistry. HER3 is essential for pertuzumab efficacy in HER2 low -expressing pancreatic cancer and HER3 expression might be a predictive biomarker of pertuzumab efficacy in such cancers. Further studies in clinical samples are required to confirm these findings and the interest of combining anti-HER2 and anti-HER3 therapeutic antibodies.

Published
2014
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5. Technetium-99m radiolabelling of anN-amino-alkyl-benzamide nitrido- and oxo-technetium bis(aminoethanethiol) derivative synthesis and biological results. Potential melanoma tracer agents

Author

Roberto Pasqualini, Michèle Borel, M. F. Moreau, Janine Papon, Philippe Auzeloux, Martine Bayle, Jean-Claud Madelmont, Imagerie Moléculaire et Thérapie Vectorisée (IMTV), ITMO ' Technologies pour la Santé '-Cancéropôle CLARA-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université d'Auvergne - Clermont-Ferrand I (UdA), Etude Métabolique des Molécules Marquées, Université d'Auvergne - Clermont-Ferrand I (UdA)-Institut National de la Santé et de la Recherche Médicale (INSERM), CIS BIOINTERNATIONAL, Inserm, and Université d'Auvergne - Clermont-Ferrand I (UdA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Cancéropôle CLARA-ITMO ' Technologies pour la Santé '

Subjects
Ligand, Stereochemistry, [SDV]Life Sciences [q-bio], Organic Chemistry, chemistry.chemical_element, Conjugated system, Technetium, Biochemistry, Medicinal chemistry, Chemical synthesis, 3. Good health, Analytical Chemistry, chemistry.chemical_compound, chemistry, Technetium-99, Drug Discovery, [CHIM]Chemical Sciences, Radiology, Nuclear Medicine and imaging, Benzamide, Technetium-99m, Spectroscopy, Derivative (chemistry)
Abstract

International audience; N-(2-diethylaminoethyl)-4-iodobenzamide has been reported to be an excellent agent for malignant melanoma diagnosis by SPECT. To obtain a 99mTc analog, we synthesized a new bis(aminothiol) (BAT) derivative. The benzamide function was conjugated onto a nitrogen of the BAT backbone. This ligand was successfully radiolabelled with both nitrido-technetium and oxo-technetium cores. These complexes were purified by HPLC. Tumour uptake was measured in intravenously injected mice bearing the B16 murine melanoma.

Published
1999
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6. Homogeneous Two-Site Immunometric Assay Kinetics as a Theoretical Tool for Data Analysis

Author

Jean-Pierre Flandrois, Emmanuel Zuber, Gérard Mathis, Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS), Cisbio, Research Department, CIS BIOINTERNATIONAL, Bioinformatique, phylogénie et génomique évolutive (BPGE), Département PEGASE [LBBE] (PEGASE), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE)

Subjects
Fluoroimmunoassay, Kinetics, Combined use, Biophysics, Analytical chemistry, [SDV.BID.SPT]Life Sciences [q-bio]/Biodiversity/Systematics, Phylogenetics and taxonomy, 01 natural sciences, Biochemistry, Antigen-Antibody Reactions, 03 medical and health sciences, [SDV.EE.ECO]Life Sciences [q-bio]/Ecology, environment/Ecosystems, medicine, Animals, Humans, Antigens, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, Chromatography, medicine.diagnostic_test, Chemistry, [SDV.BID.EVO]Life Sciences [q-bio]/Biodiversity/Populations and Evolution [q-bio.PE], 010401 analytical chemistry, Reproducibility of Results, Cell Biology, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Prolactin, 0104 chemical sciences, Models, Chemical, Nonlinear Dynamics, Evaluation Studies as Topic, Inflection point, Homogeneous, Data Interpretation, Statistical, Immunoassay, Data reduction
Abstract

The easily accessible kinetics of a new homogeneous two-site fluorometric immunoassay for prolactin was studied, in order to determine its usefulness for assay data reduction and optimization. The combined use of a simple descriptive model fitted to experimental data and a mechanistic model to simulate the kinetics revealed that (i) the kinetics curve presented an early inflexion point. Its time of occurrence was constant as long as the antigen concentration was below the smallest antibody concentration and decreased to zero for higher concentrations. It may therefore be used as an indicator of hooked samples. (ii) The kinetics steepest slope was correlated with antigen concentration. Its use as a dose–response curve variable would allow higher concentrations to be assayed than with the classical end-point dose–response curve. The results suggest that control and exploitation of kinetic parameters could help to improve the rapidity, analytical range, and reliability of homogeneous two-site immunometric assays.

