Identification of Nsp15 Inhibitors Restoring Interferon-Dependent Antiviral Activity Against SARS-CoV-2.

Publication date: May 22, 2026

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 EC value of 2 μM, and low cytopathic effect in multiple cell types (CC >28 μM). More importantly, SARS-CoV-2 replication was inhibited in cells with an intact IFN system, but not in IFN-deficient cell lines.