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1. Molecular mechanism of a triazole-containing inhibitor of Mycobacterium tuberculosis DNA gyrase

Author

Antoine Gedeon, Emilie Yab, Aurelia Dinut, Elodie Sadowski, Estelle Capton, Aurore Dreneau, Julienne Petit, Bruna Gioia, Catherine Piveteau, Kamel Djaout, Estelle Lecat, Anne Marie Wehenkel, Francesca Gubellini, Ariel Mechaly, Pedro M. Alzari, Benoît Deprez, Alain Baulard, Alexandra Aubry, Nicolas Willand, and Stéphanie Petrella

Subjects
Multidrug resistant organisms, Drugs, Molecular Structure, Microbiology, Science
Abstract

Summary: Antimicrobial resistance remains a persistent and pressing public health concern. Here, we describe the synthesis of original triazole-containing inhibitors targeting the DNA gyrase, a well-validated drug target for developing new antibiotics. Our compounds demonstrate potent antibacterial activity against various pathogenic bacteria, with notable potency against Mycobacterium tuberculosis (Mtb). Moreover, one hit, compound 10a, named BDM71403, was shown to be more potent in Mtb than the NBTI of reference, gepotidacin. Mechanistic enzymology assays reveal a competitive interaction of BDM71403 with fluoroquinolones within the Mtb gyrase cleavage core. High-resolution cryo-electron microscopy structural analysis provides detailed insights into the ternary complex formed by the Mtb gyrase, double-stranded DNA, and either BDM71403 or gepotidacin, providing a rational framework to understand the superior in vitro efficacy on Mtb. This study highlights the potential of triazole-based scaffolds as promising gyrase inhibitors, offering new avenues for drug development in the fight against antimicrobial resistance.

Published
2024
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2. Exploring the Antitubercular Activity of Anthranilic Acid Derivatives: From MabA (FabG1) Inhibition to Intrabacterial Acidification

Author

Léo Faïon, Kamel Djaout, Catalin Pintiala, Catherine Piveteau, Florence Leroux, Alexandre Biela, Stéphanie Slupek, Rudy Antoine, Monika Záhorszká, Francois-Xavier Cantrelle, Xavier Hanoulle, Jana Korduláková, Benoit Deprez, Nicolas Willand, Alain R. Baulard, and Marion Flipo

Subjects
MabA inhibitors, anthranilic acid, FabG1, tuberculosis, mycolic acids, Medicine, Pharmacy and materia medica, RS1-441
Abstract

Mycobacterium tuberculosis, the pathogen that causes tuberculosis, is responsible for the death of 1.5 million people each year and the number of bacteria resistant to the standard regimen is constantly increasing. This highlights the need to discover molecules that act on new M. tuberculosis targets. Mycolic acids, which are very long-chain fatty acids essential for M. tuberculosis viability, are synthesized by two types of fatty acid synthase (FAS) systems. MabA (FabG1) is an essential enzyme belonging to the FAS-II cycle. We have recently reported the discovery of anthranilic acids as MabA inhibitors. Here, the structure–activity relationships around the anthranilic acid core, the binding of a fluorinated analog to MabA by NMR experiments, the physico-chemical properties and the antimycobacterial activity of these inhibitors were explored. Further investigation of the mechanism of action in bacterio showed that these compounds affect other targets than MabA in mycobacterial cells and that their antituberculous activity is due to the carboxylic acid moiety which induces intrabacterial acidification.

Published
2023
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3. On the Hunt for Next-Generation Antimicrobial Agents: An Online Symposium Organized Jointly by the French Society for Medicinal Chemistry (Société de Chimie Thérapeutique) and the French Microbiology Society (Société Française de Microbiologie) on 9–10 December 2021

Author

Kevin Antraygues, Nina Compagne, Francesca Ruggieri, Kamel Djaout, Zainab Edoo, Maxime Eveque, Léo Faïon, Bruna Gioia, Salia Tangara, Anais Vieira Da Cruz, Baptiste Villemagne, Marion Flipo, Alain Baulard, and Nicolas Willand

Subjects
antimicrobial resistance, drug discovery, microbiology, Medicine, Pharmacy and materia medica, RS1-441
Abstract

The restrictions posed by the COVID-19 pandemic obliged the French Society for Medicinal Chemistry (Société de chimie thérapeutique) and the French Microbiology Society (Société Française de Microbiologie) to organize their joint autumn symposium (entitled “On the hunt for next-generation antimicrobial agents”) online on 9–10 December 2021. The meeting attracted more than 200 researchers from France and abroad with interests in drug discovery, antimicrobial resistance, medicinal chemistry, and related disciplines. This review summarizes the 13 invited keynote lectures. The symposium generated high-level scientific dialogue on the most recent advances in combating antimicrobial resistance. The University of Lille, the Institut Pasteur de Lille, the journal Pharmaceuticals, Oxeltis, and INCATE, sponsored the event.

