The interferon (IFN) system is the first line of defense against viral infections. Evasion of IFN signaling by Ebola viral protein 24 (VP24) is a critical event in the pathogenesis of the infection and, hence, VP24 is a potential target for drug development. Since no drugs target VP24, the identification of molecules able to inhibit VP24, restoring and possibly enhancing the IFN response, is a goal of concern. Accordingly, we developed a dual signal firefly and Renilla luciferase cell-based drug screening assay able to quantify IFN-mediated induction of Interferon Stimulated Genes (ISGs) and its inhibition by VP24. Human Embryonic Kidney 293T (HEK293T) cells were transiently transfected with a luciferase reporter gene construct driven by the promoter of ISGs, Interferon-Stimulated Response Element (ISRE). Stimulation of cells with IFN-α activated the IFN cascade leading to the expression of ISRE. Cotransfection of cells with a plasmid expressing VP24 cloned from a virus isolated during the last 2014 outbreak led to the inhibition of ISRE transcription, quantified by a luminescent signal. To adapt this system to test a large number of compounds, we performed it in 96-well plates; optimized the assay analyzing different parameters; and validated the system by calculating the Z'- and Z-factor, which showed values of 0.62 and 0.53 for IFN-α stimulation assay and VP24 inhibition assay, respectively, indicative of robust assay performance.

The coronavirus disease 2019 (COVID-19) pandemic is fading, however its etiologic agent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues posing - despite the availability of licensed vaccines - a global health threat, due to the potential emergence of vaccine-resistant SARS-CoV-2 variants. This makes the development of new drugs against COVID-19 a persistent urgency and sets as research priority the validation of novel therapeutic targets within the SARS-CoV-2 proteome. Among these, a promising one is the SARS-CoV-2 nucleocapsid (N) phosphoprotein, a major structural component of the virion with indispensable role in packaging the viral genome into a ribonucleoprotein (RNP) complex, which also contributes to SARS-CoV-2 innate immune evasion by inhibiting the host cell type-I interferon (IFN-I) response. By combining miniaturized differential scanning fluorimetry with microscale thermophoresis, we found that the 100-year-old drug Suramin interacts with SARS-CoV-2 N-terminal domain (NTD) and C-terminal domain (CTD), thereby inhibiting their single-stranded RNA (ssRNA) binding function with low-micromolar Kd and IC50 values. Molecular docking suggests that Suramin interacts with basic NTD cleft and CTD dimer interface groove, highlighting three potentially druggable ssRNA binding sites. Electron microscopy shows that Suramin inhibits the formation in vitro of RNP complex-like condensates by SARS-CoV-2 N with a synthetic ssRNA. In a dose-dependent manner, Suramin also reduced SARS-CoV-2-induced cytopathic effect on Vero E6 and Calu-3 cells, partially reverting the SARS-CoV-2 N-inhibited IFN-I production in 293T cells. Our findings indicate that Suramin inhibits SARS-CoV-2 replication by hampering viral genome packaging, thereby representing a starting model for design of new COVID-19 antivirals.