Identification of Myricetin as Ebolavirus VP35-dsRNA interaction inhibitor through a novel fluorescence-based assay

Gian Luca Daino;Aldo Frau;Cinzia Sanna;Daniela Rigano;Simona Distinto;Veronica Madau;Francesca Esposito;Elisa Fanunza;Giulia Bianco;Orazio Taglialatela-Scafati;Luca Zinzula;Elias Maccioni;Angela Corona

Penultimate

;Enzo Tramontano

Last

2018-01-01

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Abstract

The Filoviridae Ebola virus (EBOV) causes a severe and rapidly progressing hemorrhagic syndrome whose recent epidemic enlightened the urgent need of novel therapeutic agents, since no drug is currently approved. A key contribution to EBOV high lethality comes from viral evasion of the host antiviral innate immune response in which the viral protein VP35 plays a crucial role, blocking the interferon type I production, firstly by masking the viral dsRNA and preventing its detection by the pattern recognition receptor RIG-I. Aiming to identify inhibitors of VP35 interaction with the viral dsRNA, counteracting the VP35 viral innate immune elusion, we established a new methodology for high-yield recombinant VP35 (rVP35) expression and purification, and a novel and robust fluorescence-based rVP35-RNA interaction assay (Z'-factor of 0.69). Taking advantage of such newly-established methods, we screened a small library of Sardinian natural extracts finding Limonium morisianum as the most potent inhibitor extract. A bio-guided fractionation led to the identification of myricetin as the component able to inhibit rVP35-dsRNA interaction with an IC50 value of 2.7 µM. Molecular docking studies showed that myricetin interacts with the highly conserved region of the VP35 RNA binding domain, laying the basis for further structural optimization of potent VP35-dsRNA inhibitors.