Published
1997
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7. Time-resolved fluorescence resonance energy transfer (TR-FRET) to analyze the disruption of EGFR/HER2 dimers: a new method to evaluate the efficiency of targeted therapy using monoclonal antibodies

Author

Gaborit, Nadège, Larbouret, Christel, Vallaghe, Julie, Peyrusson, Frédéric, Bascoul-Mollevi, Caroline, Crapez, Evelyne, Azria, David, Chardes, Thierry, Poul, Marie-Alix, Mathis, Gérard, Bazin, Hervé, Pèlegrin, André, Institut de recherche en cancérologie de Montpellier (IRCM - U896 Inserm - UM1), CRLCC Val d'Aurelle - Paul Lamarque-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Montpellier 1 (UM1), CRLC Val d'Aurelle-Paul Lamarque, CRLCC Val d'Aurelle - Paul Lamarque, Cisbio, Research Department, and CIS BIOINTERNATIONAL

Subjects
MESH: Cell Line, Tumor, MESH: Humans, MESH: Phosphorylation, MESH: Fluorescence Resonance Energy Transfer, MESH: Protein Multimerization, MESH: Drug Screening Assays, Antitumor, [SDV.CAN]Life Sciences [q-bio]/Cancer, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, MESH: Receptors, Fibroblast Growth Factor, [SDV.SP]Life Sciences [q-bio]/Pharmaceutical sciences, MESH: Quinazolines, MESH: Antibodies, Neoplasm, MESH: Antibodies, Monoclonal, Murine-Derived, MESH: Receptor, erbB-2, MESH: Cell Proliferation, MESH: Protein Kinase Inhibitors, MESH: Antineoplastic Agents, [SDV.IMM]Life Sciences [q-bio]/Immunology, MESH: Animals, MESH: Neoplasms, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, skin and connective tissue diseases, neoplasms, MESH: Mice, MESH: NIH 3T3 Cells
Abstract

International audience; In oncology, simultaneous inhibition of epidermal growth factor receptor (EGFR) and HER2 by monoclonal antibodies (mAbs) is an efficient therapeutic strategy but the underlying mechanisms are not fully understood. Here, we describe a time-resolved fluorescence resonance energy transfer (TR-FRET) method to quantify EGFR/HER2 heterodimers on cell surface to shed some light on the mechanism of such therapies. First, we tested this antibody-based TR-FRET assay in NIH/3T3 cell lines that express EGFR and/or HER2 and in various tumor cell lines. Then, we used the antibody-based TR-FRET assay to evaluate in vitro the effect of different targeted therapies on EGFR/HER2 heterodimers in the ovarian carcinoma cell line SKOV-3. A simultaneous incubation with Cetuximab (anti-EGFR) and Trastuzumab (anti-HER2) disturbed EGFR/HER2 heterodimers resulting in a 72% reduction. Cetuximab, Trastuzumab or Pertuzumab (anti-HER2) alone induced a 48, 44, or 24% reduction, respectively. In contrast, the tyrosine kinase inhibitors Erlotinib and Lapatinib had very little effect on EGFR/HER2 dimers concentration. In vivo, the combination of Cetuximab and Trastuzumab showed a better therapeutic effect (median survival and percentage of tumor-free mice) than the single mAbs. These results suggest a correlation between the extent of the mAb-induced EGFR/HER2 heterodimer reduction and the efficacy of such mAbs in targeted therapies. In conclusion, quantifying EGFR/HER2 heterodimers using our antibody-based TR-FRET assay may represent a useful method to predict the efficacy and explain the mechanisms of action of therapeutic mAbs, in addition to other commonly used techniques that focus on antibody-dependent cellular cytotoxicity, phosphorylation, and cell proliferation.

