Identification of new benzofuran derivatives as STING agonists with broad-spectrum antiviral activity

•Benzofuran derivatives were shown to induce IFN-I expression in a STING-dependent luciferase assay.•Activity as STING agonist was confirmed by mutagenesis studies.•Antiviral effect of BZFs was demonstrated on HCoV-229E and SARS-CoV-2 replication.•IFN-I mediated antiviral effect was confirmed by immunofluorescent analysis. The Stimulator of Interferon Genes (STING) is involved in cytosolic DNA sensing and type I Interferons (IFN-I) induction. Aiming to identify new STING agonists with antiviral activity and given the known biological activity of benzothiazole and benzimidazole derivatives, a series of benzofuran derivatives were tested for their ability to act as STING agonists, induce IFN-I and inhibit viral replication. Compounds were firstly evaluated in a gene reporter assay measuring luciferase activity driven by the human IFN-β promoter in cells expressing exogenous STING (HEK293T). Seven of them were able to induce IFN-β transcription while no induction of the IFN promoter was observed in the presence of a mutated and inactive STING, showing specific protein-ligand interaction. Docking studies were performed to predict their putative binding mode. The best hit compounds were then tested on human coronavirus 229E replication in BEAS-2B and MRC-5 cells and three derivatives showed EC50 values in the μM range. Such compounds were also tested on SARS-CoV-2 replication in BEAS-2B cells and in Calu-3 showing they can inhibit SARS-CoV-2 replication at nanomolar concentrations. To further confirm their IFN-dependent antiviral activity, compounds were tested to verify their effect on phospho-IRF3 nuclear localization, that was found to be induced by benzofuran derivatives, and SARS-CoV-2 replication in Vero E6 cells, lacking IFN production, founding them to be inactive. In conclusion, we identified benzofurans as STING-dependent immunostimulatory compounds and host-targeting inhibitors of coronaviruses representing a novel chemical scaffold for the development of broad-spectrum antivirals.