Identification of Nsp15 inhibitors restoring interferon-dependent antiviral activity against SARS-CoV-2

The endoribonuclease Nsp15 is essential for coronavirus pathogenesis and evasion of host defenses and is therefore a promising drug target. We determined optimal parameters for an endoribonuclease FRET biochemical assay and use it for a high-throughput drug screen. Measurement of Z'-factor confirmed robust assay performance (Z' = 0.7- 0.9). We screened a commercially available library (LOPAC 1280) and identified three molecules able to inhibit the catalytic endonuclease activity of Nsp15 in the low micromolar range. Among them, a promising hit compound, Reactive Blue 2 showed also anti-SARS-CoV-2 activity with EC₅₀ value of 2 μM, and low cytopathic effect in multiple cell types (CC₅₀ > 28 μM). More importantly, SARS-CoV-2 replication was inhibited in cells with an intact IFN system, but not in IFN-deficient cell lines.