First-in-Class Covalent Inhibitors of PFKFB3: Discovery and Characterization in PDAC Models.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer, driven by metabolic reprogramming. Since direct glycolytic enzyme inhibition is limited by toxicity, indirect glycolysis modulation through inhibition of the kinase activity of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) may offer a safer therapeutic strategy. Herein, we report the first-in-class covalent PFKFB3 inhibitor (6), targeting a previously unexplored cysteine. Enzyme assays, site-directed mutagenesis, and mass spectrometry confirmed covalent binding and kinetic selectivity for PFKFB3. Compound 6 reduced viability across multiple PDAC cell lines and suppressed PDAC growth in zebrafish xenografts. Its combination with standard chemotherapeutics revealed synergistic effects. Although the limited cellular activity of 6 restricts its use as a chemical probe in biological studies, we proved for the first time the druggability of a previously unexplored cysteine in PFKFB3. Our work represents a significant achievement in the selective targeting of this kinase, paving the way for an innovative mechanism of action for PFKFB3 inhibitors.