Synergistic targeting of host malate dehydrogenase and phytoviral capsid by a new pyrimidine morpholine guanidine compound

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.