Pocket-based lead optimization strategy for the design of laccase inhibitors as potential antifungal agents.

Plant pathogenic fungi are becoming increasingly resistant to antifungal agents, necessitating the development of novel fungicides targeting new pathways. 4-chlorocinnamaldehyde thiosemicarbazide (PMDD-5Y) was the first compound reported to control plant pathogenic fungi by inhibiting laccase activity. By utilizing pocket-based lead optimization strategy, this study designed and synthesized a series of novel thiosemicarbazide derivatives containing the natural product benzophenone structure, with PMDD-5Y as the lead compound. Biological activity testing results showed that the target compounds exhibited significant inhibitory activity against various pathogenic fungi, particularly Valsa mali and Sclerotinia sclerotiorum. Among them, compound 7i had EC50 values of 0.07 μg/mL and 0.14 μg/mL against S. sclerotiorum and V. mali, respectively, which were superior to the lead compound PMDD-5Y (EC50 = 0.80 μg/mL and 0.52 μg/mL). Moreover, 7i exhibited notable curative and protective effects on S. sclerotiorum and V. mali in vivo. Scanning electron microscopy results revealed that 7i caused distortion of hyphal growth and disappearance of septa. Enzyme activity testing demonstrated that compound 7i (IC50 = 21.88 μg/mL) had a significant inhibitory effect on laccase, which was superior to PMDD-5Y (IC50 = 132.63 μg/mL) and cysteine (IC50 = 39.32 μg/mL). Additionally, molecular docking between 7i and laccase, and density functional theory (DFT) calculations were discussed. Overall, this study identified a novel lead compound that can be used to develop laccase-targeting antifungal candidates to control plant pathogenic fungi.