Discovery of Pyruvate Kinase as a Novel Target of New Fungicide Candidate 3-(4-Methyl-1,2,3-thiadiazolyl)-6-trichloromethyl-[1,2,4]-triazolo-[3,4-b][1,3,4]-thiadizole

Abstract

Target identification is an essential basis for novel-pesticide development in new molecular design and lead optimization. 3-(4-Methyl-1,2,3-thiadiazolyl)-6-trichloromethyl [1,2,4]triazolo [3,4-b][1,3,4]thiadizole (YZK-C22) is a novel fungicide candidate with specific antifungal activity. We investigated its mode of action, and our studies indicated that YZK-C22 showed no cross resistance against Saccharomyces cerevisiae mutants with classic fungicide targets. Mec1 and Rad53 are two kinases that respond to DNA-replication damage, and the efficacy test showed that YZK-C22 could not perform its fungicidal activity by inhibiting DNA repair. Target screening by drug-affinity-responsive target stability (DARTS) showed that pyruvate kinase (PK), a key enzyme in the glycolytic pathway, was the potent new fungicidal target of YZK-C22. Fifty-eight differentially expressed proteins (DEPs) primarily involved in the metabolic process were identified by isobaric tags for relative and absolute quantification analysis (iTRAQ) in S. cerevisiae, and protein expression in the citrate cycle decreased with treatment of 5 mg/L YZK-C22, which was consistent with the results of DARTS. Molecular-docking analysis further validated that YZK-C22 could dock into the active center of PK instead of phosphoenolpyruvate. The enzyme activity of PK from S. cerevisiae was competitively inhibited with a K i of 3.33 ± 0.28 μmol/L, and the cell-growth inhibition of S. cerevisiae was released by supplementation with pyruvic acid, whereas the growth of S. cerevisiae was not recovered by adding PK's substrate (phosphoenolpyruvate) or allosteric regulator (fructose-1,6-bisphosphate). The present studies uncovered and validated the primary target of the new, potent fungicidal candidate YZK-C22; our results provide a successful, valuable, and applicable case of target discovery and identification for novel-fungicide development.

Synthesis of novel N‐(2‐phenyl‐3‐pyridyl) thiadiazole/isothiazole carboxamide analogs as potent plant elicitors

“…Compounds were tested with three replicates, and the relative gene expression was calculated by GraphPad Prim version 6.02. 32 Expression level higher than Table 1. In vitro fungicidal activity of target compounds at 50 μg mL -1 (% inhibition) 1 was classified as up-regulation and lower than 1 classified as down-regulation.…”

Design, synthesis and fungicidal activity of isothiazole–thiazole derivatives

“…17 Novel fungicide development is one of the important directions and measures for fungicide resistance management. 20 On the basis of our former ndings, 4,5,[21][22][23] to continue our aim of nding novel highly anti-fungal active compounds with novel modes of action and without resistance risks, 4 types of novel target molecules were designed and synthesized for fungicidal activity and systemic acquired resistance evaluation based on PcORP1 as the target and N and S containing vemembered heterocycles as bioactive substructures by the combination of bioactive substructures of plant elicitor isotianil and fungicide oxathiapiprolin according to the active compound derivatization method 24 (Fig. 1 and Table 1).…”

Review on Structures of Pesticide Targets

“…Compound YZK-C22 inhibits pyruvate kinase by reducing the expression of pyruvate kinase proteins in the metabolic process. Pyruvate kinase is a potential bactericidal target [ 35 ] and regarded as a novel target for the discovery of new fungicides [ 36 ]. Rice bacterial blight caused by Xanthomonas oryzae is the most serious bacterial disease of rice.…”