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1. Novel Antiviral Molecules against Ebola Virus Infection

Author

Mila Collados Rodríguez, Patrick Maillard, Alexandra Journeaux, Anastassia V. Komarova, Valérie Najburg, Raul-Yusef Sanchez David, Olivier Helynck, Mingzhe Guo, Jin Zhong, Sylvain Baize, Frédéric Tangy, Yves Jacob, Hélène Munier-Lehmann, and Eliane F. Meurs

Subjects
Ebola virus, measles virus, VP35, PKR, PACT, RIG-I, Biology (General), QH301-705.5, Chemistry, QD1-999
Abstract

Infection with Ebola virus (EBOV) is responsible for hemorrhagic fever in humans with a high mortality rate. Combined efforts of prevention and therapeutic intervention are required to tackle highly variable RNA viruses, whose infections often lead to outbreaks. Here, we have screened the 2P2I3D chemical library using a nanoluciferase-based protein complementation assay (NPCA) and isolated two compounds that disrupt the interaction of the EBOV protein fragment VP35IID with the N-terminus of the dsRNA-binding proteins PKR and PACT, involved in IFN response and/or intrinsic immunity, respectively. The two compounds inhibited EBOV infection in cell culture as well as infection by measles virus (MV) independently of IFN induction. Consequently, we propose that the compounds are antiviral by restoring intrinsic immunity driven by PACT. Given that PACT is highly conserved across mammals, our data support further testing of the compounds in other species, as well as against other negative-sense RNA viruses.

Published
2023
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2. Prevalence of SARS-CoV-2 antibodies in France: results from nationwide serological surveillance

Author

Stéphane Le Vu, Gabrielle Jones, François Anna, Thierry Rose, Jean-Baptiste Richard, Sibylle Bernard-Stoecklin, Sophie Goyard, Caroline Demeret, Olivier Helynck, Nicolas Escriou, Marion Gransagne, Stéphane Petres, Corinne Robin, Virgile Monnet, Louise Perrin de Facci, Marie-Noelle Ungeheuer, Lucie Léon, Yvonnick Guillois, Laurent Filleul, Pierre Charneau, Daniel Lévy-Bruhl, Sylvie van der Werf, and Harold Noel

Subjects
Science
Abstract

The percentage of national populations infected during the first stages of the COVID-19 pandemic are unclear owing to limited early testing. Here the authors provide a nation-wide prevalence study of SARS-CoV-2 antibodies in France from the first wave of COVID-19 in 2020, including stratification based on age, sex and region.

Published
2021
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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. 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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5. 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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6. 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/OBJECTIVES: Human 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 FINDINGS: We 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/SIGNIFICANCE: Our 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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7. 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

8. Prevalence of SARS-CoV-2 antibodies in France: results from nationwide serological surveillance

Author

Louise Perrin de Facci, Virgile Monnet, Pierre Charneau, Caroline Demeret, Jean-Baptiste Richard, Nicolas Escriou, Daniel Lévy-Bruhl, Gabrielle Jones, Thierry Rose, François Anna, Yvonnick Guillois, Marion Gransagne, Corinne Robin, Sophie Goyard, Stéphane Petres, Stéphane Le Vu, Marie-Noëlle Ungeheuer, Lucie Léon, Laurent Filleul, Sylvie van der Werf, Sibylle Bernard-Stoecklin, Olivier Helynck, Harold Noel, Santé publique France - French National Public Health Agency [Saint-Maurice, France], Virologie Moléculaire et Vaccinologie / Molecular Virology and Vaccinology, Institut Pasteur [Paris], Laboratoire commun Pasteur-TheraVectys, Institut Pasteur [Paris]-TheraVectys, Biologie Cellulaire des Lymphocytes - Lymphocyte Cell Biology, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Génétique Moléculaire des Virus à ARN - Molecular Genetics of RNA Viruses (GMV-ARN (UMR_3569 / U-Pasteur_2)), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Chimie et Biocatalyse, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Laboratoire CERBA [Saint Ouen l'Aumône], Biomnis Sample Library (BSL), Eurofins Biomnis, Investigation Clinique et d’Accès aux Ressources Biologiques (Plate-forme) - Clinical Investigation and Access to BioResources (ICAReB), Centre National de Référence des virus des infections respiratoires (dont la grippe) - National Reference Center Virus Influenzae [Paris] (CNR), We thank Christine Larsen, Bruno Coignard and Jean-Claude Desenclos (Santé publiqueFrance) for their helpful contribution at setting up the study, from Institut Pasteur,Isabelle Cailleau for support in the funding process, Hélène Munier-Lehmann for accessto automate and supply management at the Unit of Chemistry and Biocatalysis, Yves L.Janin for providing the luciferase prosubstrate hikarazine 108, Philippe Souque forproduction of the lentiviral pseudo-types, the whole ICAReB team and COVID-19support staff for sample management at Institut Pasteur, Juliette Paireau (Institut Pas-teur, Santé publique France), Rodolphe Thiébaut (Bordeaux Université) and Xavier deLamballerie (Aix-Marseille Université) for valuable comments onfirst results. We alsothank the team from the Eurofins Biomnis Sample Library and from CerbaHealthcareBenedicte Roquebert (Laboratoire Cerba) and Marie Pierre Guerra (CerbaXpert) forcontributing to sample collection. Santé publique France provided funding to the NRCand to the two centralising biobanks to cover sample collection, preparation, transportand analysis costs. The funder had no role in analysis, interpretation of data or writing ofthe report. S.L.V., G.J. and H.N. had full access to all the data and had responsibility tosubmit for publication., Institut Pasteur [Paris] (IP), Institut Pasteur [Paris] (IP)-TheraVectys, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Centre National de Référence des virus des infections respiratoires (dont la grippe) - National Reference Center Virus Influenzae [Paris] (CNR - laboratoire coordonnateur), Lassailly-Bondaz, Anne, Santé publique France - French National Public Health Agency, Centre National de Référence des virus des infections respiratoires (dont la grippe) [Paris] (CNR), Santé publique France, Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris], and Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris]

