Data from: Identification and evaluation of novel acetolactate synthase inhibitors as antifungal agents

High-throughput phenotypic screening against yeast Saccharomyces cerevisiae revealed a series of triazolo-pyrimidine-sulfonamide compounds with broad-spectrum antifungal activity, no significant cytotoxicity, and low protein binding. To elucidate the target of this series we have applied a chemogenomic profiling approach using the S. cerevisiae deletion collection. All compounds of the series yielded highly similar profiles that suggested acetolactate synthase (Ilv2p, catalyzes the first common step in branched chain amino acid biosynthesis) as a possible target. High correlation to profiles of known Ilv2p inhibitors like chlorimuron-ethyl provided further evidence for a similar mechanism of action. Genome-wide mutagenesis in S. cerevisiae identified 13 resistant clones with 3 different mutations in the catalytic subunit of acetolactate synthase that also conferred cross-resistance to established Ilv2p inhibitors. Mapping the mutations into the published Ilv2p crystal structure outlined the chlorimuron-ethyl binding cavity and it was possible to dock the triazolo-pyrimidine-sulfonamide compound into this pocket in silico. However, fungal growth inhibition could be bypassed through supplementation with exogenous branched chain amino acids, or by the addition of serum to the medium in all of the fungal organisms tested except for Aspergillus fumigatus. Thus, these data support the identification of triazolo-pyrimidine-sulfonamide as inhibitors of acetolactate synthase but suggest that targeting may be compromised due to the possibility of nutrient bypass in vivo.针对酿酒酵母（Saccharomyces cerevisiae）开展的高通量表型筛选，发现了一系列兼具广谱抗真菌活性、无显著细胞毒性且蛋白结合率较低的三唑并嘧啶磺酰胺（triazolo-pyrimidine-sulfonamide）类化合物。为阐明该类化合物的作用靶点，我们采用基于酿酒酵母缺失突变库的化学基因组谱分析策略。该系列所有化合物均呈现高度相似的谱图特征，提示乙酰乳酸合酶（Ilv2p，催化支链氨基酸生物合成的首个共同步骤）可能为其作用靶点。与乙磺隆（chlorimuron-ethyl）等已知Ilv2p抑制剂的谱图具有高度相关性，进一步佐证了二者具有相似的作用机制。通过对酿酒酵母开展全基因组诱变，我们筛选得到13株耐药克隆，其乙酰乳酸合酶催化亚基存在3种不同突变，且这些突变可使菌株对已确认的Ilv2p抑制剂产生交叉耐药性。将上述突变位点定位至已发表的Ilv2p晶体结构中，可明确乙磺隆的结合口袋，且可通过计算机模拟（in silico）将三唑并嘧啶磺酰胺类化合物对接至该口袋内。然而，在所测试的所有真菌菌株中，烟曲霉（Aspergillus fumigatus）除外，通过添加外源性支链氨基酸或向培养基中加入血清，均可绕过该类化合物对真菌生长的抑制作用。综上，上述数据支持三唑并嘧啶磺酰胺类化合物为乙酰乳酸合酶抑制剂的结论，但同时提示由于体内可能存在营养旁路途径，该类化合物的靶点靶向作用或受影响。