A Fluorescence-Based High-Throughput Assay for the Identification of Anticancer Reagents Targeting Fructose-1,6-Bisphosphate Aldolase
A Fluorescence-Based High-Throughput Assay for the Identification of Anticancer Reagents Targeting Fructose-1,6-Bisphosphate Aldolase
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DOI：
10.1177/2472555217726325
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发表时间：
2018-01-01
期刊：
影响因子：
3.1
通讯作者：
Dalby, Kevin N.
中科院分区：
文献类型：
--
作者：
Cho, Eun Jeong;Devkota, Ashwini K.;Dalby, Kevin N.
高速率的糖酵解为合成代谢提供能量和物质，在广泛的肿瘤细胞中被观察到，使其成为控制癌症生长的潜在途径。ALDOA是糖酵解途径中的一种多功能酶，也能促进HIF-1 α，在缺氧实体瘤中发挥重要作用。目前测定ALDOA活性的方法包括通过偶联酶反应使用吸光度或固有荧光监测体外NADH的消耗。在这里，我们报告了一种均质生化分析的发展，可以克服当前方法的局限性，特别是在高通量药物筛选应用方面。该检测利用市售的Elite NADH检测试剂盒，该试剂盒采用酶促反应，使用荧光探针测量NADH的水平。讨论了测定方法的优化和验证。通过筛选65000个抑制ALDOA的小分子化合物，证明了其高通量筛选（HTS）的可行性。通过验证筛选和剂量反应评估，鉴定出4种IC50低于10 μ M的抑制剂。总之，我们证明了传统的ALDOA分析可以很容易地转化为基于荧光的分析，利用商业NADH检测试剂盒，快速，敏感，廉价，和HTS友好。
A high rate of glycolysis, which supplies energy and materials for anabolism, is observed in a wide range of tumor cells, making it a potential pathway to control cancer growth. ALDOA is a multifunctional enzyme in the glycolytic pathway and also promotes HIF-1 alpha, which is of importance in hypoxic solid tumors. The current method for assaying ALDOA activity involves monitoring the consumption of NADH in vitro using absorbance or intrinsic fluorescence via a coupled enzymatic reaction. Here, we report the development of a homogeneous biochemical assay that can overcome limitations of current methods, in particular for the application of high-throughput drug screening. The assay utilizes the commercially available Elite NADH Assay Kit, which incorporates an enzymatic reaction to measure the level of NADH using a fluorescent probe. Assay optimization and validation are discussed. Its feasibility for high-throughput screening (HTS) was demonstrated by screening 65,000 compounds for the identification of small molecules that inhibit ALDOA. Through a validation screen and dose-response evaluation, four inhibitors with IC50 below 10 mu M were identified. In conclusion, we demonstrate that a traditional ALDOA assay can be transformed readily into a fluorescence-based assay utilizing a commercial NADH detection kit that is rapid, sensitive, inexpensive, and HTS friendly.