A Robust and Cost-Effective Luminescent-Based High-Throughput Assay for Fructose-1, 6-Bisphosphate Aldolase A. (October 2020)

• Record Type: Journal Article
• Authors: Cho, Eun Jeong; Devkota, Ashwini K.; Stancu, Gabriel; Edupunganti, Ramakrishna; Debevec, Ginamarie; Giulianotti, Marc; Houghten, Richard; Powis, Garth; Dalby, Kevin N.
• Is Part Of: SLAS discovery. Volume 25:Number 9(2020)
• Page Start: 1038
• Page End: 1046
• Publication Date: 2020-10
• Subjects: aldolase A -- luminescence -- NADH -- anticancer -- high-throughput screening; Drugs -- Analysis -- Periodicals; Drugs -- Testing -- Periodicals; Biomolecules -- Analysis -- Periodicals; Biomolecules -- Analysis; Drugs -- Analysis; Drugs -- Testing; Drug Evaluation, Preclinical; Molecular Biology -- methods; Periodicals; Periodicals; 615.1
• DOI: 10.1177/2472555220926146
• Languages: English
• ISSNs: 2472-5552

Abstract

Hypoxic solid tumors induce the stabilization of hypoxia-inducible factor 1 alpha (HIF1α), which stimulates the expression of many glycolytic enzymes and hypoxia-responsive genes. A high rate of glycolysis supports the energetic and material needs for tumors to grow. Fructose-1, 6-bisphosphate aldolase A (ALDOA) is an enzyme in the glycolytic pathway that promotes the expression of HIF1α. Therefore, inhibition of ALDOA activity represents a potential therapeutic approach for a range of cancers by blocking two critical cancer survival mechanisms. Here, we present a luminescence-based strategy to determine ALDOA activity. The assay platform was developed by integrating a previously established ALDOA activity assay with a commercial NAD/NADH detection kit, resulting in a significant (>12-fold) improvement in signal/background (S/B) compared with previous assay platforms. A screening campaign using a mixture-based compound library exhibited excellent statistical parameters of Z′ (>0.8) and S/B (~20), confirming its robustness and readiness for high-throughput screening (HTS) application. This assay platform provides a cost-effective method for identifying ALDOA inhibitors using a large-scale HTS campaign.