Subjects
0301 basic medicine, Male, Epidemiology, [SDV]Life Sciences [q-bio], General Physics and Astronomy, Antibodies, Viral, Serology, 0302 clinical medicine, Seroepidemiologic Studies, Case fatality rate, Prevalence, Cumulative incidence, 030212 general & internal medicine, Child, Aged, 80 and over, 0303 health sciences, education.field_of_study, Multidisciplinary, biology, Transmission (medicine), Middle Aged, 3. Good health, [SDV] Life Sciences [q-bio], [SDV.IMM.IA]Life Sciences [q-bio]/Immunology/Adaptive immunology, Child, Preschool, Female, France, medicine.symptom, Antibody, Adult, medicine.medical_specialty, Adolescent, Science, Population, Asymptomatic, General Biochemistry, Genetics and Molecular Biology, Article, 03 medical and health sciences, Young Adult, medicine, Seroprevalence, Humans, education, Epidemics, 030304 developmental biology, Aged, business.industry, SARS-CoV-2, Public health, Infant, Newborn, COVID-19, Infant, General Chemistry, 030104 developmental biology, Viral infection, [SDV.SPEE] Life Sciences [q-bio]/Santé publique et épidémiologie, biology.protein, [SDV.SPEE]Life Sciences [q-bio]/Santé publique et épidémiologie, business, Demography
Abstract

Assessment of the cumulative incidence of SARS-CoV-2 infections is critical for monitoring the course and extent of the COVID-19 epidemic. Here, we report estimated seroprevalence in the French population and the proportion of infected individuals who developed neutralising antibodies at three points throughout the first epidemic wave. Testing 11,000 residual specimens for anti-SARS-CoV-2 IgG and neutralising antibodies, we find nationwide seroprevalence of 0.41% (95% CI: 0.05–0.88) mid-March, 4.14% (95% CI: 3.31–4.99) mid-April and 4.93% (95% CI: 4.02–5.89) mid-May 2020. Approximately 70% of seropositive individuals have detectable neutralising antibodies. Infection fatality rate is 0.84% (95% CI: 0.70–1.03) and increases exponentially with age. These results confirm that the nationwide lockdown substantially curbed transmission and that the vast majority of the French population remained susceptible to SARS-CoV-2 in May 2020. Our study shows the progression of the first epidemic wave and provides a framework to inform the ongoing public health response as viral transmission continues globally., The percentage of national populations infected during the first stages of the COVID-19 pandemic are unclear owing to limited early testing. Here the authors provide a nation-wide prevalence study of SARS-CoV-2 antibodies in France from the first wave of COVID-19 in 2020, including stratification based on age, sex and region.