Published
2022
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4. Targeting of Helicobacter pylori thymidylate synthase ThyX by non-mitotoxic hydroxy-naphthoquinones

Author

Stéphane Skouloubris, Kamel Djaout, Isabelle Lamarre, Jean-Christophe Lambry, Karine Anger, Julien Briffotaux, Ursula Liebl, Hilde de Reuse, and Hannu Myllykallio

Subjects
naphthoquinone, anti-microbial agents, helicobacter pylori, thymidylate synthase thyx, Biology (General), QH301-705.5
Abstract

ThyX is an essential thymidylate synthase that is mechanistically and structurally unrelated to the functionally analogous human enzyme, thus providing means for selective inhibition of bacterial growth. To identify novel compounds with anti-bacterial activity against the human pathogenic bacterium Helicobacter pylori, based on our earlier biochemical and structural analyses, we designed a series of eighteen 2-hydroxy-1,4-naphthoquinones (2-OH-1,4-NQs) that target HpThyX. Our lead-like molecules markedly inhibited the NADPH oxidation and 2′-deoxythymidine-5′-monophosphate-forming activities of HpThyX enzyme in vitro, with inhibitory constants in the low nanomolar range. The identification of non-cytotoxic and non-mitotoxic 2-OH-1,4-NQ inhibitors permitted testing their in vivo efficacy in a mouse model for H. pylori infections. Despite the widely assumed toxicity of naphthoquinones (NQs), we identified tight-binding ThyX inhibitors that were tolerated in mice and can be associated with a modest effect in reducing the number of colonizing bacteria. Our results thus provide proof-of-concept that targeting ThyX enzymes is a highly feasible strategy for the development of therapies against H. pylori and a high number of other ThyX-dependent pathogenic bacteria. We also demonstrate that chemical reactivity of NQs does not prevent their exploitation as anti-microbial compounds, particularly when mitotoxicity screening is used to prioritize these compounds for further experimentation.

Published
2015
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5. The small-molecule SMARt751 reverses Mycobacterium tuberculosis resistance to ethionamide in acute and chronic mouse models of tuberculosis

Author

Marion Flipo, Rosangela Frita, Marilyne Bourotte, María S. Martínez-Martínez, Markus Boesche, Gary W. Boyle, Geo Derimanov, Gerard Drewes, Pablo Gamallo, Sonja Ghidelli-Disse, Stephanie Gresham, Elena Jiménez, Jaime de Mercado, Esther Pérez-Herrán, Esther Porras-De Francisco, Joaquín Rullas, Patricia Casado, Florence Leroux, Catherine Piveteau, Mehdi Kiass, Vanessa Mathys, Karine Soetaert, Véronique Megalizzi, Abdalkarim Tanina, René Wintjens, Rudy Antoine, Priscille Brodin, Vincent Delorme, Martin Moune, Kamel Djaout, Stéphanie Slupek, Christian Kemmer, Marc Gitzinger, Lluis Ballell, Alfonso Mendoza-Losana, Sergio Lociuro, Benoit Deprez, David Barros-Aguirre, Modesto J. Remuiñán, Nicolas Willand, and Alain R. Baulard

