Development of FRET‐based high‐throughput screening for viral RNase III inhibitors

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 FRETcompatible 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. K E Y W O R D Sfluorescence resonance energy transfer, high-throughput screening, RNA silencing suppressor, RNase III, small interfering RNA

In Vitro Identification and In Vivo Confirmation of Inhibitors for Sweet Potato Chlorotic Stunt Virus RNA Silencing Suppressor, a Viral RNase III

“…Under native Western blotting conditions, CSR3 could be detected as a monomer and a dimer ( Fig. 2D ); under denatured conditions, CSR3 was shown to be primarily monomeric in our previous study ( 24 , 27 ), which is consistent with the results of SDS-PAGE in Fig. 2C .…”

“…These results were consistent with previous studies demonstrating that these four amino acid residues were essential for the catalytic activity of class 1 RNase III (20,23). Moreover, previous studies showed that mutations of CSR3 40E and 44D lead to a loss of function of its endoribonuclease activity on dsRNA and suppression of RNA silencing (15,18,24), confirming their critical function for CSR3 activity.…”

“…3A ). According to our previous study ( 24 ) detailing CSR3 kinetic-based HTS development and optimization, this assay was conducted using 100 nM CSR3 enzyme and 375 nM siRNA substrate over 12 plate read cycles (∼17 min total) at 37°C. In this study, a total of 6,622 compounds (including 6,400 selected from Glide docking and 222 empirically selected compounds for preliminary assay setup) were screened using kinetic-based HTS at a final concentration of 10 μM.…”

“…They all were inactive in the studies except for FIMM031755, which affected the activity of chain B of the human cytokine/receptor binary complex ( https://pubchem.ncbi.nlm.nih.gov/compound/7114450#section=Biological-Test-Results ). Moreover, FIMM031755 has also been screened in our latest article about the development of a FRET-based screening method ( 24 ). The class 2 compounds, which share the same core structure but have different R groups, had different binding affinities, providing evidence for further analyses of structure-activity relationships.…”

“…Moreover, FIMM031755 has also been screened in our latest article about development of a FRET-on April 22, 2021 by guest http://jvi.asm.org/ Downloaded from based screening (24). The class 2 compounds, which share the same core structure but have different R-groups, had different binding affinities, providing evidence for further analyses of structure-activity relationships.…”