1. Approved drugs screening against the nsP1 capping enzyme of Venezuelan equine encephalitis virus using an immuno-based assay
- Author
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Bruno Canard, Ana Sofia Ferreira Ramos, Jean-Marie Contreras, Changqing Li, Marie-Louise Jung, Christophe Morice, Gilles Querat, Etienne Decroly, María-Jesús Pérez Pérez, Bruno Coutard, Alba Gigante, Cécilia Eydoux, Jean-Claude Guillemot, European Commission, Ministerio de Economía y Competitividad (España), Architecture et fonction des macromolécules biologiques (AFMB), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Prestwick Chemical [Illkirch, Strasbourg], Unité des Virus Emergents (UVE), Institut de Recherche pour le Développement (IRD)-Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Instituto de Quimica Médica (CSIC), Consejo Superior de Investigaciones Científicas [Madrid] (CSIC), ANR-14-ASTR-0026,VMTaseIn,Identification d'inhibiteurs de méthyltransférases de virus: une nouvelle stratégégie antivirale(2014), European Project: 264286,EC:FP7:PEOPLE,FP7-PEOPLE-2010-ITN,EUVIRNA(2011), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU)-Institut de Recherche pour le Développement (IRD)-Institut National de la Santé et de la Recherche Médicale (INSERM), Consejo Superior de Investigaciones Científicas [Spain] (CSIC), Architecture et fonction des macromolécules biologiques ( AFMB ), Centre National de la Recherche Scientifique ( CNRS ) -Aix Marseille Université ( AMU ) -Institut National de la Recherche Agronomique ( INRA ), Unité des Virus Emergents ( UVE ), Institut de Recherche pour le Développement ( IRD ) -Aix Marseille Université ( AMU ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), Instituto de Quimica Médica ( CSIC ), Consejo Superior de Investigaciones Científicas [Spain] ( CSIC ), ANR-14-ASTR-0026,VMTaseIn,Identification d'inhibiteurs de méthyltransférases de virus: une nouvelle stratégégie antivirale ( 2014 ), European Project : 264286,EC:FP7:PEOPLE,FP7-PEOPLE-2010-ITN,EUVIRNA ( 2011 ), coutard, bruno, Accompagnement spécifique des travaux de recherches et d’innovation défense - Identification d'inhibiteurs de méthyltransférases de virus: une nouvelle stratégégie antivirale - - VMTaseIn2014 - ANR-14-ASTR-0026 - ASTRID - VALID, and European Training Network on (+)RNA Virus Replication and Antiviral Drug Development - EUVIRNA - - EC:FP7:PEOPLE2011-03-01 - 2015-02-28 - 264286 - VALID
- Subjects
0301 basic medicine ,[SDV]Life Sciences [q-bio] ,viruses ,Viral Nonstructural Proteins ,medicine.disease_cause ,[SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology ,Virus Replication ,Encephalitis Virus, Venezuelan Equine ,[ SDV.BBM.BC ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM] ,[SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases ,Chlorocebus aethiops ,[SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM] ,Drug Approval ,[SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology ,NSP1 ,biology ,medicine.diagnostic_test ,Drug discovery ,Chemistry ,virus diseases ,Antivirals ,mRNA capping ,3. Good health ,[SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM] ,[SDV] Life Sciences [q-bio] ,[ SDV.MHEP.MI ] Life Sciences [q-bio]/Human health and pathology/Infectious diseases ,Guanylylation ,[SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology ,[SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases ,RNA Caps ,030106 microbiology ,Enzyme-Linked Immunosorbent Assay ,Alphavirus ,[ SDV.BBM.BM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology ,[ SDV.MP.VIR ] Life Sciences [q-bio]/Microbiology and Parasitology/Virology ,Antiviral Agents ,03 medical and health sciences ,Inhibitory Concentration 50 ,Western blot ,Capping enzyme ,Virology ,medicine ,Animals ,Humans ,[SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM] ,Vero Cells ,HT screening ,Pharmacology ,Messenger RNA ,[ SDV ] Life Sciences [q-bio] ,[SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology ,biology.organism_classification ,Approved drugs ,High-Throughput Screening Assays ,030104 developmental biology ,Viral replication ,Venezuelan equine encephalitis virus - Abstract
Alphaviruses such as the Venezuelan equine encephalitis virus (VEEV) are important human emerging pathogens transmitted by mosquitoes. They possess a unique viral mRNA capping mechanism catalyzed by the viral non-structural protein nsP1, which is essential for virus replication. The alphaviruses capping starts by the methylation of a GTP molecule by the N7-guanine methyltransferase (MTase) activity; nsP1 then forms a covalent link with mGMP releasing pyrophosphate (GT reaction) and the mGMP is next transferred onto the 5′-diphosphate end of the viral mRNA to form a cap-0 structure. The cap-0 structure decreases the detection of foreign viral RNAs, prevents RNA degradation by cellular exonucleases, and promotes viral RNA translation into proteins. Additionally, reverse-genetic studies have demonstrated that viruses mutated in nsP1 catalytic residues are both impaired towards replication and attenuated. The nsP1 protein is thus considered an attractive antiviral target for drug discovery. We have previously demonstrated that the guanylylation of VEEV nsP1 can be monitored by Western blot analysis using an antibody recognizing the cap structure. In this study, we developed a high throughput ELISA screening assay to monitor the GT reaction through mGMP-nsP1 adduct quantitation. This assay was validated using known nsP1 inhibitors before screening 1220 approved compounds. 18 compounds inhibiting the nsP1 guanylylation were identified, and their IC determined. Compounds from two series were further characterized and shown to inhibit the nsP1 MTase activity. Conversely, these compounds barely inhibited a cellular MTase demonstrating their specificity towards nsP1. Analogues search and SAR were also initiated to identify the active pharmacophore features. Altogether the results show that this HT enzyme-based assay is a convenient way to select potent and specific hit compounds targeting the viral mRNA capping of Alphaviruses., This work was supported by the French research agency ANR “VMTaseIn”, grant ANR-ST14-ASTR-0026, and by the European Union (Seventh Framework Program, Marie Skłodowska-Curie ETN “EUVIRNA”, grant agreement number 264286 and Horizon 2020 Marie Skłodowska-Curie ETN “ANTIVIRALS”, grant agreement number 642434), and by the MINECO/FEDER SAF2015-64629-C2-1-R.
- Published
- 2019