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1. An equine iPSC-based phenotypic screening platform identifies pro- and anti-viral molecules against West Nile virus

Author

Marielle Cochet, François Piumi, Kamila Gorna, Noémie Berry, Gaëlle Gonzalez, Anne Danckaert, Nathalie Aulner, Odile Blanchet, Stéphan Zientara, Francesc Xavier Donadeu, Hélène Munier-Lehmann, Jennifer Richardson, Alexandra Benchoua, and Muriel Coulpier

Subjects
Equine, brain, neural progenitors, Flavivirus, antiviral, statin, Veterinary medicine, SF600-1100
Abstract

Abstract Outbreaks of West Nile virus (WNV) occur periodically, affecting both human and equine populations. There are no vaccines for humans, and those commercialised for horses do not have sufficient coverage. Specific antiviral treatments do not exist. Many drug discovery studies have been conducted, but since rodent or primate cell lines are normally used, results cannot always be transposed to horses. There is thus a need to develop relevant equine cellular models. Here, we used induced pluripotent stem cells to develop a new in vitro model of WNV-infected equine brain cells suitable for microplate assay, and assessed the cytotoxicity and antiviral activity of forty-one chemical compounds. We found that one nucleoside analog, 2′C-methylcytidine, blocked WNV infection in equine brain cells, whereas other compounds were either toxic or ineffective, despite some displaying anti-viral activity in human cell lines. We also revealed an unexpected proviral effect of statins in WNV-infected equine brain cells. Our results thus identify a potential lead for future drug development and underscore the importance of using a tissue- and species-relevant cellular model for assessing the activity of antiviral compounds.

Published
2024
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2. A journey into the regulatory secrets of the de novo purine nucleotide biosynthesis

Author

Nour Ayoub, Antoine Gedeon, and Hélène Munier-Lehmann

Subjects
allostery, antibacterial agents, chemical compounds, enzyme regulation, IMP dehydrogenase, nucleotide biosynthesis, Therapeutics. Pharmacology, RM1-950
Abstract

De novo purine nucleotide biosynthesis (DNPNB) consists of sequential reactions that are majorly conserved in living organisms. Several regulation events take place to maintain physiological concentrations of adenylate and guanylate nucleotides in cells and to fine-tune the production of purine nucleotides in response to changing cellular demands. Recent years have seen a renewed interest in the DNPNB enzymes, with some being highlighted as promising targets for therapeutic molecules. Herein, a review of two newly revealed modes of regulation of the DNPNB pathway has been carried out: i) the unprecedent allosteric regulation of one of the limiting enzymes of the pathway named inosine 5′-monophosphate dehydrogenase (IMPDH), and ii) the supramolecular assembly of DNPNB enzymes. Moreover, recent advances that revealed the therapeutic potential of DNPNB enzymes in bacteria could open the road for the pharmacological development of novel antibiotics.

Published
2024
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3. Discovery of Bis-Imidazoline Derivatives as New CXCR4 Ligands

Author

Zhicheng Zhou, Isabelle Staropoli, Anne Brelot, Peggy Suzanne, Aurélien Lesnard, Fanny Fontaine, Serge Perato, Sylvain Rault, Olivier Helynck, Fernando Arenzana-Seisdedos, Jana Sopkova-de Oliveira Santos, Bernard Lagane, Hélène Munier-Lehmann, and Philippe Colin

Subjects
CXCR4, CXCL12, bis-imidazoline, antagonist, therapeutic target, HIV-1 infection, Organic chemistry, QD241-441
Abstract

The chemokine receptor CXCR4 and its ligand CXCL12 regulate leukocyte trafficking, homeostasis and functions and are potential therapeutic targets in many diseases such as HIV-1 infection and cancers. Here, we identified new CXCR4 ligands in the CERMN chemical library using a FRET-based high-throughput screening assay. These are bis-imidazoline compounds comprising two imidazole rings linked by an alkyl chain. The molecules displace CXCL12 binding with submicromolar potencies, similarly to AMD3100, the only marketed CXCR4 ligand. They also inhibit anti-CXCR4 mAb 12G5 binding, CXCL12-mediated chemotaxis and HIV-1 infection. Further studies with newly synthesized derivatives pointed out to a role of alkyl chain length on the bis-imidazoline properties, with molecules with an even number of carbons equal to 8, 10 or 12 being the most potent. Interestingly, these differ in the functions of CXCR4 that they influence. Site-directed mutagenesis and molecular docking predict that the alkyl chain folds in such a way that the two imidazole groups become lodged in the transmembrane binding cavity of CXCR4. Results also suggest that the alkyl chain length influences how the imidazole rings positions in the cavity. These results may provide a basis for the design of new CXCR4 antagonists targeting specific functions of the receptor.

Published
2023
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4. Screening of potential antiviral molecules against equid herpesvirus-1 using cellular impedance measurement: Dataset of 2,891 compounds.

Author

Côme Thieulent, Christine Fortier, Hélène Munier-Lehmann, Peggy Suzanne, Patrick Dallemagne, Stephan Zientara, Aymeric Hans, Romain Paillot, Pierre-Olivier Vidalain, Stéphane Pronost, and Erika Hue

Subjects
Real-time cell assay, Chemical library screening, Antiviral, Equid herpesvirus-1, Computer applications to medicine. Medical informatics, R858-859.7, Science (General), Q1-390
Abstract

Data presented in this article are associated with the research article “Identification of antiviral compounds against equid herpesvirus-1 using real-time cell assay screening: efficacy of decitabine and valganciclovir alone and in combination” [1]. These data correspond to the in vitro screening of 2,891 potential antiviral compounds against equid herpesvirus-1 (EHV-1) based on impedance measurements using the xCELLigence® RTCA MP System. This dataset includes compounds from three different libraries: i) 1,199 compounds from the Prestwick® Chemical Library, which contains mostly US Food and Drug Administration approved drugs (Prestwick® Chemical, Illkirch, France); ii) 1,651 compounds from the Centre d'Etudes et de Recherche sur le Médicament de Normandie (CERMN, Caen, France); iii) 41 compounds (called herein in-house antiviral library) selected for their effects against different human viruses. Compounds effective against EHV-1 were selected using the area under normalised curves (AUCn) and the time required for the Cell Index to decrease by 50% after virus infection (CIT50). The full dataset from the screen is made publicly available for further analyses.

Published
2020
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5. 1-(Piperidin-3-yl)thymine amides as inhibitors of M. tuberculosis thymidylate kinase

Author

Yanlin Jian, Martijn D. P. Risseeuw, Mathy Froeyen, Lijun Song, Davie Cappoen, Paul Cos, Hélène Munier-Lehmann, and Serge van Calenbergh

Subjects
thymidylate kinase, inhibitors, mycobacterium tuberculosis, modelling, Therapeutics. Pharmacology, RM1-950
Abstract

A series of readily accessible 1-(piperidin-3-yl)thymine amides was designed, synthesised and evaluated as Mycobacterium tuberculosis TMPK (MtbTMPK) inhibitors. In line with the modelling results, most inhibitors showed reasonable MtbTMPK inhibitory activity. Compounds 4b and 4i were slightly more potent than the parent compound 3. Moreover, contrary to the latter, amide analogue 4g was active against the avirulent M. tuberculosis H37Ra strain (MIC50=35 µM). This finding opens avenues for future modifications.

Published
2019
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6. Identification of a small molecule that primes the type I interferon response to cytosolic DNA

Author

Samira Khiar, Marianne Lucas-Hourani, Sébastien Nisole, Nikaïa Smith, Olivier Helynck, Maryline Bourgine, Claude Ruffié, Jean-Philippe Herbeuval, Hélène Munier-Lehmann, Frédéric Tangy, and Pierre-Olivier Vidalain

Subjects
Medicine, Science
Abstract

Abstract The type I interferon response plays a pivotal role in host defense against infectious agents and tumors, and promising therapeutic approaches rely on small molecules designed to boost this system. To identify such compounds, we developed a high-throughput screening assay based on HEK-293 cells expressing luciferase under the control of Interferon-Stimulated Response Elements (ISRE). An original library of 10,000 synthetic compounds was screened, and we identified a series of 1H-benzimidazole-4-carboxamide compounds inducing the ISRE promoter sequence, specific cellular Interferon-Stimulated Genes (ISGs), and the phosphorylation of Interferon Regulatory Factor (IRF) 3. ISRE induction by ChX710, a prototypical member of this chemical series, was dependent on the adaptor MAVS and IRF1, but was IRF3 independent. Although it was unable to trigger type I IFN secretion per se, ChX710 efficiently primed cellular response to transfected plasmid DNA as assessed by potent synergistic effects on IFN-β secretion and ISG expression levels. This cellular response was dependent on STING, a key adaptor involved in the sensing of cytosolic DNA and immune activation by various pathogens, stress signals and tumorigenesis. Our results demonstrate that cellular response to cytosolic DNA can be boosted with a small molecule, and potential applications in antimicrobial and cancer therapies are discussed.

Published
2017
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7. 1-(1-Arylethylpiperidin-4-yl)thymine Analogs as Antimycobacterial TMPK Inhibitors

Author

Yanlin Jian, Fabian Hulpia, Martijn D. P. Risseeuw, He Eun Forbes, Guy Caljon, Hélène Munier-Lehmann, Helena I. M. Boshoff, and Serge Van Calenbergh

Subjects
tuberculosis, Mycobacterium tuberculosis, MtbTMPK, 1-(1-arylethylpiperidin-4-yl)thymine, Organic chemistry, QD241-441
Abstract

A series of Mycobacterium tuberculosis TMPK (MtbTMPK) inhibitors based on a reported compound 3 were synthesized and evaluated for their capacity to inhibit MtbTMPK catalytic activity and the growth of a virulent M. tuberculosis strain (H37Rv). Modifications of the scaffold of 3 failed to afford substantial improvements in MtbTMPK inhibitory activity and antimycobacterial activity. Optimization of the substitution pattern of the D ring of 3 resulted in compound 21j with improved MtbTMPK inhibitory potency (three-fold) and H37Rv growth inhibitory activity (two-fold). Moving the 3-chloro substituent of 21j to the para-position afforded isomer 21h, which, despite a 10-fold increase in IC50-value, displayed promising whole cell activity (minimum inhibitory concentration (MIC) = 12.5 μM).

Published
2020
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8. Identification of novel leishmanicidal molecules by virtual and biochemical screenings targeting Leishmania eukaryotic translation initiation factor 4A.

Author

Emna Harigua-Souiai, Yosser Zina Abdelkrim, Imen Bassoumi-Jamoussi, Ons Zakraoui, Guillaume Bouvier, Khadija Essafi-Benkhadir, Josette Banroques, Nathan Desdouits, Hélène Munier-Lehmann, Mourad Barhoumi, N Kyle Tanner, Michael Nilges, Arnaud Blondel, and Ikram Guizani

Subjects
Arctic medicine. Tropical medicine, RC955-962, Public aspects of medicine, RA1-1270
Abstract

Leishmaniases are neglected parasitic diseases in spite of the major burden they inflict on public health. The identification of novel drugs and targets constitutes a research priority. For that purpose we used Leishmania infantum initiation factor 4A (LieIF), an essential translation initiation factor that belongs to the DEAD-box proteins family, as a potential drug target. We modeled its structure and identified two potential binding sites. A virtual screening of a diverse chemical library was performed for both sites. The results were analyzed with an in-house version of the Self-Organizing Maps algorithm combined with multiple filters, which led to the selection of 305 molecules. Effects of these molecules on the ATPase activity of LieIF permitted the identification of a promising hit (208) having a half maximal inhibitory concentration (IC50) of 150 ± 15 μM for 1 μM of protein. Ten chemical analogues of compound 208 were identified and two additional inhibitors were selected (20 and 48). These compounds inhibited the mammalian eIF4I with IC50 values within the same range. All three hits affected the viability of the extra-cellular form of L. infantum parasites with IC50 values at low micromolar concentrations. These molecules showed non-significant toxicity toward THP-1 macrophages. Furthermore, their anti-leishmanial activity was validated with experimental assays on L. infantum intramacrophage amastigotes showing IC50 values lower than 4.2 μM. Selected compounds exhibited selectivity indexes between 19 to 38, which reflects their potential as promising anti-Leishmania molecules.

Published
2018
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9. Two classes of bacterial IMPDHs according to their quaternary structures and catalytic properties.

Author

Thomas Alexandre, Bertrand Raynal, and Hélène Munier-Lehmann

Subjects
Medicine, Science
Abstract

Inosine-5'-monophosphate dehydrogenase (IMPDH) occupies a key position in purine nucleotide metabolism. In this study, we have performed the biochemical and physico-chemical characterization of eight bacterial IMPDHs, among which six were totally unexplored. This study led to a classification of bacterial IMPDHs according to the regulation of their catalytic properties and their quaternary structures. Class I IMPDHs are cooperative enzymes for IMP, which are activated by MgATP and are octameric in all tested conditions. On the other hand, class II IMPDHs behave as Michaelis-Menten enzymes for both substrates and are tetramers in their apo state or in the presence of IMP, which are shifted to octamers in the presence of NAD or MgATP. Our work provides new insights into the IMPDH functional regulation and a model for the quaternary structure modulation is proposed.

Published
2015
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11. Inhibition of pyrimidine biosynthesis pathway suppresses viral growth through innate immunity.

Author

Marianne Lucas-Hourani, Daniel Dauzonne, Pierre Jorda, Gaëlle Cousin, Alexandru Lupan, Olivier Helynck, Grégory Caignard, Geneviève Janvier, Gwénaëlle André-Leroux, Samira Khiar, Nicolas Escriou, Philippe Desprès, Yves Jacob, Hélène Munier-Lehmann, Frédéric Tangy, and Pierre-Olivier Vidalain

Subjects
Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract

Searching for stimulators of the innate antiviral response is an appealing approach to develop novel therapeutics against viral infections. Here, we established a cell-based reporter assay to identify compounds stimulating expression of interferon-inducible antiviral genes. DD264 was selected out of 41,353 compounds for both its immuno-stimulatory and antiviral properties. While searching for its mode of action, we identified DD264 as an inhibitor of pyrimidine biosynthesis pathway. This metabolic pathway was recently identified as a prime target of broad-spectrum antiviral molecules, but our data unraveled a yet unsuspected link with innate immunity. Indeed, we showed that DD264 or brequinar, a well-known inhibitor of pyrimidine biosynthesis pathway, both enhanced the expression of antiviral genes in human cells. Furthermore, antiviral activity of DD264 or brequinar was found strictly dependent on cellular gene transcription, nuclear export machinery, and required IRF1 transcription factor. In conclusion, the antiviral property of pyrimidine biosynthesis inhibitors is not a direct consequence of pyrimidine deprivation on the virus machinery, but rather involves the induction of cellular immune response.

Published
2013
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12. AutomiG, a biosensor to detect alterations in miRNA biogenesis and in small RNA silencing guided by perfect target complementarity.

Author

Clément Carré, Caroline Jacquier, Anne-Laure Bougé, Fabrice de Chaumont, Corinne Besnard-Guerin, Hélène Thomassin, Josette Pidoux, Bruno Da Silva, Eleftheria Chalatsi, Sarah Zahra, Jean-Christophe Olivo-Marin, Hélène Munier-Lehmann, and Christophe Antoniewski

Subjects
Medicine, Science
Abstract

Defects in miRNA biogenesis or activity are associated to development abnormalities and diseases. In Drosophila, miRNAs are predominantly loaded in Argonaute-1, which they guide for silencing of target RNAs. The miRNA pathway overlaps the RNAi pathway in this organism, as miRNAs may also associate with Argonaute-2, the mediator of RNAi. We set up a gene construct in which a single inducible promoter directs the expression of the GFP protein as well as two miRNAs perfectly matching the GFP sequences. We show that self-silencing of the resulting automiG gene requires Drosha, Pasha, Dicer-1, Dicer-2 and Argonaute-2 loaded with the anti-GFP miRNAs. In contrast, self-silencing of the automiG gene does not involve Argonaute-1. Thus, automiG reports in vivo for both miRNA biogenesis and Ago-2 mediated silencing, providing a powerful biosensor to identify situations where miRNA or siRNA pathways are impaired. As a proof of concept, we used automiG as a biosensor to screen a chemical library and identified 29 molecules that strongly inhibit miRNA silencing, out of which 5 also inhibit RNAi triggered by long double-stranded RNA. Finally, the automiG sensor is also self-silenced by the anti-GFP miRNAs in HeLa cells and might be easily used to identify factors involved in miRNA biogenesis and silencing guided by perfect target complementarity in mammals.

Published
2013
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13. High content analysis of primary macrophages hosting proliferating Leishmania amastigotes: application to anti-leishmanial drug discovery.