Published
2021

9. High seroprevalence but short‐lived immune response to SARS‐CoV‐2 infection in Paris

Author

Pierre Charneau, Philippe Souque, Evelyne Dufour, Marion Gransagne, Stéphane Petres, Ana Ines Lalanne, Thierry Rose, Isabelle Turbiez, François Dejardin, François Anna, Odile Richard-Le Goff, Olivier Helynck, Maude Guillot-Delost, Sophie Goyard, Alexia Savignoni, François-Clément Bidard, Zaineb Choucha, Fabien Nevo, Franck Perez, Delphine Louis, Véronique Gillon, Yves L. Janin, A Gobillion, Nicolas Escriou, Olivier Lantz, Laboratoire commun Pasteur-TheraVectys, Institut Pasteur [Paris]-TheraVectys, Biologie Cellulaire des Lymphocytes - Lymphocyte Cell Biology, Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris], Institut Curie [Paris], Virologie Moléculaire et Vaccinologie / Molecular Virology and Vaccinology, Institut Pasteur [Paris], Laboratoire d’innovation : vaccins – Innovation lab : vaccines, Risques cliniques et sécurité en santé des femmes et en santé périnatale (RISCQ), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ), Immunité et cancer (U932), Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Plateforme technologique Production et purification de protéines recombinantes – Production and Purification of Recombinant Proteins Technological Platform (PPR), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Anticorps en thérapie et pathologie - Antibodies in Therapy and Pathology, Chimie et Biocatalyse, This work was made possible thanks to the financial support obtained through the URGENCE nouveau coronavirus fundraising campaign of Institut Pasteur and the financial support of the Fondation Total. This study was funded in part by a grant from Fondation de France and by Institut Curie Institutional funding. The luciferin synthesis development has been supported by DARRI (ValoExpress 2016-2018). LuLISA development has been supported by IARP Pasteur-Carnot MI (2019-2020)., Institut Pasteur [Paris] (IP)-TheraVectys, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP), Université Paris-Saclay, Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Lassailly-Bondaz, Anne, Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Curie [Paris], Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), and Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris]

Subjects
0301 basic medicine, Male, Time Factors, Antibodies, Viral, SARS‐CoV‐2, MESH: Antibodies, Neutralizing, Serology, 0302 clinical medicine, Seroepidemiologic Studies, Epidemiology, MESH: COVID-19, Immunology and Allergy, 030212 general & internal medicine, Research Articles, MESH: Immunoglobulin G, 0303 health sciences, education.field_of_study, MESH: Paris, 3. Good health, Titer, Research Article|Basic, ELISA, Female, medicine.symptom, Bioluminescence, Adult, MESH: Pandemics, medicine.medical_specialty, Paris, Population, Immunology, Anosmia, Immunity to infection, Biology, Asymptomatic, Virus, 03 medical and health sciences, COVID‐19, Internal medicine, medicine, Humans, Basic, education, Pandemics, 030304 developmental biology, LuLISA, MESH: Seroepidemiologic Studies, MESH: Humans, SARS-CoV-2, business.industry, MESH: Time Factors, COVID-19, MESH: Adult, Ageusia, Antibodies, Neutralizing, MESH: Male, 030104 developmental biology, Immunization, [SDV.SPEE] Life Sciences [q-bio]/Santé publique et épidémiologie, Immunoglobulin G, [SDV.SPEE]Life Sciences [q-bio]/Santé publique et épidémiologie, business, MESH: Female, MESH: Antibodies, Viral, 030215 immunology
Abstract

International audience; Background Although the COVID-19 pandemic peaked in March/April 2020 in France, the prevalence of infection is barely known. Herein, we assessed the serological response against the SARS-CoV-2 virus in a large population working in one institution of the Paris conurbation. We set up two high-throughput and sensitive methods to assess SARS CoV-2 Nucleoprotein and Spike protein-specific IgG response along with a pseudo-neutralization assay in sera. We studied 1847 participants who also answered a web-based survey on clinical symptoms.Methods and Results In May-July 2020, 11% (95% CI: 9.7-12.6) of serums were positive for IgG against the SARS-CoV-2 N and S protein and 9.5% (CI:8.2-11.0) were pseudo-neutralizer. The prevalence of immunization was 11.6% (CI:10.2-13.2) considering positivity in at least one assays. In 5% (CI:3.9-7.1) of RT-qPCR positive individuals, no systemic IgGs were detected. Among immune individuals, 21% had been asymptomatic.Anosmia and ageusia occurred in 52% of the IgG-positive individuals and in 3% of the negative ones. In contrast, 30% of the anosmia-ageusia cases were seronegative suggesting that the true prevalence of infection may reach 16.6%. In sera obtained 4-8 weeks after the first sampling anti-N and anti-S IgG titers and pseudo-neutralization activity declined by 31%, 17% and 53%, respectively with half-life of 35, 87 and 28 days, respectively.Conclusions The population studied being not particularly exposed to SARS-CoV-2 infection is representative of active workers in the Paris conurbation, suggesting that the current epidemiological models may underestimate the true prevalence of infection. The short lifespan of the serological systemic responses hinders retrospective assessment of the epidemic extent.

