Fluorogenic Assay for Acetohydroxyacid Synthase: Design and Applications,Analytical Chemistry

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Fluorogenic Assay for Acetohydroxyacid Synthase: Design and Applications
Analytical Chemistry ( IF 6.7 ) Pub Date : 2019-10-23 , DOI: 10.1021/acs.analchem.9b02739
Yonghui Xie ₁ , Changyu Zhang ₂ , Zhihua Wang ₃ , Chao Wei ₁ , Ningjing Liao ₁ , Xin Wen ₁ , Congwei Niu ₁ , Long Yi _{2,

4} , Zejian Wang ₃ , Zhen Xi _{1,

4}

Affiliation

Acetohydroxyacid synthase (AHAS) exists in plants and many microorganisms (including gut flora) but not in mammals, making it an attractive drug target. Fluorescent-based methods should be practical for high-throughput screening of inhibitors. Herein, we describe the development of the first AHAS fluorogenic assay based on an intramolecular charge transfer (ICT)-based fluorescent probe. The assay is facile, sensitive, and continuous and can be applied toward various AHASs from different species, AHAS mutants, and crude cell lysates. The fluorogenic assay was successfully applied for (1) high-throughput screening of commerical herbicides toward different AHASs for choosing matching herbicides, (2) identification of a Soybean AHAS gene with broad-spectrum herbicide resistance, and (3) identification of selective inhibitors toward intestinal-bacterial AHASs. Among the AHAS inhibitors, an active agent was found for selective inhibition of obesity-associated Ruminococcus torques growth, implying the possibility of AHAS inhibitors for the ultimate goal toward antiobesity therapeutics. The fluorogenic assay opens the door for high-throughput programs in AHAS-related fields, and the design principle might be applied for development of fluorogenic assays of other synthases.

中文翻译：

乙酰羟酸合酶的荧光测定：设计与应用

乙酰羟酸合酶（AHAS）存在于植物和许多微生物（包括肠道菌群）中，而在哺乳动物中不存在，因此成为有吸引力的药物靶标。基于荧光的方法应可用于抑制剂的高通量筛选。在这里，我们描述了基于分子内电荷转移（ICT）的荧光探针的第一个AHAS荧光检测的发展。该测定法简便，灵敏且连续，可用于来自不同物种的各种AHAS，AHAS突变体和粗细胞裂解液。荧光测定法已成功用于（1）针对不同AHAS的商业除草剂的高通量筛选，以选择匹配的除草剂，（2）大豆的鉴定具有广谱除草剂抗性的AHAS基因，以及（3）鉴定针对肠道细菌AHAS的选择性抑制剂。在AHAS抑制剂中，发现一种活性剂可选择性抑制与肥胖相关的Ruminococcus扭矩增长，这暗示了AHAS抑制剂有可能成为抗肥胖疗法的最终目标。荧光测定法为AHAS相关领域的高通量程序打开了大门，该设计原理可能适用于其他合酶荧光测定法的开发。

更新日期：2019-10-24

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