Author

Nathalie Aulner, Anne Danckaert, Eline Rouault-Hardoin, Julie Desrivot, Olivier Helynck, Pierre-Henri Commere, Hélène Munier-Lehmann, Gerald F Späth, Spencer L Shorte, Geneviève Milon, and Eric Prina

Subjects
Arctic medicine. Tropical medicine, RC955-962, Public aspects of medicine, RA1-1270
Abstract

Background/objectivesHuman leishmaniases are parasitic diseases causing severe morbidity and mortality. No vaccine is available and numerous factors limit the use of current therapies. There is thus an urgent need for innovative initiatives to identify new chemotypes displaying selective activity against intracellular Leishmania amastigotes that develop and proliferate inside macrophages, thereby causing the pathology of leishmaniasis.Methodology/principal findingsWe have developed a biologically sound High Content Analysis assay, based on the use of homogeneous populations of primary mouse macrophages hosting Leishmania amazonensis amastigotes. In contrast to classical promastigote-based screens, our assay more closely mimics the environment where intracellular amastigotes are growing within acidic parasitophorous vacuoles of their host cells. This multi-parametric assay provides quantitative data that accurately monitors the parasitic load of amastigotes-hosting macrophage cultures for the discovery of leishmanicidal compounds, but also their potential toxic effect on host macrophages. We validated our approach by using a small set of compounds of leishmanicidal drugs and recently published chemical entities. Based on their intramacrophagic leishmanicidal activity and their toxicity against host cells, compounds were classified as irrelevant or relevant for entering the next step in the drug discovery pipeline.Conclusions/significanceOur assay represents a new screening platform that overcomes several limitations in anti-leishmanial drug discovery. First, the ability to detect toxicity on primary macrophages allows for discovery of compounds able to cross the membranes of macrophage, vacuole and amastigote, thereby accelerating the hit to lead development process for compounds selectively targeting intracellular parasites. Second, our assay allows discovery of anti-leishmanials that interfere with biological functions of the macrophage required for parasite development and growth, such as organelle trafficking/acidification or production of microbicidal effectors. These data thus validate a novel phenotypic screening assay using virulent Leishmania amastigotes growing inside primary macrophage to identify new chemical entities with bona fide drug potential.

Published
2013
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14. Interaction network among de novo purine nucleotide biosynthesis enzymes in Escherichia coli

Author

Antoine Gedeon, Gouzel Karimova, Nour Ayoub, Julien Dairou, Quentin Giai Gianetto, Sophie Vichier‐Guerre, Pierre‐Olivier Vidalain, Daniel Ladant, Hélène Munier‐Lehmann, Chimie et Biocatalyse, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université Paris Cité (UPCité), Biochimie des Interactions Macromoléculaires / Biochemistry of Macromolecular Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques (LCBPT - UMR 8601), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Plateforme de Protéomique / Proteomics platform, Université Paris Cité (UPCité)-Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)-Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Chimie biologique épigénétique - Epigenetic Chemical Biology (EpiCBio), Centre International de Recherche en Infectiologie (CIRI), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and This work was supported in part by the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé Et de la Recherche Médicale (INSERM), and the Institut Pasteur.Antoine Gedeon and Nour Ayoub acknowledge PhD fellowships from the Médicament, Toxicologie, Chimieet Imageries PhD school (MTCI, ED 563), Université Paris Cité.

Subjects
bacterial two-hybrid system, [SDV]Life Sciences [q-bio], Escherichia coli, Cell Biology, de novo purine biosynthesis, Molecular Biology, Biochemistry, supramolecular assembly
Abstract

International audience; In human cells, de novo purine nucleotide biosynthesis is known to be regulated through the formation of a metabolon called purinosome. Here, we employed a bacterial two-hybrid approach to characterize the protein-protein interactions network among the corresponding enzymes of Escherichia coli. Our study revealed a dense network of binary interactions that connect most purine nucleotide biosynthesis enzymes. Notably, PurK, an exclusive prokaryotic enzyme, appears as one of the central hubs of this network. We further showed that modifications in PurK, which disrupted several interactions in the network, affected the purine nucleotide pools and altered the bacterial fitness. Our data suggest that the bacterial de novo purine nucleotide biosynthesis enzymes can assemble in a supramolecular complex and that proper interactions among the components of this complex can contribute to bacterial fitness.

Published
2023
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15. SigH stress response mediates killing of Mycobacterium tuberculosis by activating nitronaphthofuran prodrugs via induction of Mrx2 expression

Author

Laura Cioetto-Mazzabò, Francesca Boldrin, Claire Beauvineau, Martin Speth, Alberto Marina, Amine Namouchi, Greta Segafreddo, Mena Cimino, Sandrine Favre-Rochex, Seetha Balasingham, Beatriz Trastoy, Hélène Munier-Lehmann, Gareth Griffiths, Brigitte Gicquel, Marcelo E Guerin, Riccardo Manganelli, Noelia Alonso-Rodríguez, Università degli Studi di Padova = University of Padua (Unipd), Chimie et modélisation pour la biologie du cancer (CMBC), Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Physiopathologie et traitement des maladies du foie, Hôpital Paul Brousse-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay, University of Oslo (UiO), Basque Research and Technology Alliance (BRTA), Génétique mycobactérienne - Mycobacterial genetics, Institut Pasteur [Paris] (IP), Oslo University Hospital [Oslo], Hospital Universitario Cruces = Cruces University Hospital, Département de Biologie structurale et Chimie - Department of Structural Biology and Chemistry, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Chimie Biologique pour le Vivant / Chemistry for Life Sciences (CNRS - UMR3523 - Chem4Life), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Shenzhen Nanshan Center for Chronic Disease Control [Shenzhen, China] (ShenZhenCDC), Ikerbasque - Basque Foundation for Science, European Seventh Framework Program Nanotherapeutics against Resistant Emerging Bacterial Pathogens [NAREB Project 604237], European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie [609020 to N.A.R., 844905 to B.T.], Norwegian Research Council [275873, 273319], MINECO/FEDER EU contracts [PID2019-105649RB-I00], Severo Ochoa Excellence Accreditation [SEV-2016-0644], Basque Government [KK-2019/00076], NIH [R01AI149297 to M.E.G.], Innovative Medicines Initiative 2 Joint Undertaking (JU) [853989 to R.M.]. Funding for open access charge: University of Oslo., European Project: 604237,EC:FP7:NMP,FP7-NMP-2013-LARGE-7,NAREB(2014), and European Project: 609020,EC:FP7:PEOPLE,FP7-PEOPLE-2013-COFUND,SCIENTIA-FELLOWS(2014)

Subjects
[SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Genetics, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology
Abstract

The emergence of drug-resistant Mycobacterium tuberculosis strains highlights the need to discover anti-tuberculosis drugs with novel mechanisms of action. Here we discovered a mycobactericidal strategy based on the prodrug activation of selected chemical derivatives classified as nitronaphthofurans (nNFs) mediated by the coordinated action of the sigH and mrx2 genes. The transcription factor SigH is a key regulator of an extensive transcriptional network that responds to oxidative, nitrosative, and heat stresses in M. tuberculosis. The nNF action induced the SigH stress response which in turn induced the mrx2 overexpression. The nitroreductase Mrx2 was found to activate nNF prodrugs, killing replicating, non-replicating and intracellular forms of M. tuberculosis. Analysis of SigH DNA sequences obtained from spontaneous nNF-resistant M. tuberculosis mutants suggests disruption of SigH binding to the mrx2 promoter site and/or RNA polymerase core, likely promoting the observed loss of transcriptional control over Mrx2. Mutations found in mrx2 lead to structural defects in the thioredoxin fold of the Mrx2 protein, significantly impairing the activity of the Mrx2 enzyme against nNFs. Altogether, our work brings out the SigH/Mrx2 stress response pathway as a promising target for future drug discovery programs.

Published
2023
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16. Steroid hormone catabolites activate the pyrin inflammasome through a non-canonical mechanism

Author

Flora Magnotti, Daria Chirita, Sarah Dalmon, Amandine Martin, Pauline Bronnec, Jeremy Sousa, Olivier Helynck, Wonyong Lee, Daniel L. Kastner, Jae Jin Chae, Michael F. McDermott, Alexandre Belot, Michel Popoff, Pascal Sève, Sophie Georgin-Lavialle, Hélène Munier-Lehmann, Tu Anh Tran, Ellen De Langhe, Carine Wouters, Yvan Jamilloux, Thomas Henry, Centre International de Recherche en Infectiologie (CIRI), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Chimie et Biocatalyse, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université Paris Cité (UPCité), National Institutes of Health [Bethesda] (NIH), University of Leeds, Lyon Immunopathology Federation (LIFe), Hospices Civils de Lyon (HCL), Toxines bactériennes - Bacterial Toxins, Institut Pasteur [Paris] (IP), Centre de Recherche Saint-Antoine (CRSA), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Hôpital Universitaire Carémeau [Nîmes] (CHU Nîmes), Centre Hospitalier Universitaire de Nîmes (CHU Nîmes), University Hospitals Leuven [Leuven], Catholic University of Leuven - Katholieke Universiteit Leuven (KU Leuven), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon, This work is supported by an ANR grant (FMFgeneToDiag). This project has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement no. 779295. T.H.’s team is supported by The Fondation pour la Recherche Médicale (FRM EQU202103012640). D.C. is supported by a fellowship from The Fondation pour la Recherche Médicale (FDT202106012874). E.D.L. and C.W. are members of the European Reference Network for Rare Immunodeficiency, Autoinflammatory and Autoimmune Diseases—Project ID no. 739543., ANR-17-CE17-0021,FMFgeneToDiag,Fièvre Méditerranéenne Familiale (FMF) et maladies apparentées : des bases moléculaires et cellulaires à la mise au point de tests diagnostiques(2017), Centre International de Recherche en Infectiologie - UMR (CIRI), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Henry, Thomas, and Fièvre Méditerranéenne Familiale (FMF) et maladies apparentées : des bases moléculaires et cellulaires à la mise au point de tests diagnostiques - - FMFgeneToDiag2017 - ANR-17-CE17-0021 - AAPG2017 - VALID

Subjects
Molecular biology [CP], MESH: Mutation, [SDV.IMM] Life Sciences [q-bio]/Immunology, Inflammasomes, MESH: Testosterone, PAAND, progesterone, General Biochemistry, Genetics and Molecular Biology, catabolite, B30.2, autoinflammatory disease, familial Mediterranean fever, inflammasome, pyrin, MESH: Progesterone, Etiocholanolone, Humans, Testosterone, MESH: Humans, steroid, CP: Immunology, MESH: Pregnanolone, MESH: Etiocholanolone, Mutation, Immunology [CP], [SDV.IMM]Life Sciences [q-bio]/Immunology, CP: Molecular biology, pregnanolone
Abstract

The pyrin inflammasome acts as a guard of RhoA GTPases and is central to immune defenses against RhoA-manipulating pathogens. Pyrin activation proceeds in two steps. Yet, the second step is still poorly understood. Using cells constitutively activated for the pyrin step 1, a chemical screen identifies etiocholanolone and pregnanolone, two catabolites of testosterone and progesterone, acting at low concentrations as specific step 2 activators. High concentrations of these metabolites fully and rapidly activate pyrin, in a human specific, B30.2 domain-dependent manner and without inhibiting RhoA. Mutations in MEFV, encoding pyrin, cause two distinct autoinflammatory diseases pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND) and familial Mediterranean fever (FMF). Monocytes from PAAND patients, and to a lower extent from FMF patients, display increased responses to these metabolites. This study identifies an unconventional pyrin activation mechanism, indicates that endogenous steroid catabolites can drive autoinflammation, through the pyrin inflammasome, and explains the "steroid fever" described in the late 1950s upon steroid injection in humans. ispartof: CELL REPORTS vol:41 issue:2 ispartof: location:United States status: published

Published
2022
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17. Structural basis for the allosteric inhibition of UMP kinase from Gram‐positive bacteria, a promising antibacterial target

Author

Patrick Walter, Ariel Mechaly, Julien Bous, Ahmed Haouz, Patrick England, Joséphine Lai‐Kee‐Him, Aurélie Ancelin, Sylviane Hoos, Bruno Baron, Stefano Trapani, Patrick Bron, Gilles Labesse, Hélène Munier‐Lehmann, Chimie et Biocatalyse, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Cristallographie (Plateforme) - Crystallography (Platform), Centre de Biologie Structurale [Montpellier] (CBS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Biophysique Moléculaire (Plate-forme), Biophysique Moléculaire (plateforme) - Molecular Biophysics (platform), This work was supported by the European Union’s Framework Programme for Research and Innovation Horizon 2020 (2014-2020) under the Marie Skłodowska-Curie Grant Agreement No. 675555, Accelerated Early staGe drug discovery (AEGIS) project (to HML). The project was also supported in part by the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé Et de la Recherche Médicale (INSERM), the Institut Pasteur and the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INBS-05. PW was the recipient of a PhD fellowship from the EU H2020 (2014-2020) under the Marie Skłodowska-Curie Grant Agreement No. 675555, Accelerated Early staGe drug discovery (AEGIS)., ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), and European Project: 675555,H2020,H2020-MSCA-ITN-2015,AEGIS(2016)

Subjects
crystal structure, allostery, [SDV]Life Sciences [q-bio], Cryoelectron Microscopy, Uridine Triphosphate, Cell Biology, nucleotide metabolism, Gram-Positive Bacteria, Biochemistry, Anti-Bacterial Agents, Adenosine Triphosphate, effector, Allosteric Regulation, cryo-EM single particle analysis, Humans, Amino Acid Sequence, Guanosine Triphosphate, enzyme regulation, Nucleoside-Phosphate Kinase, Uridine Monophosphate, Molecular Biology, UMP kinase
Abstract

International audience; Tuberculosis claims significantly more than one million lives each year. A feasible way to face the issue of drug resistance is the development of new antibiotics. Bacterial uridine 5’-monophosphate (UMP) kinase is a promis- ing target for novel antibiotic discovery as it is essential for bacterial sur- vival and has no counterpart in human cells. The UMP kinase from M. tuberculosis is also a model of particular interest for allosteric regula- tion with two effectors, GTP (positive) and UTP (negative). In this study, using X-ray crystallography and cryo-electron microscopy, we report for the first time a detailed description of the negative effector UTP-binding site of a typical Gram-positive behaving UMP kinase. Comparison between this snapshot of low affinity for Mg-ATP with our previous 3D-structure of the GTP-bound complex of high affinity for Mg-ATP led to a better understanding of the cooperative mechanism and the allosteric regulation of UMP kinase. Thermal shift assay and circular dichroism experiments corroborate our model of an inhibition by UTP linked to higher flexibility of the Mg-ATP-binding domain. These new structural insights provide valuable knowledge for future drug discovery strategies targeting bacterial UMP kinases.