Published
2020
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10. Nitazoxanide Analogs Require Nitroreduction for Antimicrobial Activity in Mycobacterium smegmatis

Author

Gerald Larrouy-Maumus, Sandrine Favre-Rochex, Alessandro Cascioferro, Hélène Munier-Lehmann, Sonia Rebollo-Ramirez, Claire Beauvineau, Delphine Naud-Martin, Mena Cimino, Brigitte Gicquel, Maria Virginia Buchieri, Olivier Helynck, Génétique mycobactérienne - Mycobacterial genetics, Institut Pasteur [Paris], Imperial College London, Chimie, Modélisation et Imagerie pour la Biologie [Orsay], Université Paris-Sud - Paris 11 (UP11)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Pathogénomique mycobactérienne intégrée, Chimie et Biocatalyse, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), This work is part of the European Seventh Framework Program Nanotherapeutics against Resistant Emerging Bacterial Pathogens (NAREB Project 604237). This work was also supported by the 'Programme de Recherche sur les Nouvelles Molécules Intervenant dans la Lutte Contre la Tuberculose' of the Gabonese Republic, the Department of Life Sciences and the Faculty of Natural Sciences kick-start funding award from Imperial College London, U.K., and Agilent Technologies, U.K., concerning all LC–MS experiments performed on the Agilent 6545 QToF., We thank Yves Janin for fruitful discussions and technical support., European Project: 604237,EC:FP7:NMP,FP7-NMP-2013-LARGE-7,NAREB(2014), Institut Pasteur [Paris] (IP), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Mycobacterium smegmatis, 030106 microbiology, Down-Regulation, Mycobacterium Infections, Nontuberculous, MESH: Thiazoles, MESH: Down-Regulation, Mycobacterium aurum, Mycobacterium, Microbiology, 03 medical and health sciences, Nitroreductase, chemistry.chemical_compound, Hydroxylamine, MESH: Anti-Bacterial Agents, Drug Resistance, Bacterial, MESH: Drug Resistance, Bacterial, Drug Discovery, MESH: Mycobacterium, medicine, Humans, [CHIM]Chemical Sciences, MESH: Mycobacterium Infections, Gene, MESH: Mycobacterium smegmatis, Mycobacterium Infections, MESH: Humans, biology, Nitazoxanide, Nitroreductases, Nitro Compounds, MESH: Mycobacterium Infections, Nontuberculous, Antimicrobial, biology.organism_classification, Anti-Bacterial Agents, 3. Good health, Thiazoles, MESH: Nitroreductases, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, 030104 developmental biology, chemistry, Biochemistry, Molecular Medicine, Antibacterial activity, medicine.drug
Abstract

International audience; In this study, we aimed to decipher the natural resistance mechanisms of mycobacteria against novel compounds isolated by whole-cell-based high-throughput screening (HTS). We identified active compounds using Mycobacterium aurum. Further analyses were performed to determine the resistance mechanism of M. smegmatis against one hit, 3-bromo-N-(5-nitrothiazol-2-yl)-4-propoxybenzamide (3), which turned out to be an analog of the drug nitazoxanide (1). We found that the repression of the gene nfnB coding for the nitroreductase NfnB was responsible for the natural resistance of M. smegmatis against 3. The overexpression of nfnB resulted in sensitivity of M. smegmatis to 3. This compound must be metabolized into hydroxylamine intermediate for exhibiting antibacterial activity. Thus, we describe, for the first time, the activity of a mycobacterial nitroreductase against 1 analogs, highlighting the differences in the metabolism of nitro compounds among mycobacterial species and emphasizing the potential of nitro drugs as antibacterials in various bacterial species.

Published
2017

11. 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

12. 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

13. Original 2-(3-Alkoxy-1H-pyrazol-1-yl)pyrimidine Derivatives as Inhibitors of Human Dihydroorotate Dehydrogenase (DHODH)

Author

Marianne Lucas-Hourani, Gigliola Zanghi, Sandrine Guillou, Anne Noel, Pierre-Olivier Vidalain, Yves L. Janin, Frédéric Tangy, Olivier Helynck, Hélène Munier-Lehmann, Chimie et Biocatalyse, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Génomique Virale et Vaccination, Medicen Initiative (Chemical Library Project, Région Ile de France Grants I 06-222/R and I 09-1739/R) The Institut Carnot-Pasteur Maladies Infectieuses (Programme STING) Agence Nationale pour la Recherche (ANR-RPIB, Programme STING 2.0) The 2013 Leonardo da Vinci program, 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], and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)

Subjects
Oxidoreductases Acting on CH-CH Group Donors, Pyrimidine, Stereochemistry, Dihydroorotate Dehydrogenase, Antiviral Agents, Cofactor, law.invention, Measles virus, Structure-Activity Relationship, chemistry.chemical_compound, law, Drug Discovery, Humans, Enzyme Inhibitors, IC50, chemistry.chemical_classification, Coenzyme Q10, biology, [CHIM.ORGA]Chemical Sciences/Organic chemistry, biology.organism_classification, 3. Good health, Pyrimidines, Enzyme, chemistry, Biochemistry, Recombinant DNA, biology.protein, Dihydroorotate dehydrogenase, Molecular Medicine
Abstract

