Discovery of a New Inhibitor for PFKFB2 Kinase Activity Through Mechanistic Studies

Significance of the topic

The 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) enzymes are critical regulators of glycolysis, controlling cellular energy metabolism in insulin-sensitive tissues and cancer.
Aberrant PFKFB2 activity is linked to various diseases, including cancer, diabetes, and fibrosis, highlighting the need for selective inhibitors and reliable analytic methods to study enzyme function in a physiological context.

Objectives and overview of the study

This study aimed to develop a robust assay pipeline for identifying small-molecule inhibitors of PFKFB2 kinase activity.
Using an AI-driven virtual screen with the AtomNet platform, a focused library was tested to discover novel modulators.
The lead compound, B2, was characterized through complementary metabolic analyses to confirm its mechanism of action.

Methodology and instrumentation

A multi-tiered approach combined in vitro enzyme assays with cellular and metabolomic analyses:

• Enzymatic assay: PPi-PFK-coupled activity measurement of purified PFKFB2 and PFKFB3.
• Functional metabolism: Extracellular acidification rate (ECAR) assessed by Agilent Seahorse XFe24 glycolysis stress test in A-498 kidney cancer cells.
• Targeted metabolomics: LC/MS using Agilent 1290 Infinity II LC coupled to 6546 Q-TOF with HILIC-Z separation; data processed with Agilent MassHunter and METLIN PCDL.
• Cell normalization: Hoechst 33342 staining and live cell counts on Agilent BioTek Cytation 5.

Main results and discussion

B2 emerged as a dual inhibitor of PFKFB2 (IC50 ≈ 3.3 µM) and PFKFB3 (IC50 ≈ 11.9 µM), distinct from the benchmark inhibitor PFK158.
Treatment with B2 significantly reduced basal glycolysis and maximal glycolytic capacity in A-498 cells, as measured by ECAR.
Metabolomic profiling revealed decreased fructose-1,6-bisphosphate levels, an increased fructose-6-phosphate/fructose-1,6-bisphosphate ratio, and lowered downstream intermediates (G3P, 3-PGA, PEP), confirming PFK-1 blockage.

Benefits and practical applications of the method

The integrated workflow delivers both dynamic flux measurements and high-resolution metabolite quantification, enabling a comprehensive view of metabolic perturbations.
B2 serves as a valuable chemical probe for PFKFB-driven pathways and a promising lead for therapeutic development targeting glycolytic regulation in disease.

Future trends and potential applications

Upcoming advances include the Seahorse XF Flex analyzer with glycolytic rate assays for more physiological measurements, updated Q-TOF platforms for enhanced sensitivity, and expanded AI-driven screening to accelerate inhibitor discovery.

Conclusion

By combining Seahorse metabolic flux analysis with targeted LC/Q-TOF metabolomics, this study validated B2 as a novel PFKFB2/3 inhibitor and demonstrated a streamlined pipeline for metabolic drug discovery.

Instrumentation used

• Agilent Seahorse XFe24 extracellular flux analyzer
• Agilent 1290 Infinity II LC system
• Agilent 6546 quadrupole time-of-flight (Q-TOF) MS
• Agilent InfinityLab Poroshell 120 HILIC-Z column
• Agilent BioTek Cytation 5 cell imaging reader
• Agilent Captiva EMR-Lipid cartridges

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