Published
2022
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18. Replication of Equine arteritis virus is efficiently suppressed by purine and pyrimidine biosynthesis inhibitors

Author

Patrick Dallemagne, Pierre-Olivier Vidalain, Aymeric Hans, Anthony Madeline, D. Gaudaire, José Carlos Valle-Casuso, Lydie Martin-Faivre, Stéphane Pronost, Hélène Munier-Lehmann, Stéphan Zientara, Physiopathologie et épidémiologie des maladies équines (PhEED), Laboratoire de santé animale, sites de Maisons-Alfort et de Normandie, Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES), Centre d'Etudes et de Recherche sur le Médicament de Normandie (CERMN), Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU), LABÉO, Pôle d’analyses et de recherche de Normandie (LABÉO), Biologie, génétique et thérapies ostéoarticulaires et respiratoires (BIOTARGEN), Chimie et Biocatalyse, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Virologie UMR1161 (VIRO), École nationale vétérinaire - Alfort (ENVA)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre International de Recherche en Infectiologie (CIRI), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques (LCBPT - UMR 8601), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Fonds Eperon, AnsesFonds EperonRegion Normandie, IFCE (Institut Francais du Cheval et de l'Equitation), Région Normandie, ANSES, Laboratoire de Santé Animale - site de Dozulé, Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES), Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Université Paris-Est (UPE), Laboratoire de santé animale, sites de Maisons-Alfort et de Dozulé, Centre International de Recherche en Infectiologie - UMR (CIRI), Institut National de la Santé et de la Recherche Médicale (INSERM)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), This work was supported by Anses, IFCE (Institut Français du Cheval et de l’Equitation), Fonds Eperon and Région Normandie., AnsesIFCE (Institut Francais du Cheval et de l'Equitation)Fonds EperonRegion Normandie, Bodescot, Myriam, École nationale vétérinaire - Alfort (ENVA)-Laboratoire de santé animale, sites de Maisons-Alfort et de Normandie, Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Dihydroorotate Dehydrogenase, lcsh:Medicine, Virus Replication, chemistry.chemical_compound, Cytopathogenic Effect, Viral, Coronaviridae, lcsh:Science, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, 0303 health sciences, education.field_of_study, Multidisciplinary, Arterivirus Infections, Drug discovery, 3. Good health, [SDV.SP.PHARMA] Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, Pyrimidine metabolism, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Oxidoreductases Acting on CH-CH Group Donors, 030106 microbiology, Population, Biology, Antiviral Agents, Microbiology, Article, Cell Line, 03 medical and health sciences, Equartevirus, Ribavirin, Animals, Horses, education, 030304 developmental biology, [SDV.BA.MVSA]Life Sciences [q-bio]/Animal biology/Veterinary medicine and animal Health, 030306 microbiology, lcsh:R, [SDV.BA.MVSA] Life Sciences [q-bio]/Animal biology/Veterinary medicine and animal Health, RNA, RNA virus, biology.organism_classification, Virology, 030104 developmental biology, Pyrimidines, chemistry, Viral replication, Purines, Dihydroorotate dehydrogenase, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, Horse Diseases, lcsh:Q
Abstract

RNA viruses are responsible for a large variety of animal infections. Equine Arteritis Virus (EAV) is a positive single-stranded RNA virus member of the family Arteriviridae from the order Nidovirales like the Coronaviridae. EAV causes respiratory and reproductive diseases in equids. Although two vaccines are available, the vaccination coverage of the equine population is largely insufficient to prevent new EAV outbreaks around the world. In this study, we present a high-throughput in vitro assay suitable for testing candidate antiviral molecules on equine dermal cells infected by EAV. Using this assay, we identified three molecules that impair EAV infection in equine cells: the broad-spectrum antiviral and nucleoside analog ribavirin, and two compounds previously described as inhibitors of dihydroorotate dehydrogenase (DHODH), the fourth enzyme of the pyrimidine biosynthesis pathway. These molecules effectively suppressed cytopathic effects associated to EAV infection, and strongly inhibited viral replication and production of infectious particles. Since ribavirin is already approved in human and small animal, and that several DHODH inhibitors are in advanced clinical trials, our results open new perspectives for the management of EAV outbreaks.

Published
2020
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19. Identification of antiviral molecules against West-Nile virus by an approach combining cell imaging and equine neural cells derived from induced-pluripotent stem cells

Author

Marielle Cochet-Bernoin, François Piumi, Kamila Gorna, Gaelle Gonzalez, Anne Danckaert, Nathalie Aulner, Hélène Munier-Lehmann, Stephan Zientara, Xavier Donadeu, F., Alexandra Benchoua, Muriel Coulpier, Virologie UMR1161 (VIRO), École nationale vétérinaire d'Alfort (ENVA)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut National de la Recherche Agronomique (INRA)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-École nationale vétérinaire d'Alfort (ENVA), BioImagerie Photonique – Photonic BioImaging (UTechS PBI), Institut Pasteur [Paris], The Roslin Institute, CECS - I-STEM, Association française contre les myopathies (AFM-Téléthon), École nationale vétérinaire - Alfort (ENVA)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), École nationale vétérinaire - Alfort (ENVA)-Institut National de la Recherche Agronomique (INRA)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES), Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Institut Pasteur [Paris] (IP), Biotechnology and Biological Sciences Research Council (BBSRC), Institut des cellules souches pour le traitement et l'étude des maladies monogéniques (I-STEM), and Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay-Généthon

Subjects
[SDV.MHEP.ME]Life Sciences [q-bio]/Human health and pathology/Emerging diseases, [SDV.BA.MVSA]Life Sciences [q-bio]/Animal biology/Veterinary medicine and animal Health, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, ComputingMilieux_MISCELLANEOUS
Abstract

International audience

Published
2021

20. Screening of potential antiviral molecules against equid herpesvirus-1 using cellular impedance measurement: dataset of 2,891 compounds

Author

Christine Fortier, Erika Hue, Aymeric Hans, Pierre-Olivier Vidalain, Patrick Dallemagne, Romain Paillot, Peggy Suzanne, Stéphane Pronost, Côme Thieulent, Hélène Munier-Lehmann, Stéphan Zientara, LABÉO, Pôle d’analyses et de recherche de Normandie (LABÉO), Biologie, génétique et thérapies ostéoarticulaires et respiratoires (BIOTARGEN), Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU), ImpedanCELL, Interactions Cellules Organismes Environnement (ICORE), CHU Caen, Normandie Université (NU)-Tumorothèque de Caen Basse-Normandie (TCBN)-Normandie Université (NU)-Tumorothèque de Caen Basse-Normandie (TCBN)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-CHU Caen, Normandie Université (NU), Chimie et Biocatalyse, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Centre d'Etudes et de Recherche sur le Médicament de Normandie (CERMN), Virologie UMR1161 (VIRO), École nationale vétérinaire d'Alfort (ENVA)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Laboratoire de pathologie équine de Dozulé, Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES), Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques (LCBPT - UMR 8601), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Biologie cellulaire des infections virales – Cell biology of viral infection, Centre International de Recherche en Infectiologie - UMR (CIRI), Institut National de la Santé et de la Recherche Médicale (INSERM)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Normandie Université (NU)-Normandie Université (NU)-CHU Caen, Normandie Université (NU)-Tumorothèque de Caen Basse-Normandie (TCBN)-Tumorothèque de Caen Basse-Normandie (TCBN)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Tumorothèque de Caen Basse-Normandie (TCBN)-Tumorothèque de Caen Basse-Normandie (TCBN), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), École nationale vétérinaire - Alfort (ENVA)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Laboratoire de santé animale, sites de Maisons-Alfort et de Normandie, Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Centre International de Recherche en Infectiologie (CIRI), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), HUE, Erika, École nationale vétérinaire - Alfort (ENVA)-Laboratoire de santé animale, sites de Maisons-Alfort et de Normandie, Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Equid herpesvirus-1, Chemical library screening, Decitabine, lcsh:Computer applications to medicine. Medical informatics, Virus, Chemical library, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, medicine, Molecule, Research article, Antiviral, lcsh:Science (General), Data Article, 030304 developmental biology, Real-time cell assay, 0303 health sciences, Multidisciplinary, [SDV.BA.MVSA]Life Sciences [q-bio]/Animal biology/Veterinary medicine and animal Health, Focused Impedance Measurement, Chemistry, screening, [SDV.BA.MVSA] Life Sciences [q-bio]/Animal biology/Veterinary medicine and animal Health, Valganciclovir, Virology, In vitro, 3. Good health, lcsh:R858-859.7, 030217 neurology & neurosurgery, lcsh:Q1-390, medicine.drug
Abstract

International audience; Data presented in this article are associated with the research article “Identification of antiviral compounds against equid herpesvirus-1 using real-time cell assay screening: efficacy of decitabine and valganciclovir alone and in combination” [1]. These data correspond to the in vitro screening of 2,891 potential antiviral compounds against equid herpesvirus-1 (EHV-1) based on impedance measurements using the xCELLigence® RTCA MP System. This dataset includes compounds from three different libraries: i) 1,199 compounds from the Prestwick® Chemical Library, which contains mostly US Food and Drug Administration approved drugs (Prestwick® Chemical, Illkirch, France); ii) 1,651 compounds from the Centre d'Etudes et de Recherche sur le Médicament de Normandie (CERMN, Caen, France); iii) 41 compounds (called herein in-house antiviral library) selected for their effects against different human viruses. Compounds effective against EHV-1 were selected using the area under normalised curves (AUCn) and the time required for the Cell Index to decrease by 50% after virus infection (CIT50). The full dataset from the screen is made publicly available for further analyses.

Published
2020
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21. Identification of antiviral compounds against equid herpesvirus-1 using real-time cell assay screening: Efficacy of decitabine and valganciclovir alone or in combination

Author

Pierre-Olivier Vidalain, Patrick Dallemagne, Aymeric Hans, Côme Thieulent, Christine Fortier, Erika Hue, Stéphan Zientara, Stéphane Pronost, Romain Paillot, Hélène Munier-Lehmann, Gabrielle Sutton, LABÉO, Pôle d’analyses et de recherche de Normandie (LABÉO), Biologie, génétique et thérapies ostéoarticulaires et respiratoires (BIOTARGEN), Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU), ImpedanCELL, Interactions Cellules Organismes Environnement (ICORE), CHU Caen, Normandie Université (NU)-Tumorothèque de Caen Basse-Normandie (TCBN)-Normandie Université (NU)-Tumorothèque de Caen Basse-Normandie (TCBN)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-CHU Caen, Normandie Université (NU), Centre d'Etudes et de Recherche sur le Médicament de Normandie (CERMN), Virologie UMR1161 (VIRO), École nationale vétérinaire d'Alfort (ENVA)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Chimie et Biocatalyse, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Laboratoire de pathologie équine de Dozulé, Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES), ANSES, Laboratoire de Santé Animale - site de Dozulé, Centre International de Recherche en Infectiologie - UMR (CIRI), Institut National de la Santé et de la Recherche Médicale (INSERM)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques (LCBPT - UMR 8601), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), This work was supported by LABÉO, IFCE (Institut Français du Cheval et de l'Equitation, project AMIE), Fonds Eperon (project N87–2014, N07–2015, N07–2016, N13–2017 and N62-2017), Région Normandie (CPER R25 P3) and CENTAURE European project co-funded by Normandy County Council, European Union in the framework of the ERDF-ESF operationnal programme 2014–2020., Normandie Université (NU)-Normandie Université (NU)-CHU Caen, Normandie Université (NU)-Tumorothèque de Caen Basse-Normandie (TCBN)-Tumorothèque de Caen Basse-Normandie (TCBN)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Tumorothèque de Caen Basse-Normandie (TCBN)-Tumorothèque de Caen Basse-Normandie (TCBN), École nationale vétérinaire - Alfort (ENVA)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Physiopathologie et épidémiologie des maladies équines (PhEED), Laboratoire de santé animale, sites de Maisons-Alfort et de Normandie, Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES), Centre International de Recherche en Infectiologie (CIRI), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), HUE, Erika, École nationale vétérinaire - Alfort (ENVA)-Laboratoire de santé animale, sites de Maisons-Alfort et de Normandie, Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Aphidicolin, Ganciclovir, Equid herpesvirus-1, 030106 microbiology, Chemical library screening, Decitabine, Antiviral Agents, Cell Line, 03 medical and health sciences, chemistry.chemical_compound, Virology, Drug Discovery, Medicine, Animals, Valganciclovir, Synergism, Horses, Antiviral, Pharmacology, Real-time cell assay, [SDV.BA.MVSA]Life Sciences [q-bio]/Animal biology/Veterinary medicine and animal Health, business.industry, Idoxuridine, [SDV.BA.MVSA] Life Sciences [q-bio]/Animal biology/Veterinary medicine and animal Health, Drug Synergism, Deoxycytidine kinase, Herpesviridae Infections, 3. Good health, High-Throughput Screening Assays, Drug Combinations, 030104 developmental biology, chemistry, [SDV.SP.PHARMA] Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, Deoxycytidine, Rabbits, business, Nucleoside, medicine.drug, Herpesvirus 1, Equid
Abstract

International audience; Equid herpesvirus-1 infections cause respiratory, neurological and reproductive syndromes. Despite preventive treatments with vaccines, resurgence of EHV-1 infection still constitutes a major threat to equine industry. However, no antiviral compound is available to treat infected horses. In this study, 2891 compounds were screened against EHV-1 using impedance measurement. 22 compounds have been found to be effective in vitro against EHV-1. Valganciclovir, ganciclovir, decitabine, aphidicolin, idoxuridine and pritelivir (BAY 57-1293) are the most effective compounds identified, and their antiviral potency was further assessed on E. Derm, RK13 and EEK cells and against 3 different field strains of EHV-1 (ORF30 2254 A/G/C). We also provide evidences of synergistic interactions between valganciclovir and decitabine in our in vitro antiviral assay as determined by MacSynergy II, isobologramm and Chou-Talalay methods. Finally, we showed that deoxycytidine reverts the antiviral effect of decitabine, thus supporting some competition at the level of nucleoside phosphorylation by deoxycytidine kinase and/or DNA synthesis. Deoxycitidine analogues, like decitabine, is a family of compounds identified for the first time with promising antiviral efficacy against herpesviruses.

Published
2020
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22. Synthesis of sulfamide analogues of deoxthymidine monophosphate as potential inhibitors of mycobacterial cell wall biosynthesis

Author

Wanting Jiao, Antony J. Fairbanks, Hélène Munier-Lehmann, Kajitha Suthagar, University of Canterbury [Christchurch], Victoria University of Wellington, Chimie et Biocatalyse, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), The authors thank the University of Canterbury (PhD Scholarship to KS) and the Biomolecular Interaction Centre for financial support. Hélène Munier-Lehmann was supported by INSERM, CNRS and the Institut Pasteur., and Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris]

Subjects
Isostere, Stereochemistry, Antitubercular Agents, Protein Data Bank (RCSB PDB), Carbohydrates, 01 natural sciences, Biochemistry, Analytical Chemistry, Structure-Activity Relationship, chemistry.chemical_compound, Non-competitive inhibition, Thymidine monophosphate kinase, Biosynthesis, Cell Wall, Tuberculosis, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Sulfamide, chemistry.chemical_classification, biology, 010405 organic chemistry, Chemistry, [CHIM.ORGA]Chemical Sciences/Organic chemistry, Organic Chemistry, Active site, Mycobacteria, Mycobacterium tuberculosis, General Medicine, 3. Good health, 0104 chemical sciences, 010404 medicinal & biomolecular chemistry, Enzyme, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Docking (molecular), Deoxythymidine, biology.protein, Sulfamides, Thymidine
Abstract

International audience; The recently discovered enzyme Mycobacterium tuberculosis thymidine monophosphate kinase (TMPKmt), which catalyses the phosphorylation of deoxythymidine monophosphate (dTMP) to give deoxythymidine diphosphate (dTDP), is indispensable for the growth and survival of M. tuberculosis as it plays an essential role in DNA synthesis. Inhibition of TMPKmt is an attractive avenue for the development of novel anti-tuberculosis agents. Based on the premise that sulfamide may be a suitable isostere of phosphate, deoxythymidine analogues comprising various substituted sulfamides at C5′ were modelled in silico into the active site of TMPKmt (PDB accession code: 1N5K) using induced-fit docking methods. A selection of modelled compounds was synthesized, and their activity as inhibitors of TMPKmt was evaluated. Three compounds showed competitive inhibition of TMPKmt in the micromolar range (10–50 μM). Compounds were tested in vitro for anti-mycobacterial activity against M. smegmatis: three compounds showed weak anti-mycobacterial activity (MIC 250 μg/mL).

Published
2018
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23. A real-time cell-binding assay reveals dynamic features of STxB-Gb3 cointernalization and STxB-mediated cargo delivery into cancer cells

Author

Grzegorz M Popowicz, Jos Buijs, Valeria Napolitano, Giulia Opassi, U. Helena Danielson, Hanna Björkelund, Grzegorz Dubin, João Crispim Encarnação, Hélène Munier-Lehmann, Karl Andersson, Ridgeview Instruments AB [Uppsala, Sueden], Department of Immunology, Genetics and Pathology [Uppsala, Sueden] (IGP), Uppsala University, Uniwersytet Jagielloński w Krakowie = Jagiellonian University (UJ), Department of Chemistry [Uppsala, Sueden], Biomedical Center = Biomedicinskt centrum [Uppsala, Sueden] (BMC), Uppsala University-Uppsala University, Malopolska Centre of Biotechnology [Krakow] (MCB), Helmholtz-Zentrum München (HZM), Center for Integrated Protein Science (CIPSM), Technische Universität Munchen - Université Technique de Munich [Munich, Allemagne] (TUM)-Helmholtz-Zentrum München (HZM)-Ludwig Maximilian University of Munich [Germany] (LMU München), Chimie et Biocatalyse, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], This project has received funding from the European Union's Framework Programme for Research and Innovation Horizon 2020 (2014–2020) under the Marie Sklodowska‐Curie Grant Agreement No. 675555, Accelerated Early staGe drug discovery (AEGIS)., Microscopic imaging was performed with equipment maintained by the BioVis Platform at Uppsala University. We thank to Dr Arie Geerlof and Dr André Mourão (Institute of Structural Biology, Helmholtz Zentrum München) for providing help on protein expression and purification of the STxB constructs., European Project: 675555,H2020,H2020-MSCA-ITN-2015,AEGIS(2016), Helmholtz Zentrum München = German Research Center for Environmental Health, Technische Universität Munchen - Université Technique de Munich [Munich, Allemagne] (TUM)-Ludwig-Maximilians-Universität München (LMU)-Helmholtz Zentrum München = German Research Center for Environmental Health, and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
media_common.quotation_subject, [SDV]Life Sciences [q-bio], Cell, education, Biophysics, Globotriaosylceramide, cell surface receptor, Biological Transport, Active, Shiga Toxins, Biochemistry, real-time cell-binding assays, 03 medical and health sciences, chemistry.chemical_compound, [SPI]Engineering Sciences [physics], Structural Biology, Cell surface receptor, receptor internalization, Neoplasms, Genetics, medicine, binding kinetics, cancer, Humans, Internalization, Molecular Biology, Binding Kinetics, Cancer, Cell Surface Receptor, Real-time Cell-binding Assays, Receptor Internalization, Shiga Toxin, real-time cell- binding assays, 030304 developmental biology, media_common, 0303 health sciences, Drug Carriers, Chemistry, Trihexosylceramides, 030302 biochemistry & molecular biology, Biochemistry and Molecular Biology, Cell Biology, Shiga toxin, Small molecule, Receptor–ligand kinetics, 3. Good health, Cell biology, ddc, medicine.anatomical_structure, Cancer cell, Biological Assay, K562 Cells, HT29 Cells, Intracellular, Biokemi och molekylärbiologi
Abstract

International audience; The interaction between the Shiga toxin B-subunit (STxB) and its globotriaosylceramide receptor (Gb3) has a high potential for being exploited for targeted cancer therapy. The primary goal of this study was to evaluate the capacity of STxB to carry small molecules and proteins as cargo into cells. For this purpose, an assay was designed to provide real-time information about the StxB-Gb3 interaction as well as the dynamics and mechanism of the internalization process. The assay revealed the ability to distinguish the process of binding to the cell surface from internalization and presented the importance of receptor and STxB clustering for internalization. The overall setup demonstrated that the binding mechanism is complex, and the concept of affinity is difficult to apply. Hence, time-resolved methods, providing detailed information about the interaction of STxB with cells, are critical for the optimization of intracellular delivery.