Published
2011
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8. Asymmetric functioning of dimeric metabotropic glutamate receptors disclosed by positive allosteric modulators

Author

Goudet, Cyril, Kniazeff, Julie, Hlavackova, Veronika, Malhaire, Fanny, Maurel, Damien, Acher, Francine, Blahos, Jaroslav, Prézeau, Laurent, Pin, Jean-Philippe, Institut de Génomique Fonctionnelle (IGF), Université Montpellier 1 (UM1)-Université Montpellier 2 - Sciences et Techniques (UM2)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Czech Academy of Sciences [Prague] (CAS), CIS BIOINTERNATIONAL, Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques (LCBPT - UMR 8601), Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), This work was supported in part by grants from the CNRS, the Action Concertée Incitative 'Molécules et Cibles Thérapeutiques' of the French ministry of research and technology, the fondation Paul Hamel, the comité Parkinson from the Fondation de France, Addex pharmaceuticals and the European Community (Grant LSHB-CT-200-503337) (to J.-P. P.) and by the Grant Agency of Czech Republic (GACR 301/03/1183 and 301/03/H095), Grant Agency Academy of Science of Czech Republic (KJB5039402), and AVOZ 50390512 (to J. B.). CG is supported by a fellowship from the Fondation pour la Recherche Médicale. JK is supported by a bourse de Docteur Ingénieur from the CNRS. DM is supported by both CisBio International and the French government (CIFRE fellowship)., and Goudet, Cyril

Subjects
MESH: GTP-Binding Proteins, MESH: Fluorescence Resonance Energy Transfer, MESH: Binding, Competitive, MESH: Receptors, G-Protein-Coupled, MESH: Actins, MESH: Protein Conformation, MESH: Plasmids, MESH: Recombinant Fusion Proteins, MESH: Protein Binding, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], MESH: Allosteric Site, MESH: Receptors, GABA-B, MESH: Receptors, Metabotropic Glutamate, MESH: Point Mutation, MESH: Humans, MESH: Time Factors, MESH: Models, Biological, MESH: Enzyme-Linked Immunosorbent Assay, [SDV.SP]Life Sciences [q-bio]/Pharmaceutical sciences, MESH: Inositol Phosphates, MESH: Cell Line, [SDV.SP] Life Sciences [q-bio]/Pharmaceutical sciences, MESH: Mutagenesis, Site-Directed, MESH: Dimerization, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], MESH: Cell Membrane
Abstract

International audience; The recent discovery of positive allosteric modulators (PAMs) for G-protein-coupled receptors open new possibilities to control a number of physiological and pathological processes. Understanding the mechanism of action of such compounds will provide new information on the activation process of these important receptors. Within the last 10 years, a number of studies indicate that G-protein-coupled receptors can form dimers, but the functional significance of this phenomenon remains elusive. Here we used the metabotropic glutamate receptors as a model, because these receptors, for which PAMs have been identified, are constitutive dimers. We used the quality control system of the GABA(B) receptor to generate metabotropic glutamate receptor dimers in which a single subunit binds a PAM. We show that one PAM/dimer is sufficient to enhance receptor activity. Such a potentiation can still be observed if the subunit unable to bind the PAM is also made unable to activate G-proteins. However, the PAM acts as a non-competitive antagonist when it binds in the subunit that cannot activate G-proteins. These data are consistent with a single heptahelical domain reaching the active state per dimer during receptor activation.

