Bacterial enzymes and antibiotic resistance

Projects

Bacterial RNA polymerase and antibiotic resistance (K. Brodolin)

The DNA-dependent-RNA polymerase (RNAP) is the central enzyme of gene expression and a target for genetic regulation. Its basic structure-functional features are highly conserved among all living organisms. Bacterial RNAP is a target for a large number of regulatory proteins and small regulatory molecules. Among the antibacterial molecules inhibiting RNAP, rifampicin (Rif) remains a first-line antibiotic for treatment of tuberculosis. Recently a new class of antibiotics: lipiarmycin (Fidaxomicin, Tiacumicin B) and myxopyronin (Myx) has been characterized. The mechanism of action of these molecules remains to be elucidated. Bacteria have developed multiple mechanisms to escape from the effects of antibiotic treatment. In addition to antibiotic resistance resulting from the emergence of mutations, there are also adaptive mechanisms, such as activation of stress-response pathways and switching to the persistent (dormant) state. These adaptive mechanisms are tightly linked to the regulation of RNAP activity. An increasing amount of evidence indicates that the σ subunit of RNAP as well as other transcription factors (e.g. RbpA from M.tuberculosis) are implicated in the generation of resistance in response to the antibiotic treatment. The global objectives of our studies are (1) to understand a role of the interplay between σ subunit and the RNAP-binding factors in the modulation of gene expression; (2) to decipher mechanisms of action of the antibiotics targeting RNAP and (3) to understand how bacteria resist to these molecules. Our project is focused on the mechanism of transcription regulation in two most widespread human pathogens: Escherichia coli, which is considered as a prototype for bacterial pathogens, and Mycobacterium tuberculosis that is of great clinical importance. We also explore the role of the σ subunit in the elementary steps of gene expression such as promoter recognition, RNA synthesis and transcriptional pausing. The results of our study help to understand the molecular basis of antibiotic resistance and may provide a basis for the development of new, more efficient drugs in pharmacology.


Development and lead optimization of 5'-nucleotidase inhibitors (L. Chaloin)

The main objective is the discovery and the structural optimization of new allosteric inhibitors targeting selectively two important enzymes involved in nucleotide metabolism, the 5’-nucleotidases cN-II and CD73. Indeed, a large amount of scientific work within the field of oncology has established and confirmed their role in cancer progression or in drug resistance. Indeed, cN-II affects the phosphorylation level and the pharmacological activity of intracellular cytotoxic nucleoside analogs (used as anticancer drugs). As for CD73, by dephosphorylating extracellular AMP into adenosine, its activity has been shown to favor the blockade of the antitumor immune response and to protect cancer cells against immunosurveillance. These two enzymes are now proven targets in oncology. Indeed, the development of drug-resistant clones in acute lymphoblastic leukemia (ALL) has been attributed to high cN-II activity and the inhibition of cN-II activity restores the sensitivity to these drugs. Also, through inhibition of antitumor immunity, CD73 was reported to promote tumor growth, cancer cell invasion and metastasis in many cancer types such as breast and ovarian cancers. Our project has several objectives i) the improvement of inhibitors already identified for cN-II and ii) the development of new selective inhibitors targeting CD73 by using an original approach based on the development of allosteric inhibitors. This step includes the virtual screening of chemical libraries and also molecular dynamics of the enzyme (see figure below). These compounds will block the enzyme in an inactive conformation independently of the presence of natural ligand with high selectivity, thereby limiting the off-target effects of the future drug. Allosteric inhibitors will be evaluated using relevant and ready to use in vitro assays (recombinant enzymes and model cell lines for cN-II and CD73) to guide their structural optimization.
Virtual screening carried out on human ecto-5'-nucleotidase (CD73) in order to block its enzymatic activity and restore the anticancer immune response (Glu543 residues are shown in CPK model and the allosteric inhibiteur in green sticks)

