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SLAS Discov. 2018 Jan;23(1):1-10. doi: 10.1177/2472555217726325. Epub 2017 Aug 18.

A Fluorescence-Based High-Throughput Assay for the Identification of Anticancer Reagents Targeting Fructose-1,6-Bisphosphate Aldolase.

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1 Targeted Therapeutic Drug Discovery and Development Program, Austin, TX, USA.
2 College of Pharmacy, The University of Texas at Austin, Austin, TX, USA.
3 Division of Chemical Biology and Medicinal Chemistry, The University of Texas at Austin, Austin, TX, USA.
4 Sanford Burnham Prebys Medical Discovery Institute Cancer Center, La Jolla, CA, USA.


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-1α, 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 µM 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.


NADH; aldolase A; anticancer; fluorescence; high-throughput screening


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