Published
2005
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9. Synthesis of two novel oxocyclam-binding technetium complexes containing an analogue of cocaine

Author

Turpin, F., Masri, F., Riché, F., Berthommier, E., Emond, P., Vidal, M., Auzeloux, P., Loc'h, C., Neuquelman, N., Mauclaire, L., CIS BIOINTERNATIONAL, Laboratoire d'études dynamiques et structurales de la sélectivité (LEDSS), Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF), Imagerie et cerveau (iBrain - Inserm U1253 - UNIV Tours ), Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Eras Labo, Imagerie Moléculaire et Thérapie Vectorisée (IMTV), ITMO ' Technologies pour la Santé '-Cancéropôle CLARA-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université d'Auvergne - Clermont-Ferrand I (UdA), Etude Métabolique des Molécules Marquées, Université d'Auvergne - Clermont-Ferrand I (UdA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Service Hospitalier Frédéric Joliot (SHFJ), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, DOPIMAG, Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), Université d'Auvergne - Clermont-Ferrand I (UdA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Cancéropôle CLARA-ITMO ' Technologies pour la Santé ', Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Université de Tours-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
[CHIM]Chemical Sciences
Abstract

International audience; In order to visualize and quantify dopamine transporters, the synthesis of two novel ligands labelled with technetium-99 m (99mTc) has been investigated. A multi-step synthesis afforded two target ligands with a tropane skeleton and a macrocyclic complexing moiety. The choice and the position of substituents are in adequation with dopamine transporter structure. The radiolabelling of these ligands with 99mTc has been studied and the results make them good candidates for SPECT imaging.

Published
2002
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10. Synthesis and biodistribution of technetium-99m-labelled N-(diethylaminoethyl)benzamide via a bis(dithiocarbamate) nitridotechnetium(V) complex

Author

Auzeloux, Philippe, Papon, Janine, Masnada, Thierry, Borel, Michèle, Moreau, Marie-France, Veyre, Annie, Pasqualini, Roberto, Madelmont, Jean-Claude, Imagerie Moléculaire et Thérapie Vectorisée (IMTV), Université d'Auvergne - Clermont-Ferrand I (UdA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Cancéropôle CLARA-ITMO ' Technologies pour la Santé ', Etude Métabolique des Molécules Marquées, Université d'Auvergne - Clermont-Ferrand I (UdA)-Institut National de la Santé et de la Recherche Médicale (INSERM), CIS BIOINTERNATIONAL, Inserm, and ITMO ' Technologies pour la Santé '-Cancéropôle CLARA-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université d'Auvergne - Clermont-Ferrand I (UdA)

Subjects
[SDV]Life Sciences [q-bio], [CHIM]Chemical Sciences
Abstract

International audience; N-(2-diethylaminoethyl)-4-iodobenzamide has been reported to be an excellent agent for malignant melanoma diagnosis by SPECT. To obtain a 99mTc analogue, we synthesized a sodium N-{2-[4-(N-diethylaminoethylcarbamoyl)phenyl]ethyl}-N-methyldithiocarbamate ligand in 7 steps from methyl 4-(bromomethyl)benzoate. The corresponding nitridotechnetium complex formed by a ligand exchange method was purified and its biodistribution in mice bearing the B16 murine melanoma was determined.

Published
1999
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11. A Descriptive Model for the Kinetics of a Homogeneous Fluorometric Immunoassay

Author

Bruno Darbouret, Emmanuel Zuber, Francoise Socquet, Laurent Rosso, Gérard Mathis, Jean-Pierre Flandrois, Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS), Centre Technique Interprofessionnel des Fruits et Légumes (CTIFL), Thermo Fisher Scientific [Nimes], Cisbio, Research Department, CIS BIOINTERNATIONAL, Bioinformatique, phylogénie et génomique évolutive (BPGE), Département PEGASE [LBBE] (PEGASE), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE)

Subjects
Immunology, Gompertz function, Kinetics, Analytical chemistry, [SDV.BID.SPT]Life Sciences [q-bio]/Biodiversity/Systematics, Phylogenetics and taxonomy, 01 natural sciences, 03 medical and health sciences, [SDV.EE.ECO]Life Sciences [q-bio]/Ecology, environment/Ecosystems, medicine, Range (statistics), Statistical analysis, Fluorometry, Antigens, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Pharmacology, Immunoassay, 0303 health sciences, medicine.diagnostic_test, Chemistry, Kinetic information, [SDV.BID.EVO]Life Sciences [q-bio]/Biodiversity/Populations and Evolution [q-bio.PE], 010401 analytical chemistry, Models, Immunological, Reproducibility of Results, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 0104 chemical sciences, Prolactin, Homogeneous, Regression Analysis, Biological system
Abstract