2018

  1. Vishwakarma RK, Cao AM, Morichaud Z, Perumal AS, Margeat E, Brodolin K. "Single-molecule analysis reveals the mechanism of transcription activation in M. tuberculosis." Sci Adv. (2018) 4 (5):eaao5498. doi: 10.1126/sciadv.aao5498. Pubmed
  2. Rahimova R., Fontanel S., Lionne C., Jordheim L.P., Peyrottes S. and Chaloin L. “Identification of allosteric inhibitors of the ecto-5’-nucleotidase (CD73) targeting the dimer interface”. PLoS Comp. Biol. (2018), doi 10.1371/journal.pcbi.1005943. Pubmed (Free PMC article)

  3. Dulin D, Bauer DLV, Malinen AM, Bakermans JJW, Kaller M, Morichaud Z, Petushkov I, Depken M, Brodolin K, Kulbachinskiy A, Kapanidis AN. "Pausing controls branching between productive and non-productive pathways during initial transcription in bacteria." Nat Commun. (2018) 9 (1):1478. doi: 10.1038/s41467-018-03902-9. PubMed

2017

  1. Duchi D, Gryte K, Robb NC, Morichaud Z, Sheppard C, Brodolin K, Wigneshweraraj S, Kapanidis AN. "Conformational heterogeneity and bubble dynamics in single bacterial transcription initiation complexes." Nucleic Acids Res. (2017) Nov 21. doi: 10.1093/nar/gkx1146. Pubmed

  2. Leban, N., Kaplan E., Chaloin L., Godreuil S. and Lionne C. “Kinetic characterization and molecular docking of novel allosteric inhibitors of aminoglycoside phosphotransferases” Biochim Biophys Acta. (2017)1861, 3464-3473. Pubmed

  3. Gissot M., Hovasse A., Chaloin L., Schaeffer-Reiss C., Van Dorsselaer A. and Tomavo S. “An evolutionary conserved zinc finger protein is involved in Toxoplasma gondii mRNA nuclear export.” Cell. Microbiol. (2017) doi: 10.1111/cmi.12644. Pubmed

  4. Baud A, Aymé L, Gonnet F, Salard I, Gohon Y, Jolivet P, Brodolin K, Da Silva P, Giuliani A, Sclavi B, Chardot T, Mercère P, Roblin P, Daniel R. "SOLEIL shining on the solution-state structure of biomacromolecules by synchrotron X-ray footprinting at the Metrology beamline." J Synchrotron Radiat. (2017) 24(Pt 3):576-585. doi: 10.1107/S1600577517002478. 


2016

  1. Morichaud Z.Chaloin L. and Brodolin K. “Regions 1.2 and 3.2 of the RNA polymerase subunit promote DNA melting and attenuate action of the antibiotic lipiarmycin.” J. Mol. Biol. (2016428, 463-76. Pubmed

  2. Duchi D, Bauer DL, Fernandez L, Evans G, Robb N, Hwang LC, Gryte K, Tomescu A, Zawadzki P, Morichaud ZBrodolin K, Kapanidis AN. "RNA Polymerase Pausing during Initial Transcription". Mol Cell. (2016) - 63(6):939-50. Pubmed

  3. Nasri A., Valverde A., Roche D.B., Desrumaux C., Clair P., Beyrem H., Chaloin L., Ghysen A. and Perrier V. “Neurotoxicity of a Biopesticide Analog on Zebrafish Larvae at Nanomolar Concentrations” Int. J. of Mol. Sci., (2016)17 (12). Pubmed

  4. Kaplan E., Guichou J.F., Chaloin L., Kunzelmann S., Leban N., Serpersu E.H. and Lionne C. “Aminoglycoside binding and catalysis specificity of aminoglycoside 2''-phosphotransferase IVa: a thermodynamic, structural and kinetic study” Biochim Biophys Acta. (2016) 1860, 802-813. Pubmed

  5. Nguyen V.T., Hospital, A., Lionne C., Jordheim L.P., Dumontet C., Périgaud C., Chaloin L. and Peyrottes S. “Beta-hydroxyphosphonate ribonucleoside analogues derived from 4-substituted-1,2,3-triazoles as IMP/GMP mimics: synthesis and biological evaluation » Beilstein J. Org. (2016)121476-1486. Pubmed


