TY - JOUR
T1 - Approved drugs screening against the nsP1 capping enzyme of Venezuelan equine encephalitis virus using an immuno-based assay
AU - Ferreira-Ramos, Ana S.
AU - Li, Changqing
AU - Eydoux, Cécilia
AU - Contreras, Jean Marie
AU - Morice, Christophe
AU - Quérat, Gilles
AU - Gigante, Alba
AU - Pérez Pérez, María Jesús
AU - Jung, Marie Louise
AU - Canard, Bruno
AU - Guillemot, Jean Claude
AU - Decroly, Etienne
AU - Coutard, Bruno
N1 - Funding Information:
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 .
Funding Information:
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.
Publisher Copyright:
© 2019 Elsevier B.V.
Copyright:
Copyright 2019 Elsevier B.V., All rights reserved.
PY - 2019/3
Y1 - 2019/3
N2 - 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 m 7 GMP releasing pyrophosphate (GT reaction) and the m 7 GMP 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 m 7 GMP-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 50 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.
AB - 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 m 7 GMP releasing pyrophosphate (GT reaction) and the m 7 GMP 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 m 7 GMP-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 50 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.
UR - http://www.scopus.com/inward/record.url?scp=85060441580&partnerID=8YFLogxK
U2 - 10.1016/j.antiviral.2019.01.003
DO - 10.1016/j.antiviral.2019.01.003
M3 - Journal articles
C2 - 30639438
AN - SCOPUS:85060441580
SN - 0166-3542
VL - 163
SP - 59
EP - 69
JO - Antiviral Research
JF - Antiviral Research
ER -