A descriptive mathematical model was chosen to fit the antigen-antibody association kinetics of a new homogeneous immunometric assay for prolactin, involving time-resolved fluorescence detection (TRACE® technology, Time Resolved Amplified Cryptate Emission). We paid special attention to the methodology and criteria applied, to yield a convenient and statistically valid model, designed to allow potential exploitation of kinetic information in the data processing of the assay. We compared specific parameterizations of an hyperbolic model, the Gompertz, and the monomolecular models on the basis of morphological considerations, a statistical analysis of fit, and an assessment of the parameters estimation quality, over a wide range of antigen concentrations. The monomolecular model gave the best fit, and the most precise and stable estimation of its parameters. The study of parameter properties confirmed this choice.

Published
1997
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12. A homogeneous time-resolved fluorescence detection of telomerase activity.

Author

Gabourdes M, Bourgine V, Mathis G, Bazin H, and Alpha-Bazin B

Subjects
Anthraquinones pharmacology, Deoxyuridine chemistry, Enzyme Inhibitors pharmacology, Fluorescent Dyes chemistry, Humans, K562 Cells, Oligodeoxyribonucleotides chemistry, Organometallic Compounds chemistry, Phycocyanin chemistry, Polymerase Chain Reaction methods, Sensitivity and Specificity, Telomerase antagonists & inhibitors, Telomerase chemistry, Deoxyuridine analogs & derivatives, Fluorescence Resonance Energy Transfer methods, Telomerase metabolism
Abstract

The homogeneous time-resolved fluorescence (HTRF) technology is an assay developed to study the interaction between biomolecules. This detection system is based on a fluorescence resonance energy transfer (FRET) between a Tris-bipyridine europium cryptate used as a long-lived fluorescent donor and a chemically modified allophycocyanine as acceptor. This technology is characterized by both a spectral selectivity and a temporal selectivity (due to the time-resolved mode), ensuring a highly specific signal. Here a europium-cryptate-labeled deoxyuridine triphosphate analogue (K-11-dUTP) was used to monitor the extension reaction on a biotinylated oligonucleotide used as substrate for telomerase in a telomeric repeat amplification protocol (TRAP). After the addition of an allophycocyanine-streptavidin conjugate, the extension products give rise to a FRET between the incorporated cryptate moieties and the allophycocyanine acceptor that then displays a specific long-lived emission. The TRAP-HTRF format was validated as a screening tool by using a 2,6-diaminoanthraquinone analogue, a known inhibitor of telomerase activity. The IC(50) measured was consistent with the reported values, showing the convenience of the HTRF technology for the study of telomerase activity and inhibitors.

Published
2004
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13. A homogeneous caspase-3 activity assay using HTRF technology.

Author

Préaudat M, Ouled-Diaf J, Alpha-Bazin B, Mathis G, Mitsugi T, Aono Y, Takahashi K, and Takemoto H

Subjects
Caspase 3, Organometallic Compounds metabolism, Phycocyanin metabolism, Biological Assay, Caspases analysis, Fluorometry methods
Abstract

Caspases are cysteine proteases presenting a conserved active site that cleaves protein substrates at a highly specific position. They are involved in different aspects of the active cell death pathway. Most of them act through proteolytic degradations of cellular components. This paper describes the assay development, assay validation, and screening for inhibitors of this enzyme, which could be potential drug candidates. The assay uses homogeneous time-resolved fluorescence based on energy transfer from europium cryptate as donor to cross-linked allophycocyanin as acceptor (XL665). A double-tagged substrate, biotinyl-epsilon-aminocaproyl-L-aspartyl-L-glutamyl-L-valyl-Laspartyl-L-alanyl-L-propyl-N(epsilon)-(2,4-dinitrophenyl)-L-lysine-amide (biotin-X-DEVDAPK(dnp)-NH(2)), is conjugated with streptavidin cryptate and anti-dnp-XL665 monoclonal antibody. The close proximity between donor and acceptor induces a specific time-resolved fluorescence signal. In the presence of enzyme activity, the substrate cleavage induces an unlinking of the two fluorescent probes and, subsequently, the disappearance of the specific signal as a result of loss of proximity. Experiments to optimize the reagent concentration, incubation times, precision, reproducibility, and robustness are discussed in comparison with a fluorometric method.

