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Updated: Aug 28, 2025
Identification of Kinase-substrate Pairs Using High Throughput Screening
Published on: August 29, 2015
A high-throughput screening campaign against PFKFB3 identified potential inhibitors with novel scaffolds
Jie Li1, Yan Zhou2, Guy Eelen3
1Department of Pharmacology, School of Basic Medical Sciences, Fudan University, Shanghai, 200032, China.
Abstract:
The growth of solid tumors depends on tumor vascularization and the endothelial cells (ECs) that line the lumen of blood vessels. ECs generate a large fraction of ATP through glycolysis, and elevation of their glycolytic activity is associated with angiogenic behavior in solid tumors. 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) positively regulates glycolysis via fructose-2/6-bisphosphate, the product of its kinase activity. Partial inhibition of glycolysis in tumor ECs by targeting PFKFB3 normalizes the otherwise abnormal tumor vessels, thereby reducing metastasis and improving the outcome of chemotherapy. Although a limited number of tool compounds exist, orally available PFKFB3 inhibitors are unavailable. In this study we conducted a high-throughput screening campaign against the kinase activity of PFKFB3, involving 250,240 chemical compounds. A total of 507 initial hits showing >50% inhibition at 20 µM were identified, 66 of them plus 1 analog from a similarity search consistently displayed low IC50 values (<10 µM). In vitro experiments yielded 22 nontoxic hits that suppressed the tube formation of primary human umbilical vein ECs at 10 µM. Of them, 15 exhibited binding affinity to PFKFB3 in surface plasmon resonance assays, including 3 (WNN0403-E003, WNN1352-H007 and WNN1542-F004) that passed the pan-assay interference compounds screening without warning flags. This study provides potential leads to the development of new PFKFB3 inhibitors.
Insights
Researchers screened 250,240 compounds to find inhibitors for 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), an enzyme crucial for tumor blood vessel growth. This study identified promising PFKFB3 inhibitors, offering potential for new cancer therapies.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Tumor growth relies on vascularization by endothelial cells (ECs), which utilize glycolysis for ATP production.
- Elevated EC glycolysis is linked to tumor angiogenesis, and targeting 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) can normalize tumor vasculature.
- Orally available PFKFB3 inhibitors are currently lacking, presenting a therapeutic gap.
Purpose of the Study:
- To identify novel, orally available inhibitors of PFKFB3 kinase activity.
- To discover compounds that suppress endothelial cell tube formation and normalize tumor vasculature.
Main Methods:
- Conducted a high-throughput screening of 250,240 chemical compounds against PFKFB3 kinase activity.
- Validated initial hits through IC50 determination, in vitro tube formation assays, and surface plasmon resonance (SPR) binding assays.
- Assessed hits for pan-assay interference compounds (PAINS) to ensure compound validity.
Main Results:
- Identified 507 initial hits, with 66 compounds and 1 analog showing low IC50 values (<10 µM).
- 22 non-toxic compounds inhibited primary human umbilical vein EC tube formation.
- 15 compounds demonstrated binding affinity to PFKFB3 via SPR, with 3 compounds (WNN0403-E003, WNN1352-H007, WNN1542-F004) passing PAINS screening.
Conclusions:
- This study successfully identified novel PFKFB3 inhibitors with potential for therapeutic development.
- The identified compounds represent promising leads for creating orally available drugs to target tumor angiogenesis and improve cancer treatment outcomes.
