Discovery of a Subnanomolar Inhibitor of Protoporphyrinogen IX Oxidase via Fragment-Based Virtual Screening

Protoporphyrinogen IX oxidase (PPO, E.C. 1.3.3.4), a key functional enzyme existing in various organisms, is acknowledged to be one of the most important action targets in the development of herbicides due to its pivotal roles in chlorophyll and heme biosynthesis pathways. As our persistent research work on the discovery of novel PPO-inhibiting herbicides, a new compound methyl 2-((5-(3-chloro-4,5,6,7-tetrahydro-2H-indazol-2-yl)-6-fluorobenzo[d]thiazol-2-yl)thio)acetate (8aj, K_i = 16 nM) was screened out as a hit compound via a fragment-based virtual screening method performed in the Auto Core Fragment in silico Screening web server. Subsequently, through a fused process of "hit-to-lead" optimization guided by molecular simulation, a total of 30 3-chloro-4,5,6,7-tetrahydro-2H-indazol-benzo[d]thiazole derivatives were synthesized and characterized. The results of the enzymatic inhibition bioassay showed that more than half of the newly synthesized compounds displayed higher activity against Nicotiana tabacum PPO (NtPPO) than oxadiazon, a commercial PPO-inhibiting herbicide. In particular, compound 8ab, a subnanomolar inhibitor with a K_i value of 380 pM against NtPPO, was discovered, which showed to be 71-fold more active than the commercial control oxadiazon (K_i = 27 nM), and was proven to be the most potent PPO inhibitor so far. Furthermore, the greenhouse assay demonstrated that most of the synthetic compounds showed good herbicidal activity toward the tested weeds. Especially, compound 8ad (K_i = 670 pM) showed the most promising post-emergence herbicidal activity with a broad spectrum of weed control even at a concentration as low as 37.5 g a.i./ha and relatively safe to rice at a dosage of 150 g a.i./ha, indicating that 8ad has the greatest potential to be developed as a new herbicide for weed control in paddy fields. This work provides a paradigm for the rational design and discovery of a novel PPO-inhibiting herbicide guided by the fragment-based drug design.