Fibroblast growth factor receptor-4 (FGFR4) is a tyrosine kinase with a range of important physiological functions.
However, it is also frequently mutated in various cancers and is now generating significant interest as a potential
therapeutic target. Unfortunately, biochemical characterization of its role in disease, and further evaluation as a drug target
is hampered by lack of a specific inhibitor. We aimed to discover new inhibitors for FGFR4 ab initio using a strategy
combining in silico, in vitro and cell-based assays. We used the homologous FGFR1 to calculate docking scores of a
chemically-diverse library of approximately 2000 potential kinase inhibitors. Nineteen potential inhibitors and ten randomly-
selected negative controls were taken forward for in vitro FGFR4 kinase assays. All compounds with good docking
scores significantly inhibited FGFR4 kinase activity, some with sub-micromolar (most potent being V4-015 with an IC50
of 0.04 μM). Four of these compounds also demonstrated substantial activity in cellular assays using the FGFR4-
overexpressing breast carcinoma cell line, MDA-MB453. Through immunoblot assays, these compounds were shown to
block the phosphorylation of the FGFR4 adaptor protein, FGFR substrate protein-2α (FRS2α). The most potent compound
to date, V4-015, suppressed proliferation of MDA-MB453 cells at sub-micromolar concentrations, activated the
pro-apoptotic caspases 3/7 and inhibited cellular migration. While achieving complete selectivity of this compound for
FGFR4 will require further lead optimization, this study has successfully identified new chemical scaffolds with unprecedented
FGFR4 inhibition capacities that will support mechanism of action studies and future anti-cancer drug design.