Smithson David C, Shelat Anang A, Baldwin Jeffrey, Phillips Margaret A, Guy R Kiplin
Graduate Program in Chemistry and Chemical Biology, University of California, San Francisco, California, USA.
Assay Drug Dev Technol. 2010 Apr;8(2):175-85. doi: 10.1089/adt.2009.0249.
Herein, we describe the optimization of a linked enzyme assay suitable for high-throughput screening of decarboxylases, a target family whose activity has historically been difficult to quantify. Our approach uses a commercially available bicarbonate detection reagent to measure decarboxylase activity. The assay is performed in a fully enclosed automated screening system under inert nitrogen atmosphere to minimize perturbation by exogenous CO2. Receiver operating characteristic (ROC) analysis following a pilot screen of a small library of approximately 3,600 unique molecules for inhibitors of Trypanosoma brucei ornithine decarboxylase quantitatively demonstrates that the assay has excellent discriminatory power (area under the curve = 0.90 with 95% confidence interval between 0.82 and 0.97).
在此，我们描述了一种适用于高通量筛选脱羧酶的连锁酶测定法的优化，脱羧酶是一个目标家族，其活性历来难以量化。我们的方法使用市售的碳酸氢盐检测试剂来测量脱羧酶活性。该测定在惰性氮气气氛下的全封闭自动筛选系统中进行，以尽量减少外源二氧化碳的干扰。在对约3600个独特分子的小文库进行初步筛选以寻找布氏锥虫鸟氨酸脱羧酶抑制剂后，接受者操作特征（ROC）分析定量表明该测定具有出色的区分能力（曲线下面积 = 0.90，95% 置信区间在0.82至0.97之间）。
