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.
To know more about these projects, see below.
- Guillon R., Rahimova R., Preeti, Egron D., Rouanet S., Dumontet C., Aghajari N., Jordheim L.P., **Chaloin L. and Peyrottes S., “Lead optimization and biological evaluation of fragment-based cN-II inhibitors” J. Med. Chem. (2019), 168, 28-44 ** co-corresponding authors
- Ghoteimi R., Nguyen V.T., Rahimova R., Grosjean F., Cros-Perrial E., Uttaro J-P., Mathé C., Chaloin L., Jordheim L.P., Peyrottes S. “Synthesis of Substituted 5'-Aminoadenosine Derivatives and Evaluation of Their Inhibitory Potential toward CD73.” ChemMedChem. (2019), 14, 1431-1443. doi: 10.1002/cmdc.201900348.
- Fernandez J., Machado A., Lyonnais S., Chamontin C., Gärtner K., Léger T., Henriquet C., Garcia C., Portilho D.M., Pugnière M., Chaloin L., Muriaux D., Yamauchi Y., Blaise M., Nisole S., Arhel N.J. “Transportin-1 binds to the HIV-1 capsid via a nuclear localization signal and triggers uncoating” Nature Microbiology (2019), 10.1038/s41564-019-0575-6
- Sudalaiyadum Perumal A, Vishwakarma RK, Hu Y, Morichaud Z, Brodolin K. "RbpA relaxes promoter selectivity of M. tuberculosis RNA polymerase." Nucleic Acids Res. (2018) 46(19):10106-10118. doi: 10.1093/nar/gky714.
- 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
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)
- 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
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
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
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
- 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.
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. (2016) 428, 463-76. Pubmed
Duchi D, Bauer DL, Fernandez L, Evans G, Robb N, Hwang LC, Gryte K, Tomescu A, Zawadzki P, Morichaud Z, Brodolin K, Kapanidis AN. "RNA Polymerase Pausing during Initial Transcription". Mol Cell. (2016) - 63(6):939-50. Pubmed
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
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
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), 12, 1476-1486. Pubmed
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. (2015) 58, 9680-96. Pubmed
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
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
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
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
- 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)
- Bianca SCLAVI, (LBPA, ENS Cachan, Cachan)
- Alain BAULARD (Institut Pasteur, Lille)
- Lars Peter Jordheim & Charles Dumontet (UCBL - Lyon)
- Christine Ménétrier-Caux (CRCL- Lyon)
- 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
- Konstantin Brodolin – DR2 INSERM, HDR.
- Mickael Blaise - DR2 CNRS, HDR
- Laurent Chaloin – DR2 CNRS, HDR
- Jean-Paul Leonetti - DR2 CNRS
- Zakia Morichaud - IE CNRS
- Ondine Duverger - PhD student
- Couston Julie - PhD student
- Marty Laetitia - PhD student
They have spent some time working with us:
Husam Alsarraf - Post-doctorate researcher
Zara Msoili - Master 2 ISDD - Paris Diderot
Maria Shaldaeva - Master 1 Biotin Student
Noémie Laurent - Master 2 student
Carla Heredia (Master 2)
Charly Patrat (Master 1 Biotin)
Christopher Chevillard (AI - CNRS)
Florent MIR (Master 2 - Bio-Santé)
Kevin Gawron (Master 2 - IMHE)
Pierre Guéracher (Master 1 Bioinfo - Rennes)
Ouerdia Maskri (Post-doctorate)
Rahila Rahimova (PhD student)
Corinne Lionne (Researcher)
Elise Kaplan (PhD student)
Ayyappasamy SUDALAIYADUM PERUMAL
Sabine Lefévère (Licence Pro.)
Zsuzsana Marton (Post-doctorate)
The Team in 2016-2017:
The Team in 2015:
We recently celebrated the departure of our favorite post-doctorate Ouerdia Maskri who has already spent two years in the team. Her work was focused on MTB transcription/regulation and we wish her a lot of successes for her scientific career.
We welcome a new master 1 student in bioinformatics, Pierre Guéracher who will contribute to the Drug discovery program on nucleotidases.
An outdoor escape at "Ravin des Arcs" near St Martin de Londres
We are pleased to welcome two new Master 2 students, Kévin Gawron and Florent Mir.
A single-molecule analysis reveals the mechanism of transcription activation in Mycobacterium tuberculosis (Vishwakarma et al, 2018) - published by Konstantin BRODOLIN publishes in Science Advances (IF=11.5):
April 2018A new post-doctorante is arrived to reinforce the team, Abdennour Braka.
Konstantin BrodolinDR2 INSERM, HDR
En savoir +
at a glance
Model organism(s)Escherichia coli, Mycobacterium tuberculosis
- structural biophysics
- drug design