Subjects
General Medicine
Abstract

The sensitivity of Mycobacterium tuberculosis , the pathogen that causes tuberculosis (TB), to antibiotic prodrugs is dependent on the efficacy of the activation process that transforms the prodrugs into their active antibacterial moieties. Various oxidases of M. tuberculosis have the potential to activate the prodrug ethionamide. Here, we used medicinal chemistry coupled with a phenotypic assay to select the N-acylated 4-phenylpiperidine compound series. The lead compound, SMARt751, interacted with the transcriptional regulator VirS of M. tuberculosis , which regulates the mymA operon encoding a monooxygenase that activates ethionamide. SMARt751 boosted the efficacy of ethionamide in vitro and in mouse models of acute and chronic TB. SMARt751 also restored full efficacy of ethionamide in mice infected with M. tuberculosis strains carrying mutations in the ethA gene, which cause ethionamide resistance in the clinic. SMARt751 was shown to be safe in tests conducted in vitro and in vivo. A model extrapolating animal pharmacokinetic and pharmacodynamic parameters to humans predicted that as little as 25 mg of SMARt751 daily would allow a fourfold reduction in the dose of ethionamide administered while retaining the same efficacy and reducing side effects.

Published
2022

6. Discovery of the first Mycobacterium tuberculosis MabA (FabG1) inhibitors through a fragment-based screening

Author

Martin Moune, Florence Leroux, Léo Faïon, Abdalkarim Tanina, Alexandre Biela, Nicolas Willand, Kevin Bourbiaux, Francois-Xavier Cantrelle, Alain R. Baulard, Laurent Kremer, Catalin Pintiala, Benoit Deprez, Kamel Djaout, René Wintjens, Adrien Herledan, Marion Flipo, Mickaël Blaise, Rosangela Frita, Alexandre Vandeputte, Catherine Piveteau, Xavier Hanoulle, Médicaments et molécules pour agir sur les Systèmes Vivants - U 1177 (M2SV), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille, Réseau International des Instituts Pasteur (RIIP), Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Biologie Structurale Intégrative (ERL 9002 - BSI ), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Facteurs de Risque et Déterminants Moléculaires des Maladies liées au Vieillissement - U 1167 (RID-AGE), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Unité Microbiologie, Chimie Bioorganique et Macromoléculaire (CP206/04), Département RD3, ANR-13-JSV5-0010,2FightTb,De l'identification de fragments à la découverte d'inhibiteurs de MabA (FabG1) un nouveau challenge pour traiter la tuberculose(2013), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), HANOULLE, Xavier, and Jeunes Chercheuses et Jeunes Chercheurs - De l'identification de fragments à la découverte d'inhibiteurs de MabA (FabG1) un nouveau challenge pour traiter la tuberculose - - 2FightTb2013 - ANR-13-JSV5-0010 - JC - VALID

Subjects
Drug Evaluation, Preclinical, 01 natural sciences, Mycolic acid, chemistry.chemical_compound, [CHIM] Chemical Sciences, mycolic acid, Drug Discovery, Fragment, ortho-Aminobenzoates, Enzyme Inhibitors, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], chemistry.chemical_classification, 0303 health sciences, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Molecular Structure, INHA, General Medicine, Sciences bio-médicales et agricoles, 3. Good health, Fatty acid synthase, Biochemistry, tuberculosis, Tuberculosis, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], fragment, Mycobacterium tuberculosis, 03 medical and health sciences, Structure-Activity Relationship, Biosynthesis, Bacterial Proteins, Anthranilic acid, medicine, MabA inhibitors, [CHIM]Chemical Sciences, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], 030304 developmental biology, Pharmacology, Dose-Response Relationship, Drug, 010405 organic chemistry, Organic Chemistry, medicine.disease, biology.organism_classification, 0104 chemical sciences, Enzyme, chemistry, biology.protein, Fatty Acid Synthases, FabG1
Abstract

Mycobacterium tuberculosis (M.tb), the etiologic agent of tuberculosis, remains the leading cause of death from a single infectious agent worldwide. The emergence of drug-resistant M.tb strains stresses the need for drugs acting on new targets. Mycolic acids are very long chain fatty acids playing an essential role in the architecture and permeability of the mycobacterial cell wall. Their biosynthesis involves two fatty acid synthase (FAS) systems. Among the four enzymes (MabA, HadAB/BC, InhA and KasA/B) of the FAS-II cycle, MabA (FabG1) remains the only one for which specific inhibitors have not been reported yet. The development of a new LC-MS/MS based enzymatic assay allowed the screening of a 1280 fragment-library and led to the discovery of the first small molecules that inhibit MabA activity. A fragment from the anthranilic acid series was optimized into more potent inhibitors and their binding to MabA was confirmed by 19F ligand-observed NMR experiments., info:eu-repo/semantics/published