International audience; From a research program aimed at the design of new chemical entities followed by extensive screening on various models of infectious diseases, an original series of 2-(3-alkoxy-1H-pyrazol-1-yl)pyrimidines endowed with notable antiviral properties were found. Using a whole cell measles virus replication assay, we describe here some aspects of the iterative process that, from 2-(4-benzyl-3-ethoxy-5-methyl-1H-pyrazol-1-yl)pyrimidine, led to 2-(4-(2,6-difluorophenoxy)-3-isopropoxy-5-methyl-1H-pyrazol-1-yl)-5-ethylpyrimidine and a 4000-fold improvement of antiviral activity with a subnanomolar level of inhibition. Moreover, recent precedents in the literature describing antiviral derivatives acting at the level of the de novo pyrimidine biosynthetic pathway led us to determine that the mode of action of this series is based on the inhibition of the cellular dihydroorotate dehydrogenase (DHODH), the fourth enzyme of this pathway. Biochemical studies with recombinant human DHODH led us to measure IC50 as low as 13 nM for the best example of this original series when using 2,3-dimethoxy-5-methyl-6-(3-methyl-2-butenyl)-1,4-benzoquinone (coenzyme Q1) as a surrogate for coenzyme Q10, the cofactor of this enzyme.

Published
2015

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

Author

Marianne Lucas-Hourani, Jean-Philippe Herbeuval, Nikaïa Smith, Maryline Bourgine, Samira Khiar, Sébastien Nisole, Claude Ruffié, Pierre-Olivier Vidalain, Frédéric Tangy, Hélène Munier-Lehmann, Olivier Helynck, Génomique virale et vaccination, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Toxicité environnementale, cibles thérapeutiques, signalisation cellulaire (T3S - UMR_S 1124), Université Paris Descartes - Paris 5 (UPD5)-Centre National de la Recherche Scientifique (CNRS)-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), Chimie et Biocatalyse, Virologie moléculaire et Vaccinologie, This work was supported by the Agence National de la Recherche (ChemInnate program to POV, SN and FT), the Institut Pasteur (www.pasteur.fr), the Centre National de la Recherche Scientifique (CNRS, Institut Pasteur [Paris] - Centre National de la Recherche Scientifique (CNRS), Toxicologie, Pharmacologie et Signalisation Cellulaire (U1124), Institut National de la Santé et de la Recherche Médicale (INSERM) - Université Paris Descartes - Paris 5 (UPD5) - Centre National de la Recherche Scientifique (CNRS), Université Paris Descartes - Paris 5 (UPD5) - Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Bidault, Floran

Subjects
0301 basic medicine, Science, [SDV]Life Sciences [q-bio], Biology, Response Elements, Transfection, Article, Small Molecule Libraries, 03 medical and health sciences, chemistry.chemical_compound, Cytosol, Interferon, medicine, Humans, Promoter Regions, Genetic, Regulation of gene expression, Multidisciplinary, DNA, Molecular biology, High-Throughput Screening Assays, [SDV] Life Sciences [q-bio], HEK293 Cells, 030104 developmental biology, IRF1, Gene Expression Regulation, chemistry, Interferon Type I, Medicine, Interferon Regulatory Factor-3, IRF3, Interferon type I, Interferon regulatory factors, medicine.drug
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

