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A Robust and Cost-Effective Luminescent-Based High-Throughput Assay for Fructose-1,6-Bisphosphate Aldolase A
Eun Jeong Cho1, Ashwini K Devkota1, Gabriel Stancu2
1Targeted Therapeutic Drug Discovery and Development, College of Pharmacy, The University of Texas at Austin, Austin, TX, USA.
SLAS Discovery : Advancing Life Sciences R & D
|May 29, 2020
Summary
Researchers developed a new assay to measure the activity of fructose-1,6-bisphosphate aldolase A (ALDOA), an enzyme crucial for cancer growth. This tool enables efficient identification of potential cancer therapeutics by targeting ALDOA.
Area of Science:
- Biochemistry
- Cancer Biology
- Enzyme Assays
Background:
- Hypoxic solid tumors stabilize hypoxia-inducible factor 1 alpha (HIF1α), promoting glycolysis for tumor growth.
- Fructose-1,6-bisphosphate aldolase A (ALDOA) is a glycolytic enzyme that enhances HIF1α expression, making it a key target for cancer therapy.
Purpose of the Study:
- To develop a robust and cost-effective luminescence-based assay for quantifying ALDOA activity.
- To validate the assay's suitability for high-throughput screening (HTS) to identify ALDOA inhibitors.
Main Methods:
- Integrated a known ALDOA activity assay with a commercial NAD/NADH detection kit.
- Developed a luminescence-based detection strategy.
- Performed a compound library screen to assess assay performance.
Main Results:
- Achieved a >12-fold improvement in signal/background (S/B) ratio compared to previous methods.
- Demonstrated excellent statistical assay parameters, including Z' (>0.8) and S/B (~20).
- Confirmed the assay's robustness and readiness for high-throughput screening (HTS).
Conclusions:
- The developed luminescence-based assay is a sensitive and efficient tool for measuring ALDOA activity.
- This platform facilitates cost-effective, large-scale HTS for identifying novel ALDOA inhibitors.
- Targeting ALDOA offers a promising therapeutic strategy for various cancers by disrupting tumor glycolysis and HIF1α stabilization.
