Characterization of F-30 as an FGFR2/4-selective covalent inhibitor with activity against clinically relevant...

Fibroblast growth factor receptors FGFR2 and FGFR4 are oncogenic drivers in multiple cancers, yet most approved FGFR inhibitors lack subtype selectivity and are prone to acquired resistance. Here we identify F-30 as, to our knowledge, the first covalent inhibitor with preferential activity toward both FGFR2 and FGFR4 through covalent targeting of a conserved P-loop cysteine. Structural analyses reveal that differential engagement of the P-loop and surrounding binding environment contributes to paralog discrimination. Consistent with these structural observations, F-30 showed an approximately 12-fold lower K_I and 13-fold higher k_inact/K_I for FGFR2 than for FGFR1, whereas their k_inact values were comparable. These results indicate that the preference for FGFR2 over FGFR1 is primarily established during pre-covalent recognition. F-30 retained strong activity against multiple clinically relevant FGFR2 resistance-associated mutations but showed reduced potency against gatekeeper mutations. In cellular models, F-30 preferentially inhibited the proliferation of FGFR2/4-driven cancer cells, while substantially weaker pathway inhibition was observed in FGFR1- and FGFR3-driven models than in FGFR2-dependent cells. Taken together, these findings identify structural and kinetic features that contribute to the subtype-selective profile of F-30 and may guide the design of covalent FGFR inhibitors targeting the conserved P-loop cysteine.