Linking target-based screening to in vivo efficacy in aquaculture: identification of anti-WSSV compounds via a non-specific endonuclease (NSN) platform

A robust NSN-targeted screening platform for anti-WSSV discovery was developed. • The platform enables rapid and scalable identification of antiviral compounds. • Natural products with potent NSN inhibition were identified from a focused library. • Active compounds induced conformational changes in NSN structure. • The platform bridges biochemical screening with organism-level validation. White spot syndrome virus (WSSV), the etiological agent of white spot disease (WSD), causes rapid and high mortality in crustaceans and leads to severe economic losses in global aquaculture. The lack of permissive cell lines for WSSV propagation, together with the limitations of conventional in vivo screening approaches, has greatly constrained antiviral drug discovery. Here, we established a target-based in vitro screening platform focusing on the viral non-specific endonuclease (NSN), a key enzyme involved in WSSV replication. Recombinant WSSV-NSN was successfully expressed and purified, exhibiting a specific activity of 83.35 U/mg and a Michaelis constant (Km) of 106 mmol/L, with Mg 2+ as a strong activator. Based on enzymatic characterization, reaction conditions were optimized, and a robust screening system was developed with a Z′ factor of 0.70, indicating high reliability for high-throughput screening. Application of this platform to a natural compound library identified five NSN inhibitors, including luteolin, β-sitosterol, geniposide, cinnamic acid, and 1-hydroxyphenazine, with inhibition rates ranging from 58.5% to 96.6%. Enzyme kinetic inhibition analysis further showed that luteolin and β-sitosterol decreased Vmax with only minor changes in Km, suggesting that these compounds mainly reduced the catalytic capacity of NSN rather than simply competing with the RNA substrate. Circular dichroism analysis further revealed that luteolin and β-sitosterol directly interacted with NSN and induced conformational changes in its secondary structure, suggesting a potential mechanism of enzyme inhibition. Notably, representative compounds (luteolin and β-sitosterol) were further validated in vivo , where they significantly improved crayfish survival and reduced viral load following WSSV challenge, supporting the predictive capability of the NSN-targeted screening platform. Overall, this study establishes a reliable and efficient NSN-targeted screening system and demonstrates its effectiveness in identifying bona fide anti-WSSV agents, providing a practical strategy for target-based antiviral drug discovery in crustaceans.