Published
2020
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24. 1-(1-Arylethylpiperidin-4-yl)thymine analogs as antimycobacterial TMPK inhibitors

Author

Martijn D. P. Risseeuw, Guy Caljon, Fabian Hulpia, Serge Van Calenbergh, He Eun Forbes, Helena I. Boshoff, Yanlin Jian, and Hélène Munier-Lehmann

Subjects
Models, Molecular, 1-(1-arylethylpiperidin-4-yl)thymine, Antitubercular Agents, POTENT INHIBITORS, Pharmaceutical Science, Antimycobacterial, 01 natural sciences, Analytical Chemistry, chemistry.chemical_compound, MtbTMPK, BACTERIAL RNA-POLYMERASE, Drug Discovery, Medicine and Health Sciences, Enzyme Inhibitors, biology, Molecular Structure, TUBERCULOSIS THYMIDYLATE KINASE, Chemistry, tuberculosis, Chemistry (miscellaneous), Molecular Medicine, MYCOBACTERIUM-TUBERCULOSIS, Stereochemistry, medicine.drug_class, THYMIDINE, Substituent, Virulence, Inhibitory postsynaptic potential, Article, lcsh:QD241-441, Mycobacterium tuberculosis, Minimum inhibitory concentration, Structure-Activity Relationship, lcsh:Organic chemistry, Bacterial Proteins, medicine, Humans, Physical and Theoretical Chemistry, Biology, 010405 organic chemistry, Organic Chemistry, biology.organism_classification, 0104 chemical sciences, Thymine, 010404 medicinal & biomolecular chemistry, chemistry, DISCOVERY, CHIMERA, Thymidine, Nucleoside-Phosphate Kinase, RESISTANCE
Abstract

A series of Mycobacterium tuberculosis TMPK (MtbTMPK) inhibitors based on a reported compound 3 were synthesized and evaluated for their capacity to inhibit MtbTMPK catalytic activity and the growth of a virulent M. tuberculosis strain (H37Rv). Modifications of the scaffold of 3 failed to afford substantial improvements in MtbTMPK inhibitory activity and antimycobacterial activity. Optimization of the substitution pattern of the D ring of 3 resulted in compound 21j with improved MtbTMPK inhibitory potency (three-fold) and H37Rv growth inhibitory activity (two-fold). Moving the 3-chloro substituent of 21j to the para-position afforded isomer 21h, which, despite a 10-fold increase in IC50-value, displayed promising whole cell activity (minimum inhibitory concentration (MIC) = 12.5 &mu, M).

Published
2020

25. First-in-class allosteric inhibitors of bacterial IMPDHs

Author

Gilles Labesse, Olivier Helynck, Sophie Vichier-Guerre, Thomas Alexandre, Muriel Gelin, Hélène Munier-Lehmann, Alexandru Lupan, Laurence Dugué, Ahmed Haouz, Université Sorbonne Paris Cité (USPC), Chimie et Biocatalyse, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), 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), Cristallographie (Plateforme) - Crystallography (Platform), This work was supported in part by the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé Et de la Recherche Médicale (INSERM), the Institut Pasteur, the Conseil Régional d'Ile-de-France (Chemical Library Project, grants n° I 06-222/R and I 09-1739/R, which included a postdoctoral fellowship for Alexandru Lupan) and the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INBS-05.Thomas Alexandre was a recipient of a PhD fellowship from the Conseil Régional d'Ile-de-France and the 'D.I.M. maladies infectieuses, parasitaires et nosocomiales émergentes' 2011., We thank Emilie Dufour for participation in the chemical library screening, Zakaria Jemouai for participation in the kinetic characterization of the allosteric inhibitors and Aurélie Bourderioux for performing the fluorescence experiments. We thank the Molecular Biophysics facility (PFBMI, Institut Pasteur, Paris) for their technical assistance and helpful suggestions. The authors are grateful to the staff of the Crystallographic platform from the Institut Pasteur for robot-driven crystallization screening. We thank Alexandre Chenal for fruitful discussion and technical assistance for fluorescence experiments, and Thomas J. Dougherty for careful reading of this manuscript. We acknowledge the Synchrotron SOLEIL (St Aubin, France) staff for assistance and advice during data collection on PROXIMA-1 (X-ray diffraction data) and SWING (SAXS data) beamlines., ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), Munier-Lehmann, Hélène, Infrastructure Française pour la Biologie Structurale Intégrée - - FRISBI2010 - ANR-10-INBS-0005 - INBS - VALID, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], and Cristallographie (Plate-forme)

Subjects
MESH: IMP Dehydrogenase, Drug target, Dehydrogenase, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, 01 natural sciences, Nucleotide metabolism, MESH: Allosteric Regulation, IMPDH, MESH: Apoproteins, Low affinity, Adenosine Triphosphate, IMP Dehydrogenase, Drug Discovery, MESH: Adenosine Triphosphate, Enzyme Inhibitors, MESH: Bacterial Proteins, ComputingMilieux_MISCELLANEOUS, chemistry.chemical_classification, 0303 health sciences, Effector, [CHIM.ORGA]Chemical Sciences/Organic chemistry, General Medicine, Bateman domain, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Biochemistry, MESH: Enzyme Inhibitors, MESH: Protein Domains, Allosteric inhibitor, [CHIM.THER] Chemical Sciences/Medicinal Chemistry, Allosteric regulation, Chemical library screening, Small Molecule Libraries, 03 medical and health sciences, Allosteric Regulation, Bacterial Proteins, Protein Domains, Oxidoreductase, MESH: Small Molecule Libraries, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Quaternary structure, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, 030304 developmental biology, Pharmacology, 010405 organic chemistry, Organic Chemistry, [CHIM.ORGA] Chemical Sciences/Organic chemistry, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 0104 chemical sciences, Enzyme, chemistry, Protein quaternary structure, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Apoproteins
Abstract

International audience; Inosine-5'-monophosphate dehydrogenase (IMPDH) is an essential enzyme in many bacterial pathogens and is considered as a potential drug target for the development of new antibacterial agents. Our recent work has revealed the crucial role of one of the two structural domains (i.e. Bateman domain) in the regulation of the quaternary structure and enzymatic activity of bacterial IMPDHs. Thus, we have screened chemical libraries to search for compounds targeting the Bateman domain and identified first in-class allosteric inhibitors of a bacterial IMPDH. These inhibitors were shown to counteract the activation by the natural positive effector, MgATP, and to block the enzyme in its apo conformation (low affinity for IMP). Our structural studies demonstrate the versatility of the Bateman domain to accommodate totally unrelated chemical scaffolds and pave the way for the development of allosteric inhibitors, an avenue little explored until now.

Published
2019
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26. Real-Time monitoring of Equid alphaherpesviruses infectivity in equine dermal cell based on impedance measurements: effects of aciclovir and ganciclovir treatments

Author

Côme Thieulent, Erika Hue, Christine Fortier, Peggy Suzanne, Stephan Zientara, aymeric, Hélène Munier-Lehmann, Guillaume Fortier, Pierre-Hugues Pitel, Pierre-Olivier Vidalain, Stéphane Pronost, LABÉO, Pôle d’analyses et de recherche de Normandie (LABÉO), Biologie, génétique et thérapies ostéoarticulaires et respiratoires (BIOTARGEN), Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU), ImpedanCELL, Centre d'Etudes et de Recherche sur le Médicament de Normandie (CERMN), Virologie UMR1161 (VIRO), École nationale vétérinaire d'Alfort (ENVA)-Institut National de la Recherche Agronomique (INRA)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES), Laboratoire de pathologie équine de Dozulé, Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES), Chimie et Biocatalyse, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Interactions Cellules Organismes Environnement (ICORE), Normandie Université (NU)-Normandie Université (NU)-CHU Caen, Normandie Université (NU)-Tumorothèque de Caen Basse-Normandie (TCBN)-Tumorothèque de Caen Basse-Normandie (TCBN)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Tumorothèque de Caen Basse-Normandie (TCBN)-Tumorothèque de Caen Basse-Normandie (TCBN), École nationale vétérinaire - Alfort (ENVA)-Institut National de la Recherche Agronomique (INRA)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES), Laboratoire de santé animale, sites de Maisons-Alfort et de Normandie, Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), CHU Caen, Normandie Université (NU)-Tumorothèque de Caen Basse-Normandie (TCBN)-Normandie Université (NU)-Tumorothèque de Caen Basse-Normandie (TCBN)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-CHU Caen, Normandie Université (NU), Institut National de la Recherche Agronomique (INRA)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-École nationale vétérinaire d'Alfort (ENVA), and Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris]

Subjects
[SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, ComputingMilieux_MISCELLANEOUS
Abstract

International audience

Published
2018

27. Real-Time monitoring of Equid alphaherpesviruses infectivity in equine dermal cell based on impedance measurements: effects of aciclovir and ganciclovir treatments (IBCA)

Author

Côme Thieulent, Erika Hue, Christine Fortier, Peggy Suzanne, Stephan Zientara, Hélène Munier-Lehmann, aymeric, Pierre-Hugues Pitel, Pierre-Olivier Vidalain, Stéphane Pronost, LABÉO, Pôle d’analyses et de recherche de Normandie (LABÉO), Biologie, génétique et thérapies ostéoarticulaires et respiratoires (BIOTARGEN), Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU), ImpedanCELL, Interactions Cellules Organismes Environnement (ICORE), CHU Caen, Normandie Université (NU)-Tumorothèque de Caen Basse-Normandie (TCBN)-Normandie Université (NU)-Tumorothèque de Caen Basse-Normandie (TCBN)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-CHU Caen, Normandie Université (NU), Centre d'Etudes et de Recherche sur le Médicament de Normandie (CERMN), Virologie UMR1161 (VIRO), Institut National de la Recherche Agronomique (INRA)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-École nationale vétérinaire d'Alfort (ENVA), Chimie et Biocatalyse, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Laboratoire de pathologie équine de Dozulé, Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES), Génomique Virale et Vaccination, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Normandie Université (NU)-Normandie Université (NU)-CHU Caen, Normandie Université (NU)-Tumorothèque de Caen Basse-Normandie (TCBN)-Tumorothèque de Caen Basse-Normandie (TCBN)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Tumorothèque de Caen Basse-Normandie (TCBN)-Tumorothèque de Caen Basse-Normandie (TCBN), École nationale vétérinaire - Alfort (ENVA)-Institut National de la Recherche Agronomique (INRA)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de santé animale, sites de Maisons-Alfort et de Normandie, and Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)

Subjects
viruses, [SDV]Life Sciences [q-bio], [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, [SDV.BC]Life Sciences [q-bio]/Cellular Biology
Abstract

International audience; Five herpesvirus have been reported to infect horses and 3 belong to the Alphaherpesvirinae subfamily (equine herpesvirus-1, EHV-4 and EHV-3). Among them, EHV-1 is the most pathogenic and causes respiratory disease in young horses, abortion in mares, neonatal death and neurologic damages, also call equine herpesvirus myeloencephalopathy. EHV-4, also named the rhinopneumonia virus, is closely related to EHV-1 but mostly causes respiratory disease and only sporadic abortion and neonatal infection in horses. EHV-3 is distinct from previous viruses and is responsible for equine coital exanthema, characterized by formation of papules, pustules, ulcers and vesicles on the external genitalia of horses. The equine industry economical loss linked to EHV infection is significant, which warrants surveillance and prophylaxis. Currently, no antiviral molecule has a marketing authorization for equine species despite critically needs, warranting the development or adaptation of drugs that are effective against equine viruses.The aims of this work were 1) to investigate the efficacy of Real-Time Cell Analysis (RTCA) system to monitor EHV infections in equine dermal cells 2) to assess the effectiveness of acyclovir (ACV) and ganciclovir (GCV) against EHV-1, EHV-4 and EHV-3 by RTCA system and 3) to investigate the capacity of RTCA system for the screening of compounds library in order to identified new inhibitors of these viruses. To confirm results achieved, monitoring of cell morphology by microscopy and quantification of viral loads were perform in parallel.Antiviral effect of ACV or GCV against the EHV-1 KyD strain was evaluated by RTCA. Results showed that ACV and GCV prevented the impedance decrease induced by EHV-1 infection in a dose-dependent manner and EC50 measured were 9.88 ± 2.14 µg/ml and 0.62 ± 0.49 µg/ml, respectively. The ACV and GCV EC50 against EHV-4 were 17.38 ± 5.95 µg/ml and 2.79 ± 0.25 µg/ml, respectively. Against EHV-3, the ACV and GCV EC50 were 15.23 ± 2.85 µg/ml and 1.86 ± 0.46 µg/ml, respectively. All the data were confirm by qPCR and microscopy observation. The screening of compounds library allowed identifying several molecules with an antiviral activity needing further investigation.This study confirms the efficiency of RTCA to monitor cytopathic effect formation induced by equine alphaherpesviruses on E. Derm cells in real-time. In this model, GCV was shown to be the most effective against all viruses used to infect E. Derm cells. This technology aims to complement and support conventional methods used in the field of virology.

