Pyruvate kinase (PK) was discovered as a potent new target for novel fungicide development. A series of novel triazolothiadiazine derivatives were rationally designed and synthesized by a ring expansion strategy and computer-aided pesticide design using the 3D structure of Rhizoctonia solani PK (RsPK) obtained by homology modeling as a receptor and our previously discovered lead YZK-C22 as a ligand. The in vitro bioassay results indicated that compounds 4g, 6h, 6m, 6n, 6o, and 6p exhibited good activity against R. solani with the EC50 values falling between 10.99 and 72.76 μM. Especially, 6m showed similar potency to YZK-C22 (10.99 vs 11.97 μM of the EC50 value, respectively). The in vivo bioassay results suggested that 6m against R. solani at a concentration of 200 μg/mL displayed a numerically higher inhibition than YZK-C22 (70 vs 60%, respectively). A field experiment validated that 6m at an application rate of 120 g ai/ha showed comparable efficacy against R. solani to thifluzamide at an application rate of 80 g ai/ha (77.80 vs 84.5%, respectively). Enzymatic inhibition suggested that the potency of 6m was about twofold lower than that of YZK-C22 (67.30 vs 32.64 μM of IC50, respectively). Fluorescence quenching studies validated that RsPK was quenched by both 6m and YZK-C22, implying that they both might act at the same target site of PK. A possible binding conformation of 6m in the RsPK active site was depicted by molecular docking. Our studies suggest that 6m could be a fungicidal lead targeting PK.
Keywords: fungicidal activity; molecular docking; pyruvate kinase inhibitor; ring expansion strategy.