Published
2020

7. Reversion of antibiotic resistance in Mycobacterium tuberculosis by spiroisoxazoline SMARt-420

Author

Alexandre Wohlkonig, David Hot, Sebastien Gagneux, Marc Gitzinger, Julia Feldmann, Mireia Coscolla, Marion Flipo, Nicolas Willand, Martin Moune, Kamel Djaout, Mehdi Kiass, Priscille Brodin, Vincent Delorme, Vanessa Mathys, Rosangela Frita, Karine Soetaert, Benoit Deprez, Ludovic Huot, Nicolas Blondiaux, René Wintjens, Christian Kemmer, Alain R. Baulard, Vincent Trebosc, Camille Locht, Matthieu Desroses, Rudy Antoine, Willand, Nicolas, Appel à projets générique - Reprogrammation de la bioactivation des thioamides comme alternative antituberculeuse - - Tea-4-Two2014 - ANR-14-CE14-0027 - Appel à projets générique - VALID, Equipements d'excellence - Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution - - Imaginex BioMed2010 - ANR-10-EQPX-0004 - EQPX - VALID, EGID Diabetes Pole - - EGID2010 - ANR-10-LABX-0046 - LABX - VALID, Plate forme Lilloise de séquençage du génome humain pour une médecine personnalisée - - LIGAN PM2010 - ANR-10-EQPX-0007 - EQPX - VALID, A Chemical Genomics Approach of Intracellular Mycobacterium tuberculosis Towards Defining Specific Host Pathogen Interactions - INTRACELLTB - - EC:FP7:ERC2010-12-01 - 2015-11-30 - 260901 - VALID, Compensatory Evolution and Epistasis in Multidrug-resistant Mycobacterium tuberculosis - EVODRTB - - EC:FP7:ERC2013-04-01 - 2018-03-31 - 309540 - VALID, More Medicines for Tuberculosis - MM4TB - - EC:FP7:HEALTH2011-02-01 - 2016-01-31 - 260872 - VALID, Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), Médicaments et molécules pour agir sur les Systèmes Vivants - U 1177 (M2SV), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille, Karolinska Institutet [Stockholm], Institut Scientifique de Santé Publique [Belgique] - Scientific Institute of Public Health [Belgium] (WIV-ISP), Réseau International des Instituts Pasteur (RIIP), Tuberculosis Lab [Korea], Institut Pasteur Korea - Institut Pasteur de Corée, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Bioversys AG [Basel], University of Basel (Unibas), Université libre de Bruxelles (ULB), VIB-VUB Center for Structural Biology [Bruxelles], VIB [Belgium], Vrije Universiteit Brussel [Bruxelles] (VUB), Swiss Tropical and Public Health Institute [Basel], This work was supported by l’Agence Nationale de la Recherche (ANR), France (Tea-4-Two, ANR-14-CE14-0027-01) (ANR-10-EQPX-04-01), by EU grants ERC-STG INTRACELLTB no 260901, the Feder (12001407 (D-AL), PRIM (NewBio4Tb), European Research Council (grant 309540-EVODRTB), SystemsX.ch, Institut National de la Santé et de la Recherche Médicale, Université de Lille, Institut Pasteur de Lille, Centre National de la Recherche Scientifique, the Région Hauts-de-France (convention no. 12000080), and Société d’Accélération du Transfert de Technologie Nord. R.W. is Research Associate at the National Fund for Scientific Research (FNRS-FRS) (Belgium). M.M. was supported by PRIM (NewBio4Tb), V.D. was supported by EU grant 260872, and V.T. was suppported by the Marie Curie Initial Training Network (ITN-2013-607694-Translocation). The authors also thank the Unité Mixte de Recherche UMR 8199, Lille Integrated Genomics Network for Advanced Personalized Medicine (LIGAN-PM) Genomics platform (Lille, France), which belongs to the Federation de Recherche 3508 Labex EGID (European Genomics Institute for Diabetes, ANR-10-LABX-46) and was supported by the Agence Nationale de la Recherche (ANR) Equipex 2010 session (ANR-10-EQPX-07-01, LIGAN-PM). The LIGAN-PM Genomics platform is supported by the Fonds Européen de Développement Régional and the Region Hauts-de-France., ANR-14-CE14-0027,Tea-4-Two,Reprogrammation de la bioactivation des thioamides comme alternative antituberculeuse(2014), ANR-10-EQPX-0004,Imaginex BioMed,Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution(2010), ANR-10-LABX-0046,EGID,EGID Diabetes Pole(2010), ANR-10-EQPX-0007,LIGAN PM,Plate forme Lilloise de séquençage du génome humain pour une médecine personnalisée(2010), European Project: 260901,EC:FP7:ERC,ERC-2010-StG_20091118,INTRACELLTB(2010), European Project: 309540,EC:FP7:ERC,ERC-2012-StG_20111109,EVODRTB(2013), European Project: 260872,EC:FP7:HEALTH,FP7-HEALTH-2010-single-stage,MM4TB(2011), and Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille

