Discovery of Novel Triazolothiadiazines as Fungicidal Leads Targeting Pyruvate Kinase

Abstract

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 EC 50 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 EC 50 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 IC 50 , 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.

Novel [1,2,4]-Triazolo[3,4-b]-[1,3,4]thiadizoles as Potent Pyruvate Kinase Inhibitors for Fungal Control

“…Binding energy is an important factor for assessing the interaction between a receptor protein and its ligands. , At a computation level, the higher the binding energy, the higher the fungicidal activity of target compounds. According to the SARs found in previous studies, we evaluated the binding energy of the designed compounds 5a – 5g with RsPK using the AutoDock Vina, and then synthesized and assessed their performance against fungi. Compounds 5h – 5z were then designed according to the fungicidal activity of compounds 5a – 5g .…”

“…The procedures were modified according to the methods described in the literature. ,,, The hydrazine hydrate (12 mmol) was added to a solution of compounds 1 (10 mmol) dissolved in 80 mL of ethanol. The mixture was heated to 80 °C and monitored by TLC until the reaction was complete.…”

“…solani ( R. s ), and Sclerotinia sclerotiorum ( S. s ) was assessed in vitro at a concentration of 50 μg/mL using the mycelium growth-inhibition method. The median effective concentration (EC 50 ) of the active target compounds was assessed according to the reported method with some modifications. , Generally, a mother solution of the test compound (25,000 μg/mL) was prepared with N , N -dimethylformamide (DMF) as a solvent, and then a test solution (500 μg/mL) was made by adding 100 μL of mother solution to 4900 μL of sterile water with 0.1% Tween 80. Finally, the culture with 50 μg/mL of the test compound was prepared by mixing 4 mL of the test solution with 36 mL of potato dextrose agar (PDA).…”

“…The PK enzyme activity of 5g , 5o , and 5z was determined using a PK assay kit (G0811W, Suzhou Grace Biotechnology Co., Ltd.) as reported previously R.…”

“…A fungicidal lead [3-(4-methyl-1,2,3-thiadiazol-5-yl)-6-(trichloromethyl)-[1,2,4]-triazolo­[3,4- b ]-[1,3,4]­thiadiazole] ( YZK-C22 ) targeting pyruvate kinase (PK) was discovered in our previous study using drug affinity reaction target stability (DARTS) against Saccharomyces cerevisiae, and its mode of action was further validated by the corresponding enzymatic studies. , Afterward, its mode of action was further validated in the Botrytis cinerea system . In addition, a few chemical scaffolds such as isothiazopurines, triazolothiadiazines, and psoralen-based 1,3,4-oxadiazoles were successfully discovered as PK inhibitors by computer-aided molecular design technology. − These findings expanded the structural diversity of PK inhibitors and laid a foundation for further structural optimization of PK inhibitors.…”