Published
2018

28. A Novel Polyaminocarboxylate Compound To Treat Murine Pulmonary Aspergillosis by Interfering with Zinc Metabolism

Author

Oumaïma Ibrahim-Granet, Daniel Mansuy, Olivier Helynck, Laurence du Merle, Rocío Vicentefranqueira, Grégory Jouvion, Jean-Paul Latgé, Frédéric Buron, Jean-Marc Cavaillon, Hélène Munier-Lehmann, Paris Laskaris, Franck Suzenet, José Antonio Calera, Rodolphe Alves de Sousa, Cytokines et Inflammation, Institut Pasteur [Paris], Universidad de Salamanca, Chimie et Biocatalyse, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Histopathologie humaine et Modèles animaux, Biologie des Bactéries pathogènes à Gram-positif, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie Organique et Analytique (ICOA), Université d'Orléans (UO)-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut de Chimie du CNRS (INC)-Institut National de la Santé et de la Recherche Médicale (INSERM), 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), Aspergillus, O.I.-G., H.M.-L., and J.-P.L. were supported by the PTR468 funding program. P.L. received funding from PTR468 and CARNOT MS for a postdoctoral fellowship. J.A.C. was supported by the Spanish Ministry of Economy and Competitiveness through grant SAF2013-48382-R., We thank Yves Janin for the synthesis of the pyrazolones Pyr05 and Pyr11, Constance Bochot and Pierrette Battioni of UMR8601 for the supply of Por06 and Por07 compounds, and the National Library of National Chemistry for providing access to the complete database for screening. We thank Hervé Waxin from the Institut Pasteur education department and Marie-Anne Nicolas from the Institut Pasteur Photonic BioImaging (UTechS PBI) for their assistance in live imaging., Institut Pasteur [Paris] (IP), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Université d'Orléans (UO)-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris Descartes - Paris 5 (UPD5)-Centre National de la Recherche Scientifique (CNRS), Institut Carnot Pasteur MS, and Ministerio de Economía y Competitividad (España)

Subjects
0301 basic medicine, Fungal infection, [SDV]Life Sciences [q-bio], Aspergillosis, [SDV.MHEP.PSR]Life Sciences [q-bio]/Human health and pathology/Pulmonology and respiratory tract, Aspergillus fumigatus, HeLa, chemistry.chemical_compound, Mice, Pharmacology (medical), aspergillosis, Pyrazolones, Antifungal susceptibility testing, zinc metabolism, [SDV.MP.MYC]Life Sciences [q-bio]/Microbiology and Parasitology/Mycology, immunosuppression, biology, Chemistry, antifungal susceptibility testing, bioluminescence, animal models, 3. Good health, Animal models, Zinc, Infectious Diseases, Drug screening, Toxicity, antifungal agents, Bioluminescence, Combination therapy, 030106 microbiology, Microbial Sensitivity Tests, Microbiology, 03 medical and health sciences, In vivo, medicine, Animals, Experimental Therapeutics, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, drug screening, Antifungal agents, Pharmacology, fungal infection, Zinc metabolism, Resazurin, biology.organism_classification, medicine.disease, In vitro, Disease Models, Animal, 030104 developmental biology, Luminescent Measurements, Pulmonary Aspergillosis, Immunosuppression
Abstract

Aspergillus fumigatus can cause pulmonary aspergillosis in immunocompromised patients and is associated with a high mortality rate due to a lack of reliable treatment options. This opportunistic pathogen requires zinc in order to grow and cause disease. Novel compounds that interfere with fungal zinc metabolism may therefore be of therapeutic interest. We screened chemical libraries containing 59,223 small molecules using a resazurin assay that compared their effects on an A. fumigatus wild-type strain grown under zinc-limiting conditions and on a zinc transporter knockout strain grown under zinc-replete conditions to identify compounds affecting zinc metabolism. After a first screen, 116 molecules were selected whose inhibitory effects on fungal growth were further tested by using luminescence assays and hyphal length measurements to confirm their activity, as well as by toxicity assays on HeLa cells and mice. Six compounds were selected following a rescreening, of which two were pyrazolones, two were porphyrins, and two were polyaminocarboxylates. All three groups showed good in vitro activity, but only one of the polyaminocarboxylates was able to significantly improve the survival of immunosuppressed mice suffering from pulmonary aspergillosis. This two-tier screening approach led us to the identification of a novel small molecule with in vivo fungicidal effects and low murine toxicity that may lead to the development of new treatment options for fungal infections by administration of this compound either as a monotherapy or as part of a combination therapy., O.I.-G., H.M.-L., and J.-P.L. were supported by the PTR468 funding program. P.L. received funding from PTR468 and CARNOT MS for a postdoctoral fellowship. J.A.C. was supported by the Spanish Ministry of Economy and Competitiveness through grant SAF2013-48382-R.

Published
2018
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29. Structure Guided Lead Generation toward Nonchiral M. tuberculosis Thymidylate Kinase Inhibitors

Author

Paul Cos, Begoña Gracia, José A. Aínsa, Serge Van Calenbergh, Romain Merceron, Fabian Hulpia, Martijn D. P. Risseeuw, Ainhoa Lucía Quintana, Hélène Munier-Lehmann, Savvas N. Savvides, Lijun Song, Universiteit Gent = Ghent University (UGENT), VIB Inflammation Research Center, University of Zaragoza - Universidad de Zaragoza [Zaragoza], Centro de Investigación Biomédica en Red Enfermedades Respiratorias (CIBERES), University of Antwerp (UA), Chimie et Biocatalyse, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), L.S. thanks the financial support of the Chinese scholarship council (CSC) for a Ph.D. scholarship. F.H. is indebted to the FWO Flanders for a Ph.D. scholarship. The authors would also like to thank Aaron Vasseur for his help in the synthesis part of this project. R.M. and S.N.S. gratefully acknowledge infrastructural access and support at crystallographic beamlines PX1 at SOLEIL synchrotron (Gif-sur-Yvette, France) and P14 at PETRA3 synchrotron (Hamburg, Germany), as well as grant support from the Hercules Foundation (AUGE-11-029) and the Flanders Institute for Biotechnology (VIB)., Universiteit Gent = Ghent University [Belgium] (UGENT), and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, Tuberculosis, Cell Survival, Antitubercular Agents, Molecular Conformation, Microbial Sensitivity Tests, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, Pharmacology, 01 natural sciences, Thymidylate kinase, Mycobacterium tuberculosis, Gene Knockout Techniques, Structure-Activity Relationship, chemistry.chemical_compound, Biosynthesis, Drug Discovery, medicine, Humans, Transferase, Enzyme Inhibitors, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], chemistry.chemical_classification, Ligand efficiency, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 010405 organic chemistry, Pharmacology. Therapy, biology.organism_classification, medicine.disease, 0104 chemical sciences, 3. Good health, 010404 medicinal & biomolecular chemistry, Enzyme, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, chemistry, Drug Design, Molecular Medicine, Efflux, Nucleoside-Phosphate Kinase
Abstract

International audience; In recent years, thymidylate kinase (TMPK), an enzyme indispensable for bacterial DNA biosynthesis, has been pursued for the development of new antibacterial agents including against Mycobacterium tuberculosis, the causative agent for the widespread infectious disease tuberculosis (TB). In response to a growing need for more effective anti-TB drugs, we have built upon our previous efforts toward the exploration of novel and potent Mycobacterium tuberculosis TMPK ( MtTMPK) inhibitors, and reported here the design of a novel series of non-nucleoside inhibitors of MtTMPK. The inhibitors display hitherto unexplored interactions in the active site of MtTMPK, offering new insights into structure-activity relationships. To investigate the discrepancy between enzyme inhibitory activity and the whole-cell activity, experiments with efflux pump inhibitors and efflux pump knockout mutants were performed. The minimum inhibitory concentrations of particular inhibitors increased significantly when determined for the efflux pump mmr knockout mutant, which partly explains the observed dissonance

Published
2018
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30. Identification of novel leishmanicidal molecules by virtual and biochemical screenings targeting Leishmania eukaryotic translation initiation factor 4A

Author

Imen Bassoumi-Jamoussi, Guillaume Bouvier, Michael Nilges, Mourad Barhoumi, N. Kyle Tanner, Josette Banroques, Arnaud Blondel, Ikram Guizani, Nathan Desdouits, Khadija Essafi-Benkhadir, Hélène Munier-Lehmann, Emna Harigua-Souiai, Yosser Zina Abdelkrim, Ons Zakraoui, Laboratoire d'Epidémiologie Moléculaire et de Pathologie Expérimentale Appliquée aux Maladies Infectieuses (LR11IPT04), Université Tunis El Manar (UTM)-Institut Pasteur de Tunis, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Bioinformatique structurale, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Expression Génétique Microbienne (EGM (UMR_8261 / FRE_3630)), Institut de biologie physico-chimique (IBPC), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Faculté des Sciences de Bizerte, Université de Carthage - University of Carthage, Chimie et Biocatalyse, This work received financial support from the Institut Pasteur PTR program (grant PTR426), and partially from the Ministry of Higher Education and Research in Tunisia (LR00IPT04 & LR11IPT04, IG), MN received financial support from the Investissement d’Avenir bioinformatics programme (Grant Bip:Bip ANR-BINF-03-01) and BayCellS FP7-IDEAS-ERC 294809, NKT and JB are supported by a Centre National de la Recherche Scientifique (CNRS), HeliDEAD grant [ANR-13- BSV8-0009-01] from the Agence Nationale de la Recherche and the Initiative d’Excellence program from the French State [DYNAMO, ANR-11-LABX-0011-01]. EHS received support from the UNESCO-L’Oréal 'For Women in Science' international fellowship and the Institut Pasteur International Network (Calmette and Yersin programme). The non commercial compounds were obtained from Dr. Laïla El Kihel from Université de Caen de Basse-Normandie, Centre d’Études et de Recherche sur le Médicament de Normandie (CERMN), UFR des Sciences Pharmaceutiques, for the minimal costs of shipping fees. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript, ANR-10-BINF-03-01/10-BINF-0003,Bip:Bip,Bip:Bip(2010), ANR-13-BSV8-0009,HeliDEAD,Les ARN hélicases à boite DEAD: quels sont leurs rôles, leurs partenaires, leurs substrats ARN et comment elles fonctionnent.(2013), ANR-11-LABX-0011/11-LABX-0011,DYNAMO,Dynamique des membranes transductrices d'énergie : biogénèse et organisation supramoléculaire.(2011), European Project: 294809,EC:FP7:ERC,ERC-2011-ADG_20110310,BAYCELLS(2012), Université de Tunis El Manar (UTM)-Institut Pasteur de Tunis, Bioinformatique structurale - Structural Bioinformatics, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie physico-chimique (IBPC (FR_550)), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Faculté des Sciences de Bizerte [Université de Carthage], ANR-10-BINF-0003,Bip:Bip,Paradigme d'inference bayesienne pour la Biologie structurale in silico(2010), ANR-11-LABX-0011,DYNAMO,Dynamique des membranes transductrices d'énergie : biogénèse et organisation supramoléculaire.(2011), and Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris]

Subjects
Models, Molecular, 0301 basic medicine, Life Cycles, [SDV]Life Sciences [q-bio], Drug Evaluation, Preclinical, Protozoology, MESH: Parasitic Sensitivity Tests, Biochemistry, Chemical library, MESH: Eukaryotic Initiation Factor-4A, Database and Informatics Methods, White Blood Cells, chemistry.chemical_compound, Parasitic Sensitivity Tests, Animal Cells, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Medicine and Health Sciences, Leishmania infantum, MESH: Inhibitory Concentration 50, Protozoans, Leishmania, Adenosine Triphosphatases, biology, Chemistry, lcsh:Public aspects of medicine, Eukaryota, Adenosine triphosphatase, Parasitic diseases, MESH: Leishmania infantum, Enzymes, 3. Good health, Molecular Docking Simulation, Infectious Diseases, Helicases, MESH: Drug Evaluation, Preclinical, Protozoan Life Cycles, Cellular Types, Sequence Analysis, MESH: Models, Molecular, Research Article, Amastigotes, lcsh:Arctic medicine. Tropical medicine, Bioinformatics, lcsh:RC955-962, Immune Cells, Immunology, Antiprotozoal Agents, Research and Analysis Methods, Microbiology, Inhibitory Concentration 50, 03 medical and health sciences, Eukaryotic translation, MESH: Adenosine Triphosphatases, MESH: Molecular Docking Simulation, Initiation factor, Binding site, MESH: Antiprotozoal Agents, Amastigote, Virtual screening, Blood Cells, Binding Sites, Promastigotes, Macrophages, Phosphatases, Organisms, Public Health, Environmental and Occupational Health, Biology and Life Sciences, Proteins, lcsh:RA1-1270, Cell Biology, biology.organism_classification, RNA helicases, Parasitic Protozoans, 030104 developmental biology, MESH: Binding Sites, Sequence motif analysis, Eukaryotic Initiation Factor-4A, Enzymology, [INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM], Developmental Biology
Abstract

Leishmaniases are neglected parasitic diseases in spite of the major burden they inflict on public health. The identification of novel drugs and targets constitutes a research priority. For that purpose we used Leishmania infantum initiation factor 4A (LieIF), an essential translation initiation factor that belongs to the DEAD-box proteins family, as a potential drug target. We modeled its structure and identified two potential binding sites. A virtual screening of a diverse chemical library was performed for both sites. The results were analyzed with an in-house version of the Self-Organizing Maps algorithm combined with multiple filters, which led to the selection of 305 molecules. Effects of these molecules on the ATPase activity of LieIF permitted the identification of a promising hit (208) having a half maximal inhibitory concentration (IC50) of 150 ± 15 μM for 1 μM of protein. Ten chemical analogues of compound 208 were identified and two additional inhibitors were selected (20 and 48). These compounds inhibited the mammalian eIF4I with IC50 values within the same range. All three hits affected the viability of the extra-cellular form of L. infantum parasites with IC50 values at low micromolar concentrations. These molecules showed non-significant toxicity toward THP-1 macrophages. Furthermore, their anti-leishmanial activity was validated with experimental assays on L. infantum intramacrophage amastigotes showing IC50 values lower than 4.2 μM. Selected compounds exhibited selectivity indexes between 19 to 38, which reflects their potential as promising anti-Leishmania molecules., Author summary Leishmaniases constitute a group of neglected parasitic diseases that inflict major burden on public health. Novel drugs and targets need to be identified since current therapies have adverse side effects. Herein, we focused on Leishmania infantum translation initiation factor 4A (LieIF), as a potential drug target. LieIF, a pivotal enzyme in the translation machinery, is also implicated in host-pathogen interactions. We modeled its 3D structure and identified two pockets, which were used in virtual screenings of a chemical compound library. Therefore, we selected and purchased 305 compounds. We established a reliable ATPase screening assay to test the molecules against the enzymatic activity of LieIF and its mammalian homologue. A promising hit was retained and further characterized. It inhibited both proteins but showed different kinetic properties. It was used as a basis to identify similar analogues and two additional inhibitors were identified. All three hits reduced the viability of the extracellular promastigote form of the parasite, but they had no significant cytotoxic effects on host cells. They also affected the viability of the intracellular amastigote form and reduced the macrophage infection. This selectivity is very promising and indicates that these inhibitors would constitute an avenue to develop strategies to fight leishmaniases.

Published
2018
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31. Administration of Zinc Chelators Improves Survival of Mice Infected with Aspergillus fumigatus both in Monotherapy and in Combination with Caspofungin

Author

Oumaïma Ibrahim-Granet, Paris Laskaris, Jean-Paul Latgé, Christophe d'Enfert, Ahmad Atrouni, José Antonio Calera, Hélène Munier-Lehmann, Jean-Marc Cavaillon, Cytokines et Inflammation, Institut Pasteur [Paris], Universidad de Salamanca, Biologie et Pathogénicité fongiques, Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris], Chimie et Biocatalyse, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Aspergillus, Institut Pasteur, Institut Pasteur [Paris]-Institut National de la Recherche Agronomique (INRA), Institut Pasteur [PTR 468], Institut Pasteur [Paris] (IP), Biologie et Pathogénicité fongiques (BPF), Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris] (IP), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Male, Antifungal Agents, Pyridines, 030106 microbiology, Virulence, Aspergillosis, Aspergillus fumigatus, Microbiology, 03 medical and health sciences, chemistry.chemical_compound, Echinocandins, Immunocompromised Host, Lipopeptides, Pharmacotherapy, Caspofungin, medicine, Ethylamines, Animals, Pharmacology (medical), skin and connective tissue diseases, Pathogen, Mechanisms of Action: Physiological Effects, [SDV.MP.MYC]Life Sciences [q-bio]/Microbiology and Parasitology/Mycology, Chelating Agents, Pharmacology, Mice, Inbred BALB C, biology, Clioquinol, Immune Sera, Pneumonia, biology.organism_classification, medicine.disease, bacterial infections and mycoses, 3. Good health, Zinc, 030104 developmental biology, Infectious Diseases, Treatment Outcome, chemistry, Nasal administration, Drug Therapy, Combination, medicine.drug, Phenanthrolines
Abstract

Aspergillus fumigatus can infect immunocompromised patients, leading to high mortality rates due to the lack of reliable treatment options. This pathogen requires uptake of zinc from host tissues in order to successfully grow and cause virulence. Reducing the availability of that micronutrient could help treat A. fumigatus infections. In this study, we examined the in vitro effects of seven chelators using a bioluminescent strain of A. fumigatus. 1,10-Phenanthroline and N,N,N′,N′-tetrakis(2-pyridylmethyl) ethane-1,2-diamine (TPEN) proved to be the chelators most effective at inhibiting fungal growth. Intraperitoneal administration of either phenanthroline or TPEN resulted in a significant improvement in survival and decrease of weight loss and fungal burden for immunosuppressed mice intranasally infected with A. fumigatus. In vitro both chelators had an indifferent effect when employed in combination with caspofungin. The use of TPEN in combination with caspofungin also significantly increased survival compared to that when using these drugs individually. Our results suggest that zinc chelation may be a valid strategy for dealing with A. fumigatus infections and that both phenanthroline and TPEN could potentially be used either independently or in combination with caspofungin, indicating that their use in combination with other antifungal treatments might also be applicable., P.L., O.I.-G., H.M.-L., and J.-P.L. were supported by Institut Pasteur PTR 468 funding.

