The agricultural fungicide cymoxanil (CMX) is commonly used in the treatment of plant pathogens, such as Phytophthora infestans. Although the use of CMX is widespread throughout the agricultural industry and internationally, the exact mechanism of action behind this fungicide remains unclear. Therefore, we sought to elucidate the biocidal mechanism underlying CMX. This was accomplished by first performing a large-scale chemical-genomic screen comprising the 4000 haploid non-essential gene deletion array of the yeast Saccharomyces cerevisiae. We found that gene families related to de novo purine biosynthesis and ribonucleoside synthesis were enriched in the presence of CMX. These results were confirmed through additional spot-test and colony counting assays. We next examined whether CMX affects RNA biosynthesis. Using qRT-PCR and expression assays, we found that CMX appears to target RNA biosynthesis possibly through the yeast dihydrofolate reductase (DHFR) enzyme Dfr1. To determine whether DHFR is a target of CMX, we performed an in-silico molecular docking assay between CMX and yeast, human, and P. infestans DHFR. The results suggest that CMX directly interacts with the active site of all tested forms of DHFR using conserved residues. Using an in vitro DHFR activity assay we observed that CMX inhibits DHFR activity in a dose-dependent relationship.

农用杀菌剂霜脲氰 (CMX) 常用于治疗植物病原体，例如致病疫霉。尽管 CMX 在整个农业行业和国际上广泛使用，但这种杀菌剂背后的确切作用机制仍不清楚。因此，我们试图阐明 CMX 的杀菌机制。这是通过首先进行大规模化学基因组筛选来实现的，该筛选包括酿酒酵母的 4000 个单倍体非必需基因缺失阵列。我们发现与嘌呤从头生物合成和核糖核苷合成相关的基因家族在 CMX 存在下得到富集。这些结果通过额外的斑点测试和菌落计数测定得到证实。接下来我们检查了 CMX 是否影响 RNA 生物合成。使用 qRT-PCR 和表达测定，我们发现 CMX 似乎可能通过酵母二氢叶酸还原酶 (DHFR) Dfr1 来靶向 RNA 生物合成。为了确定 DHFR 是否是 CMX 的靶标，我们在 CMX 和酵母、人类和致病疫霉DHFR 之间进行了计算机分子对接测定。结果表明，CMX 使用保守残基直接与所有测试形式的 DHFR 的活性位点相互作用。使用体外 DHFR 活性测定，我们观察到 CMX 以剂量依赖性关系抑制 DHFR 活性。