Development of First-in-Class Covalent Inhibitors of PFKFB3 for PDAC treatment

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with poor prognosis. Metabolic reprogramming drives its malignancy, making cancer metabolism an attractive therapeutic target. However, direct inhibition of glycolytic enzymes is limited by toxicity. The indirect modulation of glycolysis through the inhibition of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB), and particularly the prevalent PDAC isoform PFKFB3, holds promise for safer therapeutic window. Here, we report on the first-in-class covalent PFKFB3 inhibitor, rationally designed to target a previously unexplored cysteine within the active site. Enzyme assays, site-directed mutagenesis, and mass spectrometry confirmed covalent binding. Our ligand reduced viability across multiple PDAC cell lines and suppressed PDAC growth in a zebrafish xenograft model. Moreover, the combination of our ligand with standard chemotherapeutics revealed synergistic effects. This work presents a novel covalent PFKFB3 inhibitor with strong and selective antitumor activity and supports indirect anti-glycolytic therapy as a promising strategy for PDAC treatment