Published
2016
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32. Real-time monitoring of Equid herpesviruses infectivity in equine dermal cell based on impedance measurements

Author

Stéphan Zientara, Aymeric Hans, Christine Fortier, Erika Hue, Hélène Munier-Lehmann, Peggy Suzanne, Stéphane Pronost, Pierre-Hugues Pitel, Guillaume Fortier, Pierre-Olivier Vidalain, LABÉO, Pôle d’analyses et de recherche de Normandie (LABÉO), Unité de Recherche Risques Microbiens (U2RM), Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU), Fondation Hippolia, Centre d'Etudes et de Recherche sur le Médicament de Normandie (CERMN), Virologie UMR1161 (VIRO), École nationale vétérinaire d'Alfort (ENVA)-Institut National de la Recherche Agronomique (INRA)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES), Laboratoire de pathologie équine de Dozulé, Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES), Chimie et Biocatalyse, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), 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), Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Institut National de la Recherche Agronomique (INRA)-École nationale vétérinaire d'Alfort (ENVA), Université Paris Descartes - Paris 5 (UPD5)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)

Subjects
Infectivity, Programmed cell death, [SDV.BA.MVSA]Life Sciences [q-bio]/Animal biology/Veterinary medicine and animal Health, 040301 veterinary sciences, Cell growth, Chemistry, Equine, viruses, 0402 animal and dairy science, 04 agricultural and veterinary sciences, 040201 dairy & animal science, Virology, Virus, 3. Good health, 0403 veterinary science, Cell culture, Cell adhesion, Viral load, Cytopathic effect
Abstract

International audience; In virology, different conventional methods are commonly used for virus titration and infectivity studies. Most of them are based on direct examination of viral cytopathic effect, which is time consuming, tedious and requires an endpoint reading. The RTCA xCELLigence system (ACEA Biosciences) has been developed to follow cellular events and their dynamics in real time using a micro-electrical biosensor technology. The aim of this study was to evaluate the feasibility of using this biosensor technology to measure the effect of different equid herpesviruses on equine dermal cell cultures, with or without antiviral acyclovir treatment. Cells were infected with EHV-1 (VR700), EHV-4 (VR2230), EHV-2 (VR701) or EHV-3 (VR352). The viral effects were monitored by 3 different methods: impedance measurements (RTCA), cytopathic effects recording (microscopy) and viral load quantification (qPCR). The RTCA technology showed a dose dependent drop of the cellular index induced by specific impedance variations measured during growth of each virus tested. These results correlated with the cytopathic effect induced by the different herpesvirus species, as well as viral loads measured by qPCR. For example, after 3 days of incubation with EHV-1, RTCA results showed an increase of the cellular index from 0% (infection without antiviral) to 45% and 100% in presence of Acyclovir (10μl/mL and 100 μg/mL, respectively). These results are correlated with the decreased of viral load measured in the presence of Acyclovir: 1010 genome copies/mL with EHV-1 only, 106 copies/mL with EHV-1 + Acyclovir 10 μg/ mL and 105 copies/mL with EHV-1 + Acyclovir 100 μg/ mL. In conclusion, the cellular impedance monitoring system is a new real-time method to monitor cell growth and viral proliferation in cell cultures. This technology is well adapted for high-throughput screening of antiviral molecules. In fact, the xCELLigence system enables continuous impedance measurement and quantification of cell adhesion, proliferation, cell death and detachment during viral infection.

Published
2016
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33. The Toxicity of a Novel Antifungal Compound Is Modulated by Endoplasmic Reticulum-Associated Protein Degradation (ERAD) Components

Author

Aureslien Lesnard, Peggy Suzanne, Jean-Paul Latgé, David S. Askew, Ute Zeidler, Olivier Helynck, Sylvain Rault, Cosmin Saveanu, Karthik Krishnan, Christophe d'Enfert, Shriya Raj, Hélène Munier-Lehmann, Aspergillus, Institut Pasteur [Paris], University of Cincinnati (UC), Chimie et Biocatalyse, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Centre d'Etudes et de Recherche sur le Médicament de Normandie (CERMN), Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU), Biologie et Pathogénicité fongiques, Institut Pasteur [Paris]-Institut National de la Recherche Agronomique (INRA), Génétique des Interactions macromoléculaires, Aviesan BAP109, Institut Carnot Pasteur Maladies Infectieuses FUNGI, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris], University of Cincinnati, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique ( CNRS ), Centre d'Etudes et de Recherche sur le Médicament de Normandie ( CERMN ), Université de Caen Normandie ( UNICAEN ), Normandie Université ( NU ) -Normandie Université ( NU ), Institut National de la Recherche Agronomique ( INRA ) -Institut Pasteur [Paris], Latgé, Jean-Paul, Munier-Lehmann, Hélène, Saveanu, Cosmin, Institut Pasteur [Paris] (IP), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Biologie et Pathogénicité fongiques (BPF), Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris] (IP), and Génétique des Interactions macromoléculaires / Genetics of Macromolecular Interactions

Subjects
0301 basic medicine, Antifungal Agents, Saccharomyces cerevisiae, Antifungal drug, Drug Evaluation, Preclinical, Mice, Inbred Strains, [ SDV.BBM.BM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Microbial Sensitivity Tests, UPR, Endoplasmic-reticulum-associated protein degradation, Biology, Protein degradation, Endoplasmic Reticulum, Aspergillus fumigatus, Microbiology, Small Molecule Libraries, 03 medical and health sciences, [ SDV.BBM.BC ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Candida albicans, Animals, Humans, Pharmacology (medical), [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], ER membrane protein complex, Mechanisms of Action: Physiological Effects, Pharmacology, Endoplasmic reticulum, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Endoplasmic Reticulum-Associated Degradation, ERAD, biology.organism_classification, Yeast, 3. Good health, Cell biology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], 030104 developmental biology, Infectious Diseases, [ SDV.BBM.GTP ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Mutation, Unfolded protein response, Cryptococcus neoformans, Unfolded Protein Response, Chemogenomics, HeLa Cells
Abstract

In a search for new antifungal compounds, we screened a library of 4,454 chemicals for toxicity against the human fungal pathogen Aspergillus fumigatus . We identified sr7575, a molecule that inhibits growth of the evolutionary distant fungi A. fumigatus , Cryptococcus neoformans , Candida albicans , and Saccharomyces cerevisiae but lacks acute toxicity for mammalian cells. To gain insight into the mode of inhibition, sr7575 was screened against 4,885 S. cerevisiae mutants from the systematic collection of haploid deletion strains and 977 barcoded haploid DAmP (decreased abundance by mRNA perturbation) strains in which the function of essential genes was perturbed by the introduction of a drug resistance cassette downstream of the coding sequence region. Comparisons with previously published chemogenomic screens revealed that the set of mutants conferring sensitivity to sr7575 was strikingly narrow, affecting components of the endoplasmic reticulum-associated protein degradation (ERAD) stress response and the ER membrane protein complex (EMC). ERAD-deficient mutants were hypersensitive to sr7575 in both S. cerevisiae and A. fumigatus , indicating a conserved mechanism of growth inhibition between yeast and filamentous fungi. Although the unfolded protein response (UPR) is linked to ERAD regulation, sr7575 did not trigger the UPR in A. fumigatus and UPR mutants showed no enhanced sensitivity to the compound. The data from this chemogenomic analysis demonstrate that sr7575 exerts its antifungal activity by disrupting ER protein quality control in a manner that requires ERAD intervention but bypasses the need for the canonical UPR. ER protein quality control is thus a specific vulnerability of fungal organisms that might be exploited for antifungal drug development.

Published
2016
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34. Respiratory syncytial virus infection in macaques is not suppressed by intranasal sprays of pyrimidine biosynthesis inhibitors

Author

Daniel Dauzonne, F. Taborik, Yves L. Janin, Astrid Vabret, Hélène Munier-Lehmann, Adeline Paturet, Hugues Contamin, Pierre-Olivier Vidalain, Clément Grandin, Frédéric Tangy, Marianne-Lucas Hourani, Génomique virale et vaccination, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Chimie et Biocatalyse, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Conception, synthèse et vectorisation de biomolécules. (CSVB), Institut Curie-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5), Cynbiose, CYNBIOSE SA, Laboratoire de Virologie Humaine et Moléculaire [Caen], CHU Caen, Normandie Université (NU)-Tumorothèque de Caen Basse-Normandie (TCBN)-Normandie Université (NU)-Tumorothèque de Caen Basse-Normandie (TCBN), This work was supported by the National Research Agency (ANR-2011-RPIB-006-01, Program STING 2.0), Lyonbiopôle, the Institut Carnot Pasteur MI (Program STING), the Institut Pasteur, the Institut National de la Santé et de la Recherche Médicale, and the Centre National de la Recherche Scientifique. CG was supported by a CIFRE fellowship (Conventions Industrielles de Formation par la Recherche, Association Nationale de la Recherche et de la Technologie, 2011/1187)., ANR-11-RPIB-0006,STING-2.0,Molécules immunostimulatrices ayant une activité antivirale à large spectre: une preuve de concept dans un modèle de primate non-humain(2011), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université Paris Descartes - Paris 5 (UPD5)-Institut Curie [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), and Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]

Subjects
0301 basic medicine, Palivizumab, Oxidoreductases Acting on CH-CH Group Donors, Cynomolgus macaques, Antimetabolites, [SDV]Life Sciences [q-bio], Dihydroorotate dehydrogenase, Respiratory Syncytial Virus Infections, Biology, Respiratory syncytial virus, Virus Replication, Antiviral Agents, Virus, 03 medical and health sciences, Pyrimidine biosynthesis inhibitors, Virology, medicine, Animals, Humans, Administration, Intranasal, Pharmacology, Hep G2 Cells, 3. Good health, Disease Models, Animal, Pyrimidines, 030104 developmental biology, medicine.anatomical_structure, Viral replication, Respiratory Syncytial Virus, Human, Pyrimidine metabolism, biology.protein, Macaca, Nasal administration, Antiviral compounds, Antibody, Respiratory tract, medicine.drug
Abstract

International audience; There is imperious need for efficient therapies against ubiquitous and life-threatening respiratory viruses, foremost among them being the human respiratory syncytial virus (hRSV). Several research groups who performed functional screens for broad-spectrum antivirals identified compounds targeting the de novo pyrimidine biosynthesis pathway. Despite their strong antiviral activity in vitro, whether such antimetabolites are effective in vivo remains highly controversial. Here, we evaluated two potent pyrimidine biosynthesis inhibitors developed in our laboratory, IPPA17-A04 and GAC50, in a model of mild hRSV-infection in cynomolgus macaques. In this model, hRSV replication is restricted to the epithelium of the upper respiratory tract, and is compatible with a topical treatment by intranasal sprays. The local administration of palivizumab, a neutralizing anti-hRSV antibody used in clinics, significantly reduced virus replication. In contrast, pyrimidine biosynthesis inhibitors did not show any inhibitory effect on hRSV growth when delivered topically as experimented in our model. Our results should help to better define the potential applications of this class of antimetabolites in the treatment of viral infections.

Published
2016
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35. Crystallographic studies of two variants of Pseudomonas aeruginosa IMPDH with impaired allosteric regulation

Author

Ahmed Haouz, Muriel Gelin, Thomas Alexandre, Hélène Munier-Lehmann, Gilles Labesse, Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Sorbonne Paris Cité (USPC), Chimie et Biocatalyse, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Cristallographie (Plateforme) - Crystallography (Platform), The authors acknowledge financial support from theCNRS, INSERM and the Institut Pasteur. TA was a recipientof a PhD fellowship from the Conseil Régional d’Ile-de-France and the ‘D.I.M. maladies infectieuses, parasitaires etnosocomiales émergentes’ 2011. The authors also acknowledgesupport from the French Infrastructure for IntegratedStructural Biology (FRISBI) ANR-10-INSB-05-01., ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Munier-Lehmann, Hélène, and Infrastructure Française pour la Biologie Structurale Intégrée - - FRISBI2010 - ANR-10-INBS-0005 - INBS - VALID

Subjects
[SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Protein Conformation, [SDV]Life Sciences [q-bio], Allosteric regulation, Mutant, Molecular Sequence Data, quaternary structure, nucleotide metabolism, medicine.disease_cause, Crystallography, X-Ray, IMPDH, 03 medical and health sciences, IMP Dehydrogenase, Structural Biology, Oxidoreductase, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, medicine, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, 030304 developmental biology, chemistry.chemical_classification, Genetics, 0303 health sciences, Mutation, biology, Sequence Homology, Amino Acid, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 030302 biochemistry & molecular biology, Active site, General Medicine, CBS module, allosteric regulation, [SDV] Life Sciences [q-bio], Enzyme, chemistry, Biochemistry, Pseudomonas aeruginosa, biology.protein, Protein quaternary structure, Function (biology)
Abstract

Inosine-5′-monophosphate dehydrogenases (IMPDHs), which are the rate-limiting enzymes in guanosine-nucleotide biosynthesis, are important therapeutic targets. Despite in-depth functional and structural characterizations of various IMPDHs, the role of the Bateman domain containing two CBS motifs remains controversial. Their involvement in the allosteric regulation ofPseudomonas aeruginosaIMPDH by Mg-ATP has recently been reported. To better understand the function of IMPDH and the importance of the CBS motifs, the structure of a variant devoid of these modules (ΔCBS) was solved at high resolution in the apo form and in complex with IMP. In addition, a single amino-acid substitution variant, D199N, was also structurally characterized: the mutation corresponds to the autosomal dominant mutant D226N of human IMPDH1, which is responsible for the onset of the retinopathy adRP10. These new structures shed light onto the possible mechanism of regulation of the IMPDH enzymatic activity. In particular, three conserved loops seem to be key players in this regulation as they connect the tetramer–tetramer interface with the active site and show significant modification upon substrate binding.

Published
2015
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36. Original 2-(3-Alkoxy-1H-pyrazol-1-yl)azines Inhibitors of Human Dihydroorotate Dehydrogenase (DHODH)

Author

Farah El Mazouni, Frédéric Tangy, Marianne Lucas-Hourani, Margaret A. Phillips, Yves L. Janin, Anne Noel, Artur Scherf, Nicholas A. Malmquist, Olivier Helynck, Jane Harpon, Sandrine Guillou, Hélène Munier-Lehmann, Eloi P. Coutant, Pierre-Olivier Vidalain, Génomique virale et vaccination, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Chimie et Biocatalyse, Department of Pharmacology, University of Texas Southwestern Medical Center [Dallas], Biologie des Interactions Hôte-Parasite - Biology of Host-Parasite Interactions, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Medicen Initiative (Chemical Library Project, Région Ile de France grants I 06-222/R and I 09-1739/R), United States National Institutes of Health (grant R01AI103947), The Agence Nationale de la Recherche, grant ANR-11- CRNT-0004, investment program 'GLOBAL CARE', an association of theInstituts Carnot 'Pasteur−Maladies Infectieuses', 'Curie−Cancer', 'Voir et Entendre', 'Institut du Cerveau et de la moelleEpiniere',and the Consortium pour l’Acceleration de l’Innovation et de son Transfert dans le domaine du Lymphome (CALYM)., The Institut Carnot−Pasteur Maladies Infectieuses(ProgrammeSTING), ANR-11-RPIB-0006,STING-2.0,Molécules immunostimulatrices ayant une activité antivirale à large spectre: une preuve de concept dans un modèle de primate non-humain(2011), Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Janin, Yves, and RECHERCHES PARTENARIALES ET INNOVATION BIOMEDICALE - Molécules immunostimulatrices ayant une activité antivirale à large spectre: une preuve de concept dans un modèle de primate non-humain - - STING-2.02011 - ANR-11-RPIB-0006 - RPIB - VALID

Subjects
Oxidoreductases Acting on CH-CH Group Donors, Pyrimidine, [CHIM.THER] Chemical Sciences/Medicinal Chemistry, Plasmodium falciparum, Dihydroorotate Dehydrogenase, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, Virus Replication, 01 natural sciences, Antiviral Agents, Article, 03 medical and health sciences, chemistry.chemical_compound, Inhibitory Concentration 50, Drug Discovery, Teriflunomide, Humans, Enzyme Inhibitors, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, biology, 010405 organic chemistry, Cell growth, [CHIM.ORGA]Chemical Sciences/Organic chemistry, In vitro toxicology, [CHIM.ORGA] Chemical Sciences/Organic chemistry, biology.organism_classification, Molecular biology, In vitro, 3. Good health, 0104 chemical sciences, Enzyme, HEK293 Cells, chemistry, Biochemistry, Measles virus, Drug Design, Dihydroorotate dehydrogenase, Molecular Medicine, Pyrazoles
Abstract

International audience; Following our discovery of human dihydroorotate dehydrogenase (DHODH) inhibition by 2-(3-alkoxy-1H-pyrazol-1-yl)pyrimidine derivatives as well as 2-(4-benzyl-3-ethoxy-5-methyl-1H-pyrazol-1-yl)-5-methylpyridine, we describe here the syntheses and evaluation of an array of azine-bearing analogues. As in our previous report, the structure−activity study of this series of human DHODH inhibitors was based on a phenotypic assay measuring measles virus replication. Among other inhibitors, this round of syntheses and biological evaluation iteration led to the highly active 5-cyclopropyl-2-(4-(2,6-difluorophenoxy)-3-isopropoxy-5-methyl-1H-pyrazol-1-yl)-3-fluoropyridine. Inhibition of DHODH by this compound was confirmed in an array of in vitro assays, including enzymatic tests and cell-based assays for viral replication and cellular growth. This molecule was found to be more active than the known inhibitors of DHODH, brequinar and teriflunomide, thus opening perspectives for its use as a tool or for the design of an original series of immunosuppressive agent. Moreover, because other series of inhibitors of human DHODH have been found to also affect Plasmodium falciparum DHODH, all the compounds were assayed for their effect on P. falciparum growth. However, the modest in vitro inhibition solely observed for two compounds did not correlate with their inhibition of P. falciparum DHODH.

