A Vanillin‐Derived Inhibitor of Aggregates via Targeting Intrinsically Disordered Regions of Phytoviral Nucleocapsid Protein

ABSTRACT Phase separation (PS) plays a fundamental role in organizing aggregates during the viral lifecycle, providing significant opportunities for in viral disease treatment by inhibiting PS. Intrinsically disordered regions (IDRs) have been extensively studied and found to be critical for PS. However, the discovery of small molecules that target residues within IDRs remains underexplored, particularly in the field of pesticides. Herein, we report a novel phytovirucide compound 29 , which was screened from a series of vanillin derivatives designed with sulfonylpiperazine motifs. The inactivation efficacy of compound 29 against tomato spotted wilt virus (TSWV) was significantly superior to that of the control agents vanisulfane and ribavirin . Mechanistically, compound 29 binds to the TSWV nucleocapsid protein (NP) at residues Lys68 (K68), Thr92 (T92), and Arg94 (R94), with T92 and R94 located in the IDRs of NP. Mutations at these sites impair the ability to form aggregates. Furthermore, a host factor, GTP (Guanosine Triphosphate)‐binding nuclear protein Ran‐like (Niben101scf08341g01001, NbRANL), which interacts with NP and promotes its aggregation, was identified. Compound 29 also suppresses the expression of NbRANL , resulting in the dual inhibition of ribonucleoprotein complexes (RNPs) formation. This unique mechanism of action provides insights into IDRs‐based virucide discovery.