Subjects
0301 basic medicine, Tuberculosis, medicine.drug_class, [CHIM.THER] Chemical Sciences/Medicinal Chemistry, 030106 microbiology, Antibiotics, Reversion, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, Microbiology, Mycobacterium tuberculosis, 03 medical and health sciences, Antibiotic resistance, [CHIM] Chemical Sciences, medicine, [CHIM]Chemical Sciences, chemistry.chemical_classification, Multidisciplinary, biology, medicine.disease, biology.organism_classification, 3. Good health, 030104 developmental biology, Enzyme, chemistry, Ethionamide, Bacteria, medicine.drug
Abstract

Countering TB prodrug resistance The arsenal of antibiotics for treating tuberculosis (TB) contains many prodrugs, such as ethionamide, which need activation by normal metabolism to release their toxic effects. Ethionamide is potentiated by small molecules. Blondiaux et al. screened for more potent analogs and identified a lead compound called SMARt-420. This small molecule inactivates a TetR-like repressor, EthR2, and boosts ethionamide activation. SMARt-420 successfully promoted clearance of multidrug-resistant strains of Mycobacterium tuberculosis from the lungs of mice. Science , this issue p. 1206

Published
2017

8. High-Throughput Sequencing Reveals Circular Substrates for an Archaeal RNA ligase

Author

Kamel Djaout, Mireille Regnier, Hubert F. Becker, Hannu Myllykallio, Alice Héliou, Roxane Lestini, Sorbonne Université (SU), Laboratoire d'Optique et Biosciences (LOB), École polytechnique (X)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Algorithms and Models for Integrative Biology (AMIB ), Laboratoire d'informatique de l'École polytechnique [Palaiseau] (LIX), École polytechnique (X)-Centre National de la Recherche Scientifique (CNRS)-École polytechnique (X)-Centre National de la Recherche Scientifique (CNRS)-Laboratoire de Recherche en Informatique (LRI), Université Paris-Sud - Paris 11 (UP11)-CentraleSupélec-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-CentraleSupélec-Centre National de la Recherche Scientifique (CNRS)-Inria Saclay - Ile de France, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria), École polytechnique (X)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-École polytechnique (X), Centre National de la Recherche Scientifique (CNRS)-École polytechnique (X)-Centre National de la Recherche Scientifique (CNRS)-École polytechnique (X)-Laboratoire de Recherche en Informatique (LRI), and Centre National de la Recherche Scientifique (CNRS)-École polytechnique (X)

Subjects
0301 basic medicine, Pyrococcus abyssi, Archaeal Proteins, RNA Stability, [SDV]Life Sciences [q-bio], RNA-dependent RNA polymerase, RNA, Archaeal, Substrate Specificity, Computational biology, 03 medical and health sciences, RNA Ligase (ATP), Genome, Archaeal, Circular RNA, Immunoprecipitation, RNA-Seq, Molecular Biology, Ligase ribozyme, ComputingMilieux_MISCELLANEOUS, RNA ligase, Genetics, biology, Sequence Analysis, RNA, RNA, Ribosomal, 5S, RNA, RNA, Circular, Cell Biology, Non-coding RNA, biology.organism_classification, Research Papers, Archaea, Alternative Splicing, 030104 developmental biology, Biochemistry
Abstract

