Rational design of dual-targeting solanesyl diphosphate synthase and protoporphyrinogen oxidase inhibitors

Herbicide resistance threatens sustainable weed control and demands new design strategies beyond conventional single-target chemistries. Protoporphyrinogen oxidase (PPO) and solanesyl diphosphate synthase (SPS) are two essential enzymes linked to chlorophyll biosynthesis and plastoquinone production, respectively, making them attractive targets for combinatorial intervention in plants. Here, we report the structure-guided development of a dual-target PPO/SPS inhibitor from the herbicide scaffold aclonifen. Among the synthesized analogs, compound 3q retained potent inhibition of At SPS1 while exhibiting a ∼60-fold improvement in Nt PPO inhibition relative to aclonifen. Differential scanning fluorimetry supported direct engagement of both targets by 3q . A co-crystal structure of the SPS- 3q complex revealed binding at the dimer interface and stabilization through π-π interactions with Phe211, whereas molecular dynamics simulation and mutational analysis supported the binding mode of 3q in PPO. In planta, 3q strongly inhibited primary root growth, triggered reactive oxygen species accumulation, and caused characteristic bleaching and burning-curling phenotypes. Notably, 3q remained active against an R128G PPO-resistant Amaranthus retroflexus biotype under greenhouse conditions. Together, these results establish 3q as a dual-target herbicide and provide a proof-of-concept that simultaneous inhibition of PPO and SPS can be exploited to overcome resistance and expand herbicide design strategies.