Approved drugs screening against the nsP1 capping enzyme of Venezuelan equine encephalitis virus using an immuno-based assay

• Published in: Antiviral research Vol. 163; pp. 59 - 69
• Main Authors: Ferreira-Ramos, Ana S., Li, Changqing, Eydoux, Cécilia, Contreras, Jean Marie, Morice, Christophe, Quérat, Gilles, Gigante, Alba, Pérez Pérez, María-Jesús, Jung, Marie-Louise, Canard, Bruno, Guillemot, Jean-Claude, Decroly, Etienne, Coutard, Bruno
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-03-2019; Elsevier Masson
• Subjects: Alphavirus; Animals; Antiviral Agents - pharmacology; Antivirals; Approved drugs; Biochemistry; Biochemistry, Molecular Biology; Chlorocebus aethiops; Drug Approval; Encephalitis Virus, Venezuelan Equine - drug effects; Encephalitis Virus, Venezuelan Equine - enzymology; Enzyme-Linked Immunosorbent Assay; Guanylylation; High-Throughput Screening Assays; HT screening; Human health and pathology; Humans; Infectious diseases; Inhibitory Concentration 50; Life Sciences; Microbiology and Parasitology; Molecular biology; mRNA capping; RNA Caps; Vero Cells; Viral Nonstructural Proteins - antagonists & inhibitors; Virology; Virus Replication - drug effects; Alphavirus; Guanylylation; mRNA capping; HT screening; Approved drugs; Antivirals
• ISSN: 0166-3542; 1872-9096
• DOI: 10.1016/j.antiviral.2019.01.003

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 m7GMP releasing pyrophosphate (GT reaction) and the m7GMP 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 m7GMP-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 IC50 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.
•Emergence or re-emergence of alphaviruses such as CHIKV or VEEV represents a serious health concern.•Alphaviruses have a peculiar RNA capping mechanism suitable for the development of inhibitors with antiviral effects.•We developed an enzyme-based screening of approved drugs on VEEV capping enzyme nsP1.•The screening resulted in the selection of 18 compounds and two series of compounds were further characterized for SAR.•This work provide a first basis towards the development of more potent inhibitors with antiviral effect.