2015

  1. Marton Z., Guillon R., Krimm I., Preeti, Rahimova R., Egron D., Jordheim L.P., Aghajari N.,  Dumontet C., Périgaud C., Lionne C., Peyrottes S. and Chaloin L. “Identification of non-competitive inhibitors of cytosolic 5'-nucleotidase II using a fragment-based approach.” J. Med Chem. (201558, 9680-96. Pubmed

  2. Borel S., Robert-Hebmann V., Alfaisal J., Jain A., Faure M., Espert L., Chaloin L., Paillart J.-C., Johansen T. and Biard-Piechaczyk M. “HIV-1 Vif interacts with LC3 and inhibits autophagy” AIDS (2015) 29, 275-286. Pubmed

  3. Cividini F., Pesi R., Chaloin L., Allegrini S., Camici M., Cros-Perrial E., Dumontet C., Jordheim L.P., Tozzi M.G. “The purine analog fludarabine acts as a cytosolic 5'-nucleotidase II inhibitor” Biochem Pharmacol. (2015) 94, 63-68. Pubmed

  4. Briolotti P., Chaloin L., Balaguer P., Da Silva F., Tománková V., Pascussi J.-M., Duret C., Fabre J.-M., Ramos J., Klieber S., Maurel P., Daujat-Chavanieu M., Gerbal-Chaloin S. “Analysis of glycogen synthase kinase inhibitors that regulate cytochrome P450 expression in primary human hepatocytes by activation of β-catenin, aryl hydrocarbon receptor and pregnane X receptor signaling” Toxicol Sci. (2015) 148, 261-75. Pubmed

  5. Ramya L., Gautham N., Chaloin L., Kajava A.V. “Restricted mobility of side chains on concave surfaces of solenoid proteins may impart heightened potential for intermolecular interactions.” Proteins (2015) 83, 1654-64. Pubmed

Local

  • Emmanuel MARGEAT (CBS, Montpellier)
  • Patrick BRON (CBS, Montpellier)
  • Suzanne PEYROTTES (IBMM, Montpellier)
  • Jean-Marie PELOPONESE (IRIM, Montpellier)
  • Bruno BEAUMELLE (IRIM, Montpellier)
  • D. MURIAUX & C. FAVARD (IRIM, Montpellier)
  • Sébastien GRANIER (IGF, Montpellier)
  • Andrey KAJAVA (CRBM, Montpellier)

National

  • Bianca SCLAVI, (LBPA, ENS Cachan, Cachan)
  • Alain BAULARD (Institut Pasteur, Lille)
  • Lars Peter Jordheim & Charles Dumontet (UCBL - Lyon)
  • Christine Ménétrier-Caux (CRCL- Lyon)

International

  • Andrey KULBACHINSKII, (Institute of Molecular Genetics, Russia)
  • Yangbo HU (Whuan Institute of Virology, China)
  • Achillefs KAPANIDIS (University of Oxford, UK)
  • Vladimir KATANAEV (University of Lausanne, CH)

Current team members

 

They have spent some time working with us:

Rahila Rahimova
Corinne Lionne
Elise Kaplan
Rishi VISHWAKARMA
Ayyappasamy SUDALAIYADUM PERUMAL
Sabine Lefévère
Zsuzsana Marton ....

The Team in 2016-2017:


The Team in 2015:



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Team leader

Konstantin Brodolin

CR1 INSERM, HDR
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at a glance

Model organism(s)

Escherichia coli, Mycobacterium tuberculosis

Biological process

transcription

Methods

  • biochemistry
  • structural biophysics
  • bacteriology

Medical applications

  • drug design
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Grant support

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IRIM
Institut de Recherche en Infectiologie de Montpellier
UMR 9004 - CNRS / UM
1919 route de Mende - 34293 Montpellier cedex 5
FRANCE

 

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