Published
2015
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37. Activation de la réponse innée antivirale par des inhibiteurs de la biosynthèse des pyrimidines : les surprises d'un criblage phénotypique

Author

Pierre-Olivier, Vidalain, Marianne, Lucas-Hourani, Olivier, Helynck, Frédéric, Tangy, Hélène, Munier-Lehmann, Génomique virale et vaccination, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), and Chimie et Biocatalyse

Subjects
Oxidoreductases Acting on CH-CH Group Donors, [SDV]Life Sciences [q-bio], Dihydroorotate Dehydrogenase, Drug Evaluation, Preclinical, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, Antiviral Agents, Immunity, Innate, High-Throughput Screening Assays, Small Molecule Libraries, Phenotype, Pyrimidines, Viruses, Humans, Enzyme Inhibitors
Abstract

International audience; RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. In particular, several academic laboratories and private companies are now seeking molecules that stimulate the host innate antiviral response. One appealing strategy is to identify molecules that induce the large cluster of antiviral genes known as Interferon-Stimulated Genes (ISGs). To reach this goal, we have developed a phenotypic assay based on human cells transfected with a luciferase reporter gene under control of an interferon-stimulated response element (ISRE). This system was used in a high-throughput screening of chemical libraries comprising around 54,000 compounds. Among validated hits, compound DD264 was shown to boost the innate immune response in cell cultures, and displayed a broad-spectrum antiviral activity. While deciphering its mode of action, DD264 was found to target the fourth enzyme of de novo pyrimidine biosynthesis, namely the dihydroorotate dehydrogenase (DHODH). Thus, our data unraveled a yet unsuspected link between pyrimidine biosynthesis and the innate antiviral response.

Published
2015
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38. Correction: Two Classes of Bacterial IMPDHs according to Their Quaternary Structures and Catalytic Properties

Author

Hélène Munier-Lehmann, Thomas Alexandre, and Bertrand Raynal

Subjects
Multidisciplinary, business.industry, lcsh:R, lcsh:Medicine, computer.software_genre, Bioinformatics, Correct name, lcsh:Q, Artificial intelligence, Analysis tools, business, lcsh:Science, computer, Natural language processing, Mathematics
Abstract

The second author’s name is spelled incorrectly. The correct name is: Bertrand Raynal. The correct citation is: Alexandre T, Raynal B, Munier-Lehmann H (2015) Two Classes of Bacterial IMPDHs according to Their Quaternary Structures and Catalytic Properties. PLoS ONE 10(2): e0116578. doi: 10.1371/journal.pone.0116578 There is an error in the Author Contributions. The correct contributions are: Conceived and designed the experiments: HML. Performed the experiments: TA BR HML. Analyzed the data: TA BR HML. Contributed reagents/materials/analysis tools: BR HML. Wrote the paper: BR HML.

Published
2015

39. High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

Author

Anastassia V. Komarova, Hélène Munier-Lehmann, Marianne Lucas-Hourani, Frédéric Tangy, Pierre-Olivier Vidalain, Olivier Helynck, Philippe Desprès, Génomique Virale et Vaccination, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Chimie et Biocatalyse, Interactions Moléculaires Flavivirus-Hôtes, Institut Pasteur [Paris] (IP), This work was supported by the Institut Pasteur, the Centre National de la Recherche Scientifique (CNRS), the InstitutNational de la Santé Et de la Recherche Médicale (INSERM), the Institut Carnot - Pasteur Maladies Infectieuses (Programme STING to P.O.Vand H.M.-L), the Agence Nationale pour la Recherche (ANR-RPIB, Programme STING 2.0 to P.O.V), and the 'Conseil Régional d'Ile-de-France' (Chemical Library Project, grants n° I 06-222/R and I 09-1739/R to H.M.-L.). The work on CHIKV/Ren was supported by the projectArbOAS (ANR grant 2010-INTB-1601-02)., Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris], and Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris]

Subjects
General Chemical Engineering, viruses, [SDV]Life Sciences [q-bio], Immunology, Drug Evaluation, Preclinical, Biology, medicine.disease_cause, Antiviral Agents, Virus, General Biochemistry, Genetics and Molecular Biology, Dengue fever, Microbiology, Measles virus, Luciferases, Firefly, High-Throughput Screening Assays, medicine, Humans, Chikungunya, General Immunology and Microbiology, General Neuroscience, RNA, medicine.disease, biology.organism_classification, Virology, 3. Good health, Viral replication, Viral evolution, Chikungunya virus
Abstract

International audience; RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

Published
2014
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40. On dihydroorotate dehydrogenases and their inhibitors and uses

Author

Frédéric Tangy, Pierre-Olivier Vidalain, Hélène Munier-Lehmann, Yves L. Janin, Chimie et Biocatalyse, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Génomique Virale et Vaccination, Institut Carnot-Pasteur Maladies Infectieuses (Programme STING), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris]

Subjects
Oxidoreductases Acting on CH-CH Group Donors, Pyrimidine, Dihydroorotate Dehydrogenase, Biology, Virus, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Drug Discovery, Teriflunomide, medicine, Animals, Humans, Enzyme Inhibitors, 030304 developmental biology, Leflunomide, Dihydroorotate Dehydrogenase Inhibitor, chemistry.chemical_classification, 0303 health sciences, Bacteria, biology.organism_classification, Coccidia, 3. Good health, Enzyme, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, chemistry, Biochemistry, 030220 oncology & carcinogenesis, Dihydroorotate dehydrogenase, Coccidiostats, Molecular Medicine, medicine.drug
Abstract

International audience; Proper nucleosides availability is crucial for the proliferation of living entities (eukaryotic cells, parasites, bacteria, and virus). Accordingly, the uses of inhibitors of the de novo nucleosides biosynthetic pathways have been investigated in the past. In the following we have focused on dihydroorotate dehydrogenase (DHODH), the fourth enzyme in the de novo pyrimidine nucleosides biosynthetic pathway. We first described the different types of enzyme in terms of sequence, structure, and biochemistry, including the reported bioassays. In a second part, the series of inhibitors of this enzyme along with a description of their potential or actual uses were reviewed. These inhibitors are indeed used in medicine to treat autoimmune diseases such as rheumatoid arthritis or multiple sclerosis (leflunomide and teriflunomide) and have been investigated in treatments of cancer, virus, and parasite infections (i.e., malaria) as well as in crop science.

Published
2013
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41. Synthesis and evaluation of 5'-modified thymidines and 5-hydroxymethyl-2'-deoxyuridines as Mycobacterium tuberculosis thymidylate kinase inhibitors

Author

Kiran S. Toti, Frederick Verbeke, Hélène Munier-Lehmann, Vladimir Frecer, Serge Van Calenbergh, Martijn D. P. Risseeuw, Laboratory for Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Department of Physical Chemistry of Drugs, Faculty of Pharmacy, Comenius University, Chimie et Biocatalyse, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Laboratory for Medicinal Chemistry-FWW, Universiteit Gent = Ghent University [Belgium] (UGENT), Comenius University in Bratislava, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Universiteit Gent = Ghent University (UGENT), and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)

Subjects
MESH: Mycobacterium tuberculosis, [SDV]Life Sciences [q-bio], Clinical Biochemistry, Antitubercular Agents, Pharmaceutical Science, 01 natural sciences, Biochemistry, chemistry.chemical_compound, Thymidine monophosphate kinase, DESIGN, Nucleoside analogues, Drug Discovery, Moiety, Hydroxymethyl, MESH: Tuberculosis, biology, Drug discovery, 3. Good health, Chemistry, Molecular Medicine, Inhibitor, Stereochemistry, 010402 general chemistry, Thymidylate kinase, Mycobacterium tuberculosis, MONOPHOSPHATE KINASE, Humans, Tuberculosis, Molecular Biology, Phosphate bioisosteres, MESH: Humans, ANALOGS, 010405 organic chemistry, Organic Chemistry, 5-Hydroxymethyl pyrimidines, IN-VITRO, DNA, biology.organism_classification, MESH: Antitubercular Agents, Deoxyuridine, In vitro, 0104 chemical sciences, DRUG DISCOVERY, REDUCTION, chemistry, Nucleoside-Phosphate Kinase, MESH: Nucleoside-Phosphate Kinase, MESH: Thymidine, Thymidine
Abstract

International audience; We report the synthesis of 5'-modified thymidines (16, 18, 21, 23) and 5,5'-bis-substituted 2'-deoxyuridine analogues (30, 47) as inhibitors of thymidine monophosphate kinase of Mycobacterium tuberculosis (TMPKmt). These analogues were evaluated for their capacity to inhibit TMPKmt and solely two 5'-modified thymidines were found to possess moderate inhibitory activity. In addition, a feasibility study of protecting groups for the 5-CH(2)OH moiety of 2'-deoxyuridines is described that enables to introduce the desired 5'-modification.

Published
2013
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42. Drug design and identification of potent leads against Mycobacterium tuberculosis thymidine monophosphate kinase

Author

Sylvie Pochet, S. Van Calenbergh, Hélène Munier-Lehmann, Laboratory for Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Chimie et Biocatalyse - Chemistry and Biocatalysis (group), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Chimie et Biocatalyse, and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, MECHANISM, AZT ACTIVATION, Antitubercular Agents, nucleotide metabolism, 01 natural sciences, chemistry.chemical_compound, Drug Discovery, inhibitors, Medicine and Health Sciences, COMPLETE GENOME SEQUENCE, RATIONAL DESIGN, CRYSTAL-STRUCTURE, Enzyme Inhibitors, Polymerase, HUMAN THYMIDYLATE KINASE, chemistry.chemical_classification, 0303 health sciences, Molecular Structure, biology, [CHIM.ORGA]Chemical Sciences/Organic chemistry, Kinase, structure-activity relationship, General Medicine, 3. Good health, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Biochemistry, NUCLEOSIDE ANALOGS, medicine.drug, kinase, Mycobacterium tuberculosis, Structure-Activity Relationship, 03 medical and health sciences, medicine, Humans, Tuberculosis, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, BIOLOGICAL EVALUATION, 030304 developmental biology, DNA synthesis, Nucleoside analogue, 010405 organic chemistry, molecular modeling, biology.organism_classification, 0104 chemical sciences, Enzyme, chemistry, Drug Design, biology.protein, EFFICIENT PHOSPHORYLATION, Nucleoside-Phosphate Kinase, Thymidine, Nucleoside
Abstract

International audience; Antiviral chemotherapy often relies on nucleoside analogues, which, once phophorylated by intracellular kinases, target viral polymerases impeding DNA synthesis. In contrast, nucleoside analogues are much less explored as antibacterial drugs. Thymidine monophosphate kinase from Mycobacterium tuberculosis (TMPKmt), which is essential to DNA replication, was selected as a promising target for the design of new inhibitors. This review describes stepwise modifications of the TMPKmt substrate, guided by the feedback of enzyme assays and crystallographic analysis to afford potent enzyme inhibitors some of which also exhibited antitubercular activity. More importantly, several of the reported thymidine analogues provided a deeper understanding of the structure and catalytic mechanism of this intriguing enzyme.

Published
2012
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43. Synthesis and inhibitory activity of thymidine analogues targeting Mycobacterium tuberculosis thymidine monophosphate kinase

Author

Sara Van Poecke, Olivier Helynck, Matheus Froeyen, Serge Van Calenbergh, Hélène Munier-Lehmann, Laboratory for Medicinal Chemistry-FWW, Universiteit Gent = Ghent University [Belgium] (UGENT), Chimie et Biocatalyse, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Rega Institute for Medical Research [Leuven, België], Catholic University of Leuven - Katholieke Universiteit Leuven (KU Leuven), Universiteit Gent = Ghent University (UGENT), Chimie et Biocatalyse - Chemistry and Biocatalysis (group), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)

Subjects
Models, Molecular, NUCLEOSIDES, TERMINAL ALKYNES, Clinical Biochemistry, Pharmaceutical Science, Inhibitory postsynaptic potential, 01 natural sciences, Biochemistry, Thymidylate kinase, Mycobacterium tuberculosis, 03 medical and health sciences, chemistry.chemical_compound, AZIDES, Drug Discovery, Thymidine Monophosphate, Humans, Tuberculosis, ASSAY, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Protein Kinase Inhibitors, Molecular Biology, 030304 developmental biology, alpha- and beta-Nucleosides, 0303 health sciences, Thymidine monophosphate, Nucleoside-phosphate kinase, biology, Spectrophotometric binding assay, 010405 organic chemistry, Chemistry, Organic Chemistry, Biology and Life Sciences, Inhibitory activity, Thymidine monophosphate kinase, biology.organism_classification, Molecular biology, THYMIDYLATE KINASE, 3. Good health, 0104 chemical sciences, Inhibitory potency, TMPKmt, Thymidine analogues, Molecular Medicine, Nucleoside-Phosphate Kinase, Thymidine
Abstract

International audience; We report on Mycobacterium tuberculosis thymidine monophosphate kinase (TMPKmt) inhibitory activities of a series of new 3'- and 5'-modified thymidine analogues including α- and β-derivatives. In addition, several analogues were synthesized in which the 4-oxygen was replaced by a more lipophilic sulfur atom to probe the influence of this modification on TMPKmt inhibitory activity. Several compounds showed an inhibitory potency in the low micromolar range, with the 5'-arylthiourea 4-thio-α-thymidine analogue being the most active one (K(i)=0.17μM). This compound was capable of inhibiting mycobacteria growth at a concentration of 25μg/mL.

Published
2011
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44. Structural and functional characterization of the Mycobacterium tuberculosis uridine monophosphate kinase: insights into the allosteric regulation

Author

Isabelle Salard-Arnaud, Anne-Marie Gilles, Khaled Benkali, Gilles Labesse, Hélène Munier-Lehmann, 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), Chimie et Biocatalyse - Chemistry and Biocatalysis (group), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Membranes bactériennes, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Région Ile de France(Chemical Library Project, grants I 06-222/R and I 09-1739/ R, which included a fellowship for I.S.-A.), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Chimie et Biocatalyse, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), and Munier-Lehmann, Hélène

Subjects
Models, Molecular, GTP', Molecular Sequence Data, Allosteric regulation, Cooperativity, Biology, Crystallography, X-Ray, 03 medical and health sciences, chemistry.chemical_compound, Allosteric Regulation, Bacterial Proteins, Structural Biology, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Genetics, Uridine monophosphate, Transferase, Nucleotide, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, Nucleoside-phosphate kinase, Sequence Homology, Amino Acid, 030302 biochemistry & molecular biology, Mycobacterium tuberculosis, 3. Good health, chemistry, Biochemistry, Allosteric enzyme, Mutagenesis, Site-Directed, biology.protein, Nucleoside-Phosphate Kinase
Abstract

Nucleoside Monophosphate Kinases (NMPKs) family are key enzymes in nucleotide metabolism. Bacterial UMPKs depart from the main superfamily of NMPKs. Having no eukaryotic counterparts they represent attractive therapeutic targets. They are regulated by GTP and UTP, while showing different mechanisms in Gram(+), Gram(–) and archaeal bacteria. In this work, we have characterized the mycobacterial UMPK (UMPKmt) combining enzymatic and structural investigations with site-directed mutagenesis. UMPKmt exhibits cooperativity toward ATP and an allosteric regulation by GTP and UTP. The crystal structure of the complex of UMPKmt with GTP solved at 2.5 Å, was merely identical to the modelled apo-form, in agreement with SAXS experiments. Only a small stretch of residues was affected upon nucleotide binding, pointing out the role of macromolecular dynamics rather than major structural changes in the allosteric regulation of bacterial UMPKs. We further probe allosteric regulation by site-directed mutagenesis. In particular, a key residue involved in the allosteric regulation of this enzyme was identified.