It is only recently that the abundant presence of circular RNAs (circRNAs) in all kingdoms of Life, including the hyperthermophilic archaeon Pyrococcus abyssi, has emerged. This led us to investigate the physiologic significance of a previously observed weak intramolecular ligation activity of Pab1020 RNA ligase. Here we demonstrate that this enzyme, despite sharing significant sequence similarity with DNA ligases, is indeed an RNA-specific polynucleotide ligase efficiently acting on physiologically significant substrates. Using a combination of RNA immunoprecipitation assays and RNA-seq, our genome-wide studies revealed 133 individual circRNA loci in P. abyssi. The large majority of these loci interacted with Pab1020 in cells and circularization of selected C/D Box and 5S rRNA transcripts was confirmed biochemically. Altogether these studies revealed that Pab1020 is required for RNA circularization. Our results further suggest the functional speciation of an ancestral NTase domain and/or DNA ligase toward RNA ligase activity and prompt for further characterization of the widespread functions of circular RNAs in prokaryotes. Detailed insight into the cellular substrates of Pab1020 may facilitate the development of new biotechnological applications e.g. in ligation of preadenylated adaptors to RNA molecules.

Published
2017

9. Reversion of antibiotic resistance in

Author

Nicolas, Blondiaux, Martin, Moune, Matthieu, Desroses, Rosangela, Frita, Marion, Flipo, Vanessa, Mathys, Karine, Soetaert, Mehdi, Kiass, Vincent, Delorme, Kamel, Djaout, Vincent, Trebosc, Christian, Kemmer, René, Wintjens, Alexandre, Wohlkönig, Rudy, Antoine, Ludovic, Huot, David, Hot, Mireia, Coscolla, Julia, Feldmann, Sebastien, Gagneux, Camille, Locht, Priscille, Brodin, Marc, Gitzinger, Benoit, Déprez, Nicolas, Willand, and Alain R, Baulard

Subjects
Oxadiazoles, Extensively Drug-Resistant Tuberculosis, Antitubercular Agents, DNA, Isoxazoles, Mycobacterium tuberculosis, Repressor Proteins, Mice, Piperidines, Drug Resistance, Multiple, Bacterial, Mutation, Animals, Humans, Spiro Compounds, Ethionamide, Protein Binding
Abstract

Antibiotic resistance is one of the biggest threats to human health globally. Alarmingly, multidrug-resistant and extensively drug-resistant

Published
2016

10. Predictive modeling targets thymidylate synthase ThyX in Mycobacterium tuberculosis

Author

Peter B. Madrid, Kamel Djaout, Victoria Katawera, Hannu Myllykallio, Natassja G. Bush, Hubert F. Becker, Sean Ekins, Stephen J. Hearnshaw, Vinayak Singh, Valerie Mizrahi, Yap Boum, Anthony Maxwell, Pauline Bourbon, Jennifer E. Pritchard, Laboratoire d'Optique et Biosciences (LOB), École polytechnique (X)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institute of Infectious Diseases and Molecular Medicine and Division of Medical Microbiology, University of Cape Town, Epicentre Ouganda [Mbarara] [Médecins Sans Frontières], Epicentre [Paris] [Médecins Sans Frontières], Microbiology Department [Mbarara], Mbarara University of Science and Technology [Mbarara] (MUST), Université Pierre et Marie Curie - Paris 6 (UPMC), John Innes Centre [Norwich], Biotechnology and Biological Sciences Research Council (BBSRC), Norwich Bioincubator, Norwich Research Park, Birkbeck College [University of London], SRI International [Menlo Park] (SRI), Collaborative Drug Discovery, Collaborations in Chemistry, and HAL-UPMC, Gestionnaire

Subjects
0301 basic medicine, Models, Molecular, Tuberculosis, Ubiquinone, [CHIM.THER] Chemical Sciences/Medicinal Chemistry, 030106 microbiology, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, Biology, DNA gyrase, Thymidylate synthase, [SDV.MHEP.PSR]Life Sciences [q-bio]/Human health and pathology/Pulmonology and respiratory tract, Article, Microbiology, Mycobacterium tuberculosis, Machine Learning, 03 medical and health sciences, User-Computer Interface, Bacterial Proteins, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, medicine, [CHIM.CHEM] Chemical Sciences/Cheminformatics, Enzyme Inhibitors, chemistry.chemical_classification, Multidisciplinary, Topoisomerase, Bayes Theorem, Thymidylate Synthase, medicine.disease, biology.organism_classification, Virology, 3. Good health, 030104 developmental biology, Enzyme, chemistry, Infectious disease (medical specialty), DNA Gyrase, biology.protein, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, [SDV.MHEP.PSR] Life Sciences [q-bio]/Human health and pathology/Pulmonology and respiratory tract, Bacteria, [CHIM.CHEM]Chemical Sciences/Cheminformatics, Naphthoquinones
Abstract

