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Development of FRET-based high-throughput screening for viral RNase III inhibitors.
Wang, Linping; Saarela, Jani; Poque, Sylvain; Valkonen, Jari P T.
Afiliación
  • Wang L; Department of Agricultural Sciences, University of Helsinki, Helsinki, Finland.
  • Saarela J; Institute for Molecular Medicine Finland, University of Helsinki, Helsinki, Finland.
  • Poque S; Department of Agricultural Sciences, University of Helsinki, Helsinki, Finland.
  • Valkonen JPT; Department of Agricultural Sciences, University of Helsinki, Helsinki, Finland.
Mol Plant Pathol ; 21(7): 961-974, 2020 07.
Article en En | MEDLINE | ID: mdl-32436305
ABSTRACT
The class 1 ribonuclease III (RNase III) encoded by Sweet potato chlorotic stunt virus (CSR3) suppresses RNA silencing in plant cells and thereby counters the host antiviral response by cleaving host small interfering RNAs, which are indispensable components of the plant RNA interference (RNAi) pathway. The synergy between sweet potato chlorotic stunt virus and sweet potato feathery mottle virus can reduce crop yields by 90%. Inhibitors of CSR3 might prove efficacious to counter this viral threat, yet no screen has been carried out to identify such inhibitors. Here, we report a novel high-throughput screening (HTS) assay based on fluorescence resonance energy transfer (FRET) for identifying inhibitors of CSR3. For monitoring CSR3 activity via HTS, we used a small interfering RNA substrate that was labelled with a FRET-compatible dye. The optimized HTS assay yielded 109 potential inhibitors of CSR3 out of 6,620 compounds tested from different small-molecule libraries. The three best inhibitor candidates were validated with a dose-response assay. In addition, a parallel screen of the selected candidates was carried out for a similar class 1 RNase III enzyme from Escherichia coli (EcR3), and this screen yielded a different set of inhibitors. Thus, our results show that the CSR3 and EcR3 enzymes were inhibited by distinct types of molecules, indicating that this HTS assay could be widely applied in drug discovery of class 1 RNase III enzymes.
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Texto completo: 1 Colección: 01-internacional Banco de datos: MEDLINE Asunto principal: Antivirales / Pruebas de Sensibilidad Microbiana / Crinivirus / Transferencia Resonante de Energía de Fluorescencia / Ribonucleasa III / Inhibidores Enzimáticos Idioma: En Revista: Mol Plant Pathol Año: 2020 Tipo del documento: Article País de afiliación: Finlandia

Texto completo: 1 Colección: 01-internacional Banco de datos: MEDLINE Asunto principal: Antivirales / Pruebas de Sensibilidad Microbiana / Crinivirus / Transferencia Resonante de Energía de Fluorescencia / Ribonucleasa III / Inhibidores Enzimáticos Idioma: En Revista: Mol Plant Pathol Año: 2020 Tipo del documento: Article País de afiliación: Finlandia