Published
2011
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45. Metalloprotease Vsm Is the Major Determinant of Toxicity for Extracellular Products of Vibrio splendidus

Author

Denis Saulnier, Didier Mazel, Hélène Munier-Lehmann, Monique Zagorec, Claude Delsert, Marie-Christine Champomier-Vergès, Frédérique Le Roux, Johan Binesse, Biologie systémique - Systems Biology, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Génétique et Pathologie des Mollusques Marins, 17390 La Tremblade, France. (LGPMM), Santé, Génétique et Microbiologie des Mollusques (IFREMER SG2M), Institut Français de Recherche pour l'Exploitation de la Mer - Atlantique (IFREMER Atlantique), Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER)-Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER)-Institut Français de Recherche pour l'Exploitation de la Mer - Atlantique (IFREMER Atlantique), Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER)-Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER), Centre de recherche en Biologie Cellulaire (CRBM), Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Université Montpellier 1 (UM1), Unité de recherche Flore Lactique et Environnement Carné (UFLEC), Institut National de la Recherche Agronomique (INRA), Institut Pasteur [Paris], Plasticité du Génome Bactérien (PGB), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Génétique et Pathologie (LGP), Amélioration génétique, du contrôle des performances et de la santé des mollusques marins (AGSAE), Chimie Organique, This study was carried out with financial assistance from the Institut Pasteur, the Centre National de la Recherche Scientifique (CNRS-URA 2171), the Institut Françcais de Recherche pour l'Exploitation de la Mer (IFREMER), and the Institut de Génomique Marine (Ministère de la Recherche contract no. 0425). Johan Binesse acknowledges a Ph.D. scholarship from the French Ministry of Research and Technology (MRT)., and Université Montpellier 1 (UM1)-Université Montpellier 2 - Sciences et Techniques (UM2)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
MESH: Sequence Analysis, DNA, Proteome, MESH: Sequence Homology, Amino Acid, [SDV]Life Sciences [q-bio], Mutant, species, Applied Microbiology and Biotechnology, Virulence factor, Mice, MESH: Animals, Pathogen, MESH: Bacterial Proteins, Cells, Cultured, 0303 health sciences, Metalloproteinase, Ecology, MESH: Genomics, Genomics, musculoskeletal system, MESH: Mollusca, MESH: Proteome, cardiovascular system, MESH: Metalloproteases, Biotechnology, MESH: Cells, Cultured, DNA, Bacterial, Virulence Factors, education, Molecular Sequence Data, Bacillus thuringiensis, Virulence, Biology, Microbiology, 03 medical and health sciences, MESH: Vibrio, Bacterial Proteins, Extracellular, Invertebrate Microbiology, Animals, MESH: Mice, 030304 developmental biology, MESH: Virulence Factors, Vibrio, MESH: Bacillus thuringiensis, MESH: Molecular Sequence Data, Sequence Homology, Amino Acid, 030306 microbiology, Wild type, Sequence Analysis, DNA, Fibroblasts, biology.organism_classification, Molecular biology, MESH: DNA, Bacterial, proteins, MESH: Fibroblasts, MESH: Gene Deletion, Mollusca, Metalloproteases, Genomic, Gene Deletion, Food Science
Abstract

Genomic data combined with reverse genetic approaches have contributed to the characterization of major virulence factors of Vibrio species; however, these studies have targeted primarily human pathogens. Here, we investigate virulence factors in the oyster pathogen Vibrio splendidus LGP32 and show that toxicity is correlated to the presence of a metalloprotease and its corresponding vsm gene. Comparative genomics showed that an avirulent strain closely related to LGP32 lacked the metalloprotease. The toxicity of LGP32 metalloprotease was confirmed by exposing mollusk and mouse fibroblastic cell lines to extracellular products (ECPs) of the wild type (wt) and a vsm deletion mutant (Δ vsm mutant). The ECPs of the wt induced a strong cytopathic effect whose severity was cell type dependent, while those of the Δ vsm mutant were much less toxic, and exposure to purified protein demonstrated the direct toxicity of the Vsm metalloprotease. Finally, to investigate Vsm molecular targets, a proteomic analysis of the ECPs of both LGP32 and the Δ vsm mutant was performed, revealing a number of differentially expressed and/or processed proteins. One of these, the VSA1062 metalloprotease, was found to have significant identity to the immune inhibitor A precursor, a virulence factor of Bacillus thuringiensis . Deletion mutants corresponding to several of the major proteins were constructed by allelic exchange, and the ECPs of these mutants proved to be toxic to both cell cultures and animals. Taken together, these data demonstrate that Vsm is the major toxicity factor in the ECPs of V. splendidus .

Published
2008
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46. Substituted benzyl-pyrimidines targeting thymidine monophosphate kinase of Mycobacterium tuberculosis: Synthesis and in vitro anti-mycobacterial activity

Author

Valérie Huteau, Sylvie Pochet, Gilles Marchal, Cécile Gasse, Dominique Douguet, Hélène Munier-Lehmann, Chimie Organique, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), 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), Immunothérapie, Institut Pasteur [Paris] (IP), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Institut Pasteur [Paris]

Subjects
Pyrimidine, Stereochemistry, Clinical Biochemistry, Antitubercular Agents, dTMP kinase, Pharmaceutical Science, 010402 general chemistry, 01 natural sciences, Biochemistry, Chemical synthesis, Mycobacterium tuberculosis, chemistry.chemical_compound, Drug Delivery Systems, Chlorocebus aethiops, Drug Discovery, Animals, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Vero Cells, Molecular Biology, Antibacterial agent, Mycobacterium bovis, biology, 010405 organic chemistry, [CHIM.ORGA]Chemical Sciences/Organic chemistry, Organic Chemistry, Biological activity, biology.organism_classification, 3. Good health, 0104 chemical sciences, Pyrimidines, chemistry, Enzyme inhibitor, Drug Design, biology.protein, Molecular Medicine, Nucleoside-Phosphate Kinase
Abstract

International audience; A series of N(1)-(4-substituted-benzyl)-pyrimidines were synthesized as potential inhibitors of thymidine monophosphate kinase of Mycobacterium tuberculosis (TMPKmt). Key SAR parameters included the chain length substitution in para position of the benzyl ring, the functional group terminating the alkyl chain, and the substituent on the C-5 pyrimidine ring. Synthesized molecules were assayed against both recombinant enzyme and mycobacteria cultures. The most potent compounds have K(i) values in the micromolar range and an MIC(50) of 50microg/mL against Mycobacterium bovis. These results will guide the design of a new generation of lead compounds.

Published
2008
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47. Acyclic phosphonate nucleotides and human adenylate kinases: impact of a borano group on alpha-P position

Author

Hélène Munier-Lehmann, Karine Barral, Dimitrios Topalis, C El-Amri, Karine Alvarez, Michel Véron, Bruno Canard, Dominique Deville-Bonne, Benoit Schneider, Laurence A. Mulard, Catherine Guerreiro, Enzymologie Moléculaire et Fonctionnelle, Université Pierre et Marie Curie - Paris 6 (UPMC), Architecture et fonction des macromolécules biologiques (AFMB), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Chimie Organique (UCO), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Régulation Enzymatique des Activités Cellulaires, Peptidome de la Peau des Amphibiens (FRE 2852), Chimie Organique, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA)

Subjects
Models, Molecular, MESH: Catalytic Domain, Biochemistry, MESH: Recombinant Proteins, chemistry.chemical_compound, 0302 clinical medicine, Adenosine Triphosphate, MESH: Boranes, Catalytic Domain, MESH: Adenosine Triphosphate, MESH: Soman, Nucleotide, Cloning, Molecular, Phosphorylation, Boranes, chemistry.chemical_classification, 0303 health sciences, biology, MESH: Kinetics, Chemistry, Kinase, Nucleotides, MESH: Gene Expression Regulation, Enzymologic, General Medicine, Phosphonate, Recombinant Proteins, Isoenzymes, MESH: Adenylate Kinase, 030220 oncology & carcinogenesis, MESH: Isoenzymes, Molecular Medicine, MESH: Models, Molecular, MESH: Adenine, Stereochemistry, Organophosphonates, Adenylate kinase, MESH: Phosphonic Acids, Gene Expression Regulation, Enzymologic, 03 medical and health sciences, Genetics, Humans, MESH: Cloning, Molecular, Binding site, Tenofovir, 030304 developmental biology, Binding Sites, MESH: Humans, MESH: Phosphorylation, Adenine, Adenylate Kinase, Active site, MESH: Nucleotides, Kinetics, MESH: Binding Sites, biology.protein, Adenosine triphosphate
Abstract

International audience; Adenylate kinases are involved in the activation of antiviral drugs such as the acyclic phosphonates analogs PMEA and (R)PMPA. We examine the in vitro phosphorylation of PMEA and PMPA bearing a borano- or a H- group on the phosphorus atom. The alpha-borano or alpha-H on PMEA and PMPA were detrimental to the activity of recombinant human AMP kinases 1 and 2. Docking PMEA to the active site of AMP kinase 1 indicated that the borano group may prevent two conserved critical Arg interactions with the alpha-phosphate, resulting in substrate bad positioning.

Published
2008
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48. Exploring acyclic nucleoside analogues as inhibitors of Mycobacterium tuberculosis thymidylate kinase

Author

Ana-Isabel Hernández, Federico Gago, María-Jesús Pérez-Pérez, María-José Camarasa, Andrea Negri, Dominique Douguet, Olga Familiar, Leen Rigouts, Hélène Munier-Lehmann, Instituto de Quimica Médica (CSIC), Consejo Superior de Investigaciones Científicas [Madrid] (CSIC), Chimie Organique, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Departamento de Farmacologica, Universidad de Alcalá - University of Alcalá (UAH), 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), Mycobacteriology Unit, Prince Leopold Institute of Tropical Medicine, Institut Pasteur [Paris]-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
Stereochemistry, Bacterial diseases, Naphtholactam, Antitubercular Agents, Substituent, Thymidylate kinase, Drug development, Naphthalenes, 01 natural sciences, Biochemistry, Mycobacterium tuberculosis, Structure-Activity Relationship, 03 medical and health sciences, chemistry.chemical_compound, Drug Discovery, Tuberculosis, Moiety, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Enzyme Inhibitors, General Pharmacology, Toxicology and Pharmaceutics, 030304 developmental biology, Pharmacology, chemistry.chemical_classification, 0303 health sciences, biology, 010405 organic chemistry, [CHIM.ORGA]Chemical Sciences/Organic chemistry, Acyclic nucleoside, Organic Chemistry, Nucleosides, Acyclic nucleosides, biology.organism_classification, 0104 chemical sciences, 3. Good health, Thymine, Enzyme, chemistry, Docking (molecular), Molecular Medicine, Human medicine, Nucleoside-Phosphate Kinase
Abstract

In the search for novel inhibitors of the enzyme thymidine monophosphate kinase of Mycobacterium tuberculosis (TMPKmt), an attractive target for novel antituberculosis agents, we report herein the discovery of the first acyclic nucleoside analogues that potently and selectively inhibit TMPKmt. The most potent compounds in this series are (Z)-butenylthymines carrying a naphtholactam or naphthosultam moiety at position 4, which display Ki values of 0.42 and 0.27 μM, respectively. Docking studies followed by molecular dynamics simulations performed to rationalize the interaction of this new family of inhibitors with the target enzyme revealed a key interaction between the distal substituent and Arg 95 in the target enzyme. The fact that these inhibitors are more easily synthesizable than previously identified TMPKmt inhibitors, together with their potency against the target enzyme, makes them attractive lead compounds for further optimization. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA.

Published
2008
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49. Synthesis and biological evaluation of bicyclic nucleosides as inhibitors of M. tuberculosis thymidylate kinase

Author

Serge Van Calenbergh, Li Qing, Pieter M. S. Hendrickx, Ineke Van Daele, P Chavarot, Hélène Munier-Lehmann, José C. Martins, Gilles Marchal, Matheus Froeyen, Laboratory for Medicinal Chemistry-FWW, Universiteit Gent = Ghent University (UGENT), Chimie Organique, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), NMR and Structure Analysis Unit, Immunothérapie, Institut Pasteur [Paris] (IP), Laboratory of Medicinal Chemistry, Rega Institute-Catholic University of Leuven, Universiteit Gent = Ghent University [Belgium] (UGENT), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris]

Subjects
Models, Molecular, MESH: Mycobacterium bovis, MESH: Mycobacterium tuberculosis, Stereochemistry, Molecular Conformation, Stereoisomerism, Microbial Sensitivity Tests, Biology, 010402 general chemistry, Crystallography, X-Ray, 01 natural sciences, Biochemistry, Thymidylate kinase, MESH: Bicyclo Compounds, Heterocyclic, Mycobacterium tuberculosis, chemistry.chemical_compound, Structure-Activity Relationship, MESH: Structure-Activity Relationship, Drug Discovery, Structure–activity relationship, General Pharmacology, Toxicology and Pharmaceutics, Pharmacology, MESH: Microbial Sensitivity Tests, MESH: Molecular Conformation, Nucleoside-phosphate kinase, Binding Sites, Bicyclic molecule, 010405 organic chemistry, Organic Chemistry, biology.organism_classification, Bridged Bicyclo Compounds, Heterocyclic, MESH: Crystallography, X-Ray, Mycobacterium bovis, MESH: Stereoisomerism, 3. Good health, 0104 chemical sciences, Thymine, chemistry, MESH: Binding Sites, Molecular Medicine, Thymidine, Nucleoside-Phosphate Kinase, MESH: Nucleoside-Phosphate Kinase, MESH: Models, Molecular, MESH: Thymidine
Abstract

International audience; Herein we describe the synthesis and conformational analysis of a series of bicyclic thymidine derivatives and their evaluation as inhibitors of thymidine monophosphate kinase from Mycobacterium tuberculosis (TMPKmt), based on previously discovered bicyclic sugar nucleosides. With a K(i) value of 2.3 microm, 1-[3-aminomethyl-3,5-dideoxy-2-O,6-N-(thiocarbonyl)-beta-D-ribofuranosyl]thymine emerged as the most potent TMPK inhibitor of this series. Moreover, this promising compound displays inhibitory potency against Mycobacteria cultures with an IC(99) value of 100 microg mL(-1), thus promoting TMPKmt for the first time as a validated target for further inhibitory design. Attempts to rationalise the observed structure-activity relationship (SAR) involving molecular modelling and conformational analysis are described.

Published
2006
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50. Multiple microfermentor battery: a versatile tool for use with automated parallel cultures of microorganisms producing recombinant proteins and for optimization of cultivation protocols

Author

Jacques Bellalou, Vincent Bondet, Emmanuel Frachon, Hélène Munier-Lehmann, Production de Protéines Recombinantes et d'Anticorps (Plate-Forme), Institut Pasteur [Paris] (IP), Chimie Organique, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris], and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)

Subjects
0106 biological sciences, Battery (electricity), MESH: Hydrogen-Ion Concentration, Materials science, Thermoelectric cooling, Interface (computing), Biomass, 01 natural sciences, 7. Clean energy, Applied Microbiology and Biotechnology, Online Systems, MESH: Fermentation, MESH: Recombinant Proteins, 03 medical and health sciences, Automation, MESH: Bioreactors, Bioreactors, MESH: Automation, 010608 biotechnology, Escherichia coli, Methods, Process optimization, Process engineering, 030304 developmental biology, 0303 health sciences, Ecology, business.industry, MESH: Escherichia coli, Temperature, Hydrogen-Ion Concentration, Recombinant Proteins, MESH: Temperature, MESH: Online Systems, Culture Media, Oxygen, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Volume (thermodynamics), Chemical engineering, Personal computer, Fermentation, MESH: Biotechnology, MESH: Culture Media, Aeration, business, MESH: Oxygen, Food Science, Biotechnology
Abstract

A multiple microfermentor battery was designed for high-throughput recombinant protein production in Escherichia coli . This novel system comprises eight aerated glass reactors with a working volume of 80 ml and a moving external optical sensor for measuring optical densities at 600 nm (OD 600 ) ranging from 0.05 to 100 online. Each reactor can be fitted with miniature probes to monitor temperature, dissolved oxygen (DO), and pH. Independent temperature regulation for each vessel is obtained with heating/cooling Peltier devices. Data from pH, DO, and turbidity sensors are collected on a FieldPoint (National Instruments) I/O interface and are processed and recorded by a LabVIEW program on a personal computer, which enables feedback control of the culture parameters. A high-density medium formulation was designed, which enabled us to grow E. coli to OD 600 up to 100 in batch cultures with oxygen-enriched aeration. Accordingly, the biomass and the amount of recombinant protein produced in a 70-ml culture were at least equivalent to the biomass and the amount of recombinant protein obtained in a Fernbach flask with 1 liter of conventional medium. Thus, the microfermentor battery appears to be well suited for automated parallel cultures and process optimization, such as that needed for structural genomics projects.

Published
2006
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