Mechanism-informed identification of FDA-approved topoisomerase inhibitors disrupting SARS-CoV-2 nucleocapsid-RNA interactions.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (NP) interaction with viral RNA is vital for viral replication and immune evasion, making it an attractive target for antiviral development. Inspired by adenosine triphosphate (ATP)'s competitive inhibition of NP-RNA binding, we screened 121 FDA-approved ATP-competitive kinase inhibitors, identifying mitoxantrone (IC₅₀ = 1.22 µM) as a potent inhibitor. Considering its known topoisomerase inhibitory activity, we further screened 23 additional topoisomerase inhibitors, uncovering four active compounds-pixantrone (IC₅₀ = 5.67 µM), doxorubicin (IC₅₀ = 20.19 µM), epirubicin (IC₅₀ = 7.23 µM), and suramin (IC₅₀ = 0.44 µM). Biolayer interferometry (BLI) revealed distinct inhibition mechanisms: suramin bound directly to the NP C-terminal domain (CTD) with a KD of 0.26 µM, whereas the other compounds primarily targeted RNA, with pixantrone showing the highest RNA affinity (KD = 0.63 µM). Complementary molecular docking analyses supported these observations, indicating suramin's preference for NP binding and anthracycline derivatives engaging RNA. Our findings demonstrate the feasibility of a mechanism-informed repurposing strategy and identify FDA-approved topoisomerase inhibitors and suramin as valuable chemical starting points. Although these compounds are not directly suitable as antivirals due to toxicity concerns, they provide promising scaffolds for further optimization aimed at selective disruption of SARS-CoV-2 NP-RNA interactions.