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A high-throughput screening campaign against PFKFB3 identified potential inhibitors with novel scaffolds

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.

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Fig. 1: Documented tool compounds that inhibit PFKFB3 activities.
Fig. 2: Optimization of the PFKFB3 biochemical assay (ADP-Glo kinase assay).
Fig. 3: HTS campaign.
Fig. 4: Hit compounds without PAINS alerts showed binding affinity to PFKFB3 and exhibited tube formation inhibiting property.
Fig. 5: Docking poses and interaction modes of the three PFKFB3 inhibitors.

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Acknowledgements

We are indebted to Nico Callewaert and Wim Nerinckx for valuable discussions and technical assistance. This work was partially supported by National Natural Science Foundation of China 81961138001 (MWW), 81872915 (MWW), 82073904 (MWW), 82121005 (DHY) and 81973373 (DHY); National Science & Technology Major Project of China–Key New Drug Creation and Manufacturing Program 2018ZX09735-001 (MWW) and 2018ZX09711002-002-005 (DHY); the National Key Basic Research Program of China 2018YFA0507000 (MWW); the Major Science and Technology Program of Hainan Province ZDKJ2021028 (DHY and QTZ); Kom Op Tegen Kanker (Stand up to Cancer, Flemish Cancer Society) (PC); Methusalem funding (Flemish Government); and Fund for Scientific Research-Flanders (FWO-Vlaanderen G0E4419N) (PC).

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PC, MD and MWW designed research; JL, MD, GE, YZ, WBF, VL, FFM and QTZ performed research; JL, GE, MD, HPL and DHY analyzed data; and JL, MD, GE, QTZ and MWW wrote the paper with inputs from all the co-authors.

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Correspondence to Ming-wei Wang or De-hua Yang.

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Li, J., Zhou, Y., Eelen, G. et al. A high-throughput screening campaign against PFKFB3 identified potential inhibitors with novel scaffolds. Acta Pharmacol Sin (2022). https://doi.org/10.1038/s41401-022-00989-1

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Keywords

  • solid tumors
  • angiogenesis
  • glycolysis
  • high-throughput screening
  • PFKFB3 inhibitors

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