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

  • J Med Chem. 2026 Jun 11;69(11):13248-13271. doi: 10.1021/acs.jmedchem.6c00235.
Alessandra Fiore  1 ,  Antonio Scarano  2 ,  Giulia Antonini  3 ,  Alexandra Ioana Corfù  3 ,  Lea Sicuro  4  5 ,  Serena Faggiano  2  6 ,  Adriana Celesia  1  3 ,  Chiara Tesoriero  7 ,  Raffaella Pacchiana  1 ,  Andrea Vettori  7 ,  Liaisan Arslanbaeva  8 ,  Saverio Minucci  8  9 ,  Isabella Pallavicini  8 ,  Luca Mollica  4 ,  Lucia Tamborini  3 ,  Massimo Donadelli  1 ,  Paola Conti  3 ,  Stefano Bruno  2 ,  Chiara Borsari  3
Affiliations
  • 1. Department of Neurosciences, Biomedicine and Movement Sciences, Section of Biochemistry, University of Verona, 37134 Verona, Italy.
  • 2. Department of Food and Drug, University of Parma, Parco Area delle Scienze 23/a, 43124 Parma, Italy.
  • 3. Department of Pharmaceutical Sciences, University of Milan, Via Mangiagalli 25, 20133 Milan, Italy.
  • 4. Department of Medical Biotechnologies and Translational Medicine c/o L.I.T.A/University of Milan, Via F.lli Cervi 93, 20090 Segrate (MI), Italy.
  • 5. Fondazione IRCCS Ca'Granda Ospedale Maggiore Policlinico, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, 20133 Milan, Italy.
  • 6. Institute of Biophysics, National Research Council (CNR), Via G. Moruzzi 1, 56124 Pisa, Italy.
  • 7. Department of Biotechnology, University of Verona, 37134 Verona, Italy.
  • 8. Department of Experimental Oncology, IEO European Institute of Oncology IRCSS, 20139 Milan, Italy.
  • 9. Department of Oncology and Hematology-Oncology, University of Milan, 20122 Milan, Italy.
PMID: 42186262 DOI: 10.1021/acs.jmedchem.6c00235
Abstract

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.

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