Identifying phase separation modulators mediated by the SARS-CoV-2 nucleocapsid protein using a structure-based drug discovery approach

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve through mutations and acquire resistance to existing antiviral agents, the persistence of post-pandemic infections remains a significant global health concern. To address these challenges, it is crucial to develop novel therapeutic agents that target previously unexplored viral or host mechanisms.

The nucleocapsid (N) protein of SARS-CoV-2 plays a critical role in viral RNA replication and genome packaging through its ability to undergo liquid-liquid phase separation (LLPS). Targeting the condensation of N-RNA complex could potentially serve as an effective therapeutic strategy against coronavirus infections. The present study provides a successful exemplification of antiviral agent discovery by targeting the viral N protein on the basis of its structural characteristics.

A total of 23 virtual screening compounds and 16 hit derivatives were prepared. Their binding affinity and antiviral activities were evaluated using surface plasmon resonance and immunofluorescence assay, respectively. The impact of these compounds on condensate formation was verified through fluorescence recovery after photobleaching, as well as the inhibition of N condensate formation in cells.

Structure-based virtual screening, target protein binding affinity quantification, and cell culture-based anti-SARS-CoV-2 assays enabled the identification a hit compound, D21. Subsequent structural optimization of series I and II yielded compound II8, which exhibits stronger binding affinity for the N protein and effectively inhibits the formation of N-RNA condensates, thereby demonstrating its potential to modulate LLPS, a mechanism underlying its antiviral activity. In vivo evaluation showed that II8 displays antiviral activity in a human ACE2-expressing mouse model when administered intranasally as a pre-incubated mixture with SARS-CoV-2. Due to the pharmacokinetic profile of compound II8, further structural modifications are still required.

Our findings suggest that compound II8 may serve as a localized prophylactic nasal spray, providing valuable insights for further development of LLPS modulators as next-generation antiviral agents.