15. Original Chemical Series of Pyrimidine Biosynthesis Inhibitors That Boost the Antiviral Interferon Response

Author

Marianne Lucas-Hourani, Philippe V. Afonso, Julien Dairou, Frédéric Tangy, Olivier Helynck, Sébastien Nisole, Daniel Dauzonne, Hélène Munier-Lehmann, Samira Khiar, Pierre-Olivier Vidalain, Génomique virale et vaccination, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Institut Curie, Chimie et Biocatalyse, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut de Recherche en Infectiologie de Montpellier (IRIM), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques (LCBPT - UMR 8601), Université Paris Descartes - Paris 5 (UPD5)-Centre National de la Recherche Scientifique (CNRS), Epidémiologie et Physiopathologie des Virus Oncogènes (EPVO (UMR_3569 / U-Pasteur_3)), Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Institut Pasteur [Paris], This work was supported by the Agence National de la Recherche (ChemInnate program to P.-O.V., S.N., and F.T.), the Institut Pasteur (https://www.pasteur.fr/fr), the Centre National de la Recherche Scientifique (CNRS [www.cnrs.fr]), the Institut National de la Santé Et de la Recherche Médicale (INSERM [www.inserm.fr]), the Institut Carnot–Pasteur Maladies Infectieuses (STING program to P.-O.V. and H.M.-L.), and the Fondation BNP-Paribas (to M.L.-H., S.K., and F.T.)., We thank Nicolas Vanthyne for the separation of chiral enantiomers (Laboratoire de Synthèse et Physico-Chimie de Molécules d'Intérêt Biologique, Plateforme de Chromatographie Chirale, Marseille, France). We thank Gaëlle Cousin, Pierre Jorda, and Chantal Combredet for their technical assistance and Stéphanie Dupuy from the flow cytometry core facility at Université Paris Descartes. We thank Pierre Talbot and Marc Desforges (INRS-Institut Armand-Frappier, Canada) for providing antibodies against the spike protein of HCoV-229E. We thank Philippe Desprès (Université de La Réunion, France) for providing the CHIKV/Ren recombinant virus. We thank Anastassia Komarova for the discussions on ssRNA structures., Institut Curie [Paris], Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Dicumarol, Oxidoreductases Acting on CH-CH Group Donors, Indoles, Pyrimidine, [SDV]Life Sciences [q-bio], Dihydroorotate Dehydrogenase, Antiviral Agents, Cell Line, Interferon Lambda, 03 medical and health sciences, chemistry.chemical_compound, Structure-Activity Relationship, Interferon, medicine, [CHIM]Chemical Sciences, Humans, Pharmacology (medical), Enzyme Inhibitors, Gene, innate immunity, Pharmacology, Dicoumarol, Immunity, Innate, pyrimidine metabolism, 3. Good health, interferons, Metabolic pathway, 030104 developmental biology, Infectious Diseases, HEK293 Cells, Pyrimidines, chemistry, Biochemistry, Chromones, Measles virus, Pyrimidine metabolism, Interferon Type I, Dihydroorotate dehydrogenase, Chikungunya virus, medicine.drug
Abstract

De novo pyrimidine biosynthesis is a key metabolic pathway involved in multiple biosynthetic processes. Here, we identified an original series of 3-(1 H -indol-3-yl)-2,3-dihydro-4 H -furo[3,2- c ]chromen-4-one derivatives as a new class of pyrimidine biosynthesis inhibitors formed by two edge-fused polycyclic moieties. We show that identified compounds exhibit broad-spectrum antiviral activity and immunostimulatory properties, in line with recent reports linking de novo pyrimidine biosynthesis with innate defense mechanisms against viruses. Most importantly, we establish that pyrimidine deprivation can amplify the production of both type I and type III interferons by cells stimulated with retinoic acid-inducible gene 1 (RIG-I) ligands. Altogether, our results further expand the current panel of pyrimidine biosynthesis inhibitors and illustrate how the production of antiviral interferons is tightly coupled to this metabolic pathway. Functional and structural similarities between this new chemical series and dicoumarol, which was reported before to inhibit pyrimidine biosynthesis at the dihydroorotate dehydrogenase (DHODH) step, are discussed.

Published
2017

16. 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

17. 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

18. 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

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

Author

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

Subjects
2. Zero hunger, 0303 health sciences, 03 medical and health sciences, 030306 microbiology, [SDV]Life Sciences [q-bio], General Medicine, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, Biology, Molecular biology, General Biochemistry, Genetics and Molecular Biology, 030304 developmental biology, 3. Good health
Abstract

Les virus a ARN sont responsables de maladies graves telles que la grippe, la dengue, l’hepatite C, la rougeole ou la bronchiolite des nourrissons. Ils representent egalement une menace emergente en raison des echanges internationaux et des bouleversements ecologiques lies aux activites humaines. Notre comprehension des mecanismes cellulaires qui controlent la replication virale a fortement progresse ces dernieres annees, et il apparait que des composes capables de cibler la cellule hote, plutot que le virus lui-meme, pourraient inhiber un large eventail de virus a ARN. Ainsi, differents laboratoires prives et universitaires sont en quete de molecules stimulant la reponse antivirale innee des cellules hotes. Une strategie interessante consiste a rechercher des molecules induisant l’expression des ISG (interferon-stimulated genes), lesquels constituent un vaste ensemble de genes antiviraux actives en particulier par les interferons de type I. Pour atteindre cet objectif, nous avons mis au point un test phenotypique base sur des cellules humaines transfectees avec un gene rapporteur luciferase sous controle d’un element de reponse aux interferons (ISRE). Ce systeme a ete utilise pour cribler une chimiotheque d’environ 54 000 composes. Parmi les molecules selectionnees, nous avons montre que le compose DD264 stimule la reponse immunitaire innee dans les cultures cellulaires, et presente une activite antivirale a large spectre. En recherchant la cible de ce compose, nous avons montre qu’il inhibe la voie de biosynthese de novo des pyrimidines, et notamment la quatrieme enzyme de cette voie metabolique, la dihydroorotate deshydrogenase (DHODH). Ainsi, nos resultats etablissent de facon surprenante un lien fonctionnel nouveau entre la voie de biosynthese des pyrimidines et la reponse innee antivirale.