Published
2002
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14. Assessment of chromogranin A using two-site immunoassay. Selection of a monoclonal antibody pair unaffected by human chromogranin A processing.

Author

Degorce F

Subjects
Biomarkers, Tumor analysis, Biomarkers, Tumor blood, Blood Chemical Analysis, Chromogranin A, Chromogranins blood, Chromogranins metabolism, Epitopes chemistry, Humans, Immunohistochemistry methods, Immunoradiometric Assay, Neuroendocrine Tumors chemistry, Neuroendocrine Tumors diagnosis, Protein Processing, Post-Translational, Antibodies, Monoclonal, Chromogranins analysis, Chromogranins immunology, Immunoassay methods
Published
2000
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15. A new human chromogranin A (CgA) immunoradiometric assay involving monoclonal antibodies raised against the unprocessed central domain (145-245).

Author

Degorce F, Goumon Y, Jacquemart L, Vidaud C, Bellanger L, Pons-Anicet D, Seguin P, Metz-Boutigue MH, and Aunis D

Subjects
Amino Acid Sequence, Animals, Antibodies, Monoclonal chemistry, Chromogranin A, Chromogranins chemistry, Chromogranins isolation & purification, Humans, Metalloendopeptidases metabolism, Mice, Mice, Inbred BALB C, Recombinant Proteins chemistry, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Antibodies, Monoclonal immunology, Chromogranins metabolism, Immunoradiometric Assay methods, Neuroendocrine Tumors metabolism, Peptide Fragments immunology
Abstract

Chromogranin A (CgA), a major protein of chromaffin granules, has been described as a potential marker for neuroendocrine tumours. Because of an extensive proteolysis which leads to a large heterogeneity of circulating fragments, its presence in blood has been assessed in most cases either by competitive immunoassays or with polyclonal antibodies. In the present study, 24 monoclonal antibodies were raised against native or recombinant human CgA. Their mapping with proteolytic peptides showed that they defined eight distinct epitopic groups which spanned two-thirds of the C-terminal part of human CgA. All monoclonal antibodies were tested by pair and compared with a reference radioimmunoassay (RIA) involving CGS06, one of the monoclonal antibodies against the 198-245 sequence. It appears that CgA C-terminal end seems to be highly affected by proteolysis and the association of C-terminal and median-part monoclonal antibodies is inadequate for total CgA assessment. Our new immunoradiometric assay involves two monoclonal antibodies, whose contiguous epitopes lie within the median 145-245 sequence. This assay allows a sensitive detection of total human CgA and correlates well with RIA because dibasic cleavage sites present in the central domain do not seem to be affected by degradation. It has been proved to be efficient in measuring CgA levels in patients with neuroendocrine tumours.

Published
1999
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16. Europium(III) cryptate: a fluorescent label for the detection of DNA hybrids on solid support.

Author

Prat O, Lopez E, and Mathis G

Subjects
Bacterial Proteins analysis, Biotin analysis, Streptavidin, Bridged Bicyclo Compounds chemistry, DNA Probes analysis, Europium chemistry, Fluorescent Dyes, Nucleic Acid Hybridization, Organometallic Compounds chemistry
Abstract

We report here a new detection method for DNA hybrids on dot blots. The process utilizes DNA or oligonucleotide probes labeled with biotin, followed by recognition with a conjugate of streptavidin and europium cryptate, a time-resolved fluorescent label. Unlike the other lanthanide chelates, this label is an organic molecule embedding a europium ion into an intramolecular cavity. This structure has a better stability in diluted assay media, a good sensitivity even on solid support, and an elevated fluorescence lifetime which allows elimination of most of the background generated by other species present in the assay medium. This procedure is quantitative and detects down to 2 amol of a model DNA, which is similar to other nonisotopic (especially colorimetric) methods. The main advantages of this method are easy automation, quantitation, and rapidity of measurements.

Published
1991
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