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

Maloccu, Stefania;Otsuka, Yuka;Molchan, Elizabeth;Foti, Michelle;Paulis, Annalaura;Vishwakarma, Jyoti;Malune, Paolo;Cottiglia, Filippo;Corona, Angela;Scampavia, Louis;Spicer, Timothy P.;Harris, Reuben S.;Tramontano, Enzo

;Fanunza, Elisa

2026-01-01

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Abstract

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.