Published
2015

20. 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

21. Synergy of the antibiotic colistin with echinocandin antifungals in Candida species

Author

Cécile Clavaud, Christophe d'Enfert, Zacarias Garcia, Olivier Helynck, Natacha Sertour, Alexandru Lupan, Ute Zeidler, Hélène Munier-Lehmann, Cosmin Saveanu, Marie-Elisabeth Bougnoux, Antonia Doyen, Biologie et Pathogénicité fongiques, Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris], Service de Microbiologie, Université Paris Descartes - Paris 5 (UPD5)-CHU Necker - Enfants Malades [AP-HP], Chimie et Biocatalyse, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Génétique des Interactions macromoléculaires, Dynamiques des Réponses Immunes, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Aspergillus, Institut Pasteur [Paris], This work was supported by a grant from the 'Conseil Régional d'Ile-de-France' (Chemical Library Project, grants I 06-222/R and I 09-1739/R) to H. M.-L., a grant (Programme Fungi) from Institut Carnot--Pasteur Maladies Infectieuses to H. M.-L. and C. d'E. and a grant from 'Agence Nationale de la Recherche' (ANR-08-JCJC-0019-01, GENO-GIM project) to C. S. U. Z. and C. C. were the recipients of postdoctoral grants in the framework of the Programme Fungi. A. L. was the recipient of a post-doctoral fellowship from the 'Conseil Régional d'Ile-de-France'., ANR-08-JCJC-0019,GENO-GIM,Obtention d'une carte d'interactions génétiques à l'échelle génomique chez la levure(2008), Institut Pasteur [Paris]-Institut National de la Recherche Agronomique (INRA), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Biologie et Pathogénicité fongiques (BPF), Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris] (IP), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Génétique des Interactions macromoléculaires / Genetics of Macromolecular Interactions, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP), Munier-Lehmann, Hélène, Jeunes chercheuses et jeunes chercheurs - Obtention d'une carte d'interactions génétiques à l'échelle génomique chez la levure - - GENO-GIM2008 - ANR-08-JCJC-0019 - JCJC - VALID, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Institut National de la Recherche Agronomique (INRA) - Institut Pasteur [Paris], Université Paris Descartes - Paris 5 (UPD5) - CHU Necker - Enfants Malades [AP-HP], Institut Pasteur [Paris] - Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] - Institut National de la Santé et de la Recherche Médicale (INSERM), and ANR-08-JCJC-0019, GENO-GIM, Obtention d'une carte d'interactions génétiques à l'échelle génomique chez la levure(2008)

Subjects
Antifungal Agents, Cell Membrane Permeability, [SDV]Life Sciences [q-bio], Antibiotics, Indicator Dilution Techniques, Chitin, Pharmacology, combination therapy, Echinocandins, Mice, chemistry.chemical_compound, Cell Wall, Candida albicans, polycyclic compounds, Pharmacology (medical), caspofungin, Coloring Agents, [SDV.MP.MYC]Life Sciences [q-bio]/Microbiology and Parasitology/Mycology, Candida, Mice, Inbred BALB C, 0303 health sciences, Candidiasis, Drug Synergism, Corpus albicans, 3. Good health, [SDV] Life Sciences [q-bio], Infectious Diseases, [SDV.SP.PHARMA] Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, Propidium, medicine.drug, Microbiology (medical), Genotype, Echinocandin, medicine.drug_class, Microbial Sensitivity Tests, Saccharomyces cerevisiae, Biology, Microbiology, Lipopeptides, 03 medical and health sciences, medicine, Animals, Gene Library, 030304 developmental biology, sphingolipids, Colistin, 030306 microbiology, polymyxin, biology.organism_classification, bacterial infections and mycoses, [SDV.MP.MYC] Life Sciences [q-bio]/Microbiology and Parasitology/Mycology, Aminocandin, chemistry, Mutation, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, Genetic Fitness, Caspofungin
Abstract

