A Novel Multiple-Read Screen for Metabolically Active Compounds Based on a Genetically Encoded FRET Sensor for ATP

Area of Science:

• Biochemistry
• Molecular Biology
• Cancer Therapeutics

Background:

• Glycolysis and mitochondrial energetics are key targets for antiproliferative cancer drug development.
• Existing assays may not efficiently differentiate between inhibitors of distinct metabolic pathways.

Purpose of the Study:

• To develop and validate a novel assay for screening compounds targeting cellular ATP production.
• To identify inhibitors of glycolysis and oxidative phosphorylation.
• To evaluate the antiproliferative potential of targeting these metabolic pathways in cancer cells.

Main Methods:

• Genetically encoded intramolecular Förster resonance energy transfer (FRET) sensor for adenosine triphosphate (ATP) expressed in K562 cells.
• Long-term expression and fluorescent plate reader-based assay for compound screening.
• Screening of a National Cancer Institute (NCI) compound library.
• Analysis of K562 cell proliferation data.

Main Results:

• Identified novel inhibitors of oxidative phosphorylation-dependent ATP production and glycolysis.
• Demonstrated the assay's capability to identify glycolysis inhibitors targeting hexokinase activity.
• Found that glycolysis inhibitors were significantly more antiproliferative than inhibitors of oxidative phosphorylation.

Conclusions:

• The developed FRET-based assay is a powerful tool for screening compounds affecting cellular metabolism.
• Inhibiting glycolysis is a highly effective antiproliferative strategy in K562 cancer cells.
• The assay facilitates the identification of compounds with specific mechanisms of metabolic inhibition.