ABSTRACT Identification of pesticide targets is of great significance for the development of new pesticides. The new compound GLY‐15, containing a pyrimidine heterocycle and a moroxydine skeleton structure, has good anti‐TMV activity, but the underlying molecular targets and mechanism of action remain elusive. Here, host malate dehydrogenase (MDH), glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), and tobacco mosaic virus (TMV) coat protein (CP) were identified as potential targets of GLY‐15 using activity‐based protein profiling (ABPP) and drug affinity responsive target stability (DARTS), and their interactions with GLY‐15 were validated by microscale thermophoresis (MST) and pull‐down analysis. Functional analyses demonstrate that MDH silencing significantly reduces TMV accumulation, while transient overexpression of MDH results in elevated viral infection. Meanwhile, yeast two‐hybrid (Y2H), co‐immunoprecipitation (Co‐IP), and bimolecular fluorescence complementation (BiFC) analysis uncover that MDH interacts with CP, and their interaction is effectively inhibited by GLY‐15. Site‐directed mutagenesis identifies E225 as a critical residue for both GLY‐15/MDH binding and MDH/CP interaction. Further investigations reveal that GLY‐15 functions as an MDH inhibitor and affects its interaction with CP. Meanwhile, we showed that GLY‐15 targeting MDH indicates broad antiviral activity against pepper mild mottle virus (PMMoV) and potato virus Y (PVY). This investigation systematically reveals novel insights into the anti‐TMV mechanisms of GLY‐15, establishing a valuable theoretical basis for antiviral target discovery and plant disease resistance breeding.
Yu et al. (2026) studied this question.