International audience; OBJECTIVES: Candida albicans is the most prevalent fungal pathogen of humans, causing a wide range of infections from harmless superficial to severe systemic infections. Improvement of the antifungal arsenal is needed since existing antifungals can be associated with limited efficacy, toxicity and antifungal resistance. Here we aimed to identify compounds that act synergistically with echinocandin antifungals and that could contribute to a faster reduction of the fungal burden. METHODS: A total of 38 758 compounds were tested for their ability to act synergistically with aminocandin, a β-1,3-glucan synthase inhibitor of the echinocandin family of antifungals. The synergy between echinocandins and an identified hit was studied with chemogenomic screens and testing of individual Saccharomyces cerevisiae and C. albicans mutant strains. RESULTS: We found that colistin, an antibiotic that targets membranes in Gram-negative bacteria, is synergistic with drugs of the echinocandin family against all Candida species tested. The combination of colistin and aminocandin led to faster and increased permeabilization of C. albicans cells than either colistin or aminocandin alone. Echinocandin susceptibility was a prerequisite to be able to observe the synergy. A large-scale screen for genes involved in natural resistance of yeast cells to low doses of the drugs, alone or in combination, identified efficient sphingolipid and chitin biosynthesis as necessary to protect S. cerevisiae and C. albicans cells against the antifungal combination. CONCLUSIONS: These results suggest that echinocandin-mediated weakening of the cell wall facilitates colistin targeting of fungal membranes, which in turn reinforces the antifungal activity of echinocandins.

Published
2013

22. Inhibition of pyrimidine biosynthesis pathway suppresses viral growth through innate immunity

Author

Frédéricic Tangy, Gwenaëlle André-Leroux, Olivier Helynck, Alexandru Lupan, Gaëlle Cousin, Grégory Caignard, Daniel Dauzonne, Geneviève Janvier, Philippe Desprès, Hélène Munier-Lehmann, Pierre Jorda, Yves Jacob, Samira Khiar, Marianne Lucas-Hourani, Pierre-Olivier Vidalain, Nicolas Escriou, Génomique Virale et Vaccination, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Conception, synthèse et vectorisation de biomolécules. (CSVB), Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris], Chimie et Biocatalyse, Biochimie Structurale, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Interactions Moléculaires Flavivirus-Hôtes, Institut Pasteur [Paris], Génétique moléculaire des virus à ARN, This work was supported by the Institut Pasteur, the Centre National de la Recherche Scientifique, the Institut National de la Santé Et de la Recherche Médicale, the Institut Carnot – Pasteur Maladies Infectieuses (Programme STING to POV and HML), the Agence Nationale pour la Recherche (ANR-RPIB, Programme STING 2.0 to POV), and the ‘‘Conseil Régional d’Ile-de-France’’ (Chemical Library Project, grants n° I 06-222/R and I 09-1739/R to HML). The work on CHIKV/Ren was supported by the project ArbOAS (ANR grant 2010-INTB-1601-02)., Lucas-Hourani, Marianne, 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), Institut Pasteur [Paris] (IP), and Institut Curie-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)

Subjects
immunosuppressive agent leflunomide, toll-like receptors, virus-infection, gene induction, dihydroorotate dehydrogenase, antiviral activity, mycophenolic-acid, in-vitro, [SDV]Life Sciences [q-bio], 0302 clinical medicine, Gene expression, Chlorocebus aethiops, Biology (General), 0303 health sciences, 3. Good health, Cell biology, 030220 oncology & carcinogenesis, Pyrimidine metabolism, Chikungunya virus, Research Article, QH301-705.5, Immunology, Biology, Microbiology, Antiviral Agents, 03 medical and health sciences, Immune system, Virology, Genetics, Animals, Humans, Molecular Biology, Transcription factor, Vero Cells, 030304 developmental biology, Reporter gene, Innate immune system, Alphavirus Infections, RC581-607, Immunity, Innate, Metabolic pathway, Pyrimidines, Dihydroorotate dehydrogenase, Chikungunya Fever, Parasitology, Immunologic diseases. Allergy, HeLa Cells, Interferon Regulatory Factor-1
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., Author Summary Our therapeutic arsenal to treat viral diseases is extremely limited, and there is a critical need for molecules that could be used against multiple viruses. Among possible strategies, there is a growing interest for molecules stimulating cellular defense mechanisms. We recently developed a functional assay to identify stimulators of antiviral genes, and selected compound DD264 from a chemical library using this approach. While searching for its mode of action, we identified this molecule as an inhibitor of pyrimidine biosynthesis, a metabolic pathway that fuels the cell with pyrimidine nucleobases for both DNA and RNA synthesis. Interestingly, it was recently shown that inhibitors of this metabolic pathway prevent the replication of RNA viruses. Here, we established a functional link between pyrimidine biosynthesis pathway and the induction of antiviral genes, and demonstrated that pyrimidine biosynthesis inhibitors like DD264 or brequinar critically rely on cellular immune response to inhibit virus growth. Thus, pyrimidine deprivation is not directly responsible for the antiviral activity of pyrimidine biosynthesis inhibitors, which rather involves the induction of a metabolic stress and subsequent triggering of cellular defense mechanisms.

Published
2013

23. 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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