There is an urgent need to identify new treatments for tuberculosis (TB), a major infectious disease caused by Mycobacterium tuberculosis (Mtb), which results in 1.5 million deaths each year. We have targeted two essential enzymes in this organism that are promising for antibacterial therapy and reported to be inhibited by naphthoquinones. ThyX is an essential thymidylate synthase that is mechanistically and structurally unrelated to the human enzyme. DNA gyrase is a DNA topoisomerase present in bacteria and plants but not animals. The current study set out to understand the structure-activity relationships of these targets in Mtb using a combination of cheminformatics and in vitro screening. Here, we report the identification of new Mtb ThyX inhibitors, 2-chloro-3-(4-methanesulfonylpiperazin-1-yl)-1,4-dihydronaphthalene-1,4-dione) and idebenone, which show modest whole-cell activity and appear to act, at least in part, by targeting ThyX in Mtb.

Published
2016

11. Substrate interaction dynamics and oxygen control in the active site of thymidylate synthase ThyX

Author

Marten H. Vos, Hubert F. Becker, Hannu Myllykallio, Ursula Liebl, Delphine Schaming, Isabelle Lamarre, Jonathan E. Ulmer, Véronique Balland, Kamel Djaout, Université Pierre et Marie Curie - Paris 6 (UPMC), Laboratoire d'optique et biosciences (LOB), École polytechnique (X)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Electrochimie Moléculaire (LEM (UMR_7591)), Université Paris Diderot - Paris 7 (UPD7)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), and Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Oxidative phosphorylation, Molecular Dynamics Simulation, Reductase, 010402 general chemistry, 01 natural sciences, Biochemistry, Substrate Specificity, 03 medical and health sciences, Oxygen Consumption, Bacterial Proteins, Catalytic Domain, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, 030304 developmental biology, 0303 health sciences, Oxidase test, Binding Sites, NADPH oxidase, DNA synthesis, biology, Active site, Thymidylate Synthase, Cell Biology, biology.organism_classification, 0104 chemical sciences, Chlorella, Paramecium bursaria, biology.protein, Cattle, Protein Binding
Abstract

Thymidylate synthase ThyX, required for DNA synthesis in many pathogenic bacteria, is considered a promising antimicrobial target. It binds FAD and three substrates, producing dTMP (2′-deoxythymidine-5′-monophosphate) from dUMP (2′-deoxyuridine-5′-monophosphate). However, ThyX proteins also act as NADPH oxidase by reacting directly with O2. In the present study we investigated the dynamic interplay between the substrates and their role in competing with this wasteful and potentially harmful oxidase reaction in catalytically efficient ThyX from Paramecium bursaria Chlorella virus-1. dUMP binding accelerates the O2-insensitive half-reaction between NADPH and FAD by over four orders of magnitude to ~30 s−1. Thus, although dUMP does not have a direct role in FAD reduction, any turnover with molecular O2 requires its presence. Inversely, NADPH accommodation accelerates dUMP binding ~3-fold and apparently precedes dUMP binding under physiological conditions. In the oxidative half-reaction, excess CH2H4folate (N5,N10-methylene-5,6,7,8-tetrahydrofolate) was found to re-oxidize FADH2 within 1 ms, thus very efficiently competing with FADH2 oxidation by O2 (1.5 s−1 under aerobic conditions). The resulting reaction scheme points out how the interplay between the fast reactions with the native substrates, although not rate-limiting for overall catalysis, avoids NADPH oxidase activity in aerobic micro-organisms, including many pathogens. These observations also explain why ThyX proteins are also present in aerobic micro-organisms. Abbreviations: CH2H4folate, N5,N10-methylene-5,6,7,8-tetrahydrofolate; dTMP, 2′-deoxythymidine-5′-monophosphate; dUMP, 2′-deoxyuridine-5′-monophosphate; MetF, methylenetetrahyrofolate reductase; NHE, normal hydrogen electrode; PBCV-1, Paramecium bursaria Chlorella virus-1

Published
2014

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