Characterizing and Overriding the Structural Mechanism of the Quizartinib-resistant FLT3 “Gatekeeper” F691L Mutation with PLX3397. Homo sapiens

Tyrosine kinase domain mutations are a common cause of acquired resistance to tyrosine kinase inhibitors (TKIs) used to treat cancer, including the FLT3 inhibitor quizartinib. Mutation of kinase “gatekeeper” residues, which control access to an allosteric pocket adjacent to the ATP-binding site, has been frequently implicated in TKI resistance. The molecular underpinnings of gatekeeper mutation resistance are incompletely understood. We report the first co-crystal structure of FLT3 with the TKI quizartinib, which demonstrates that quizartinib binding relies on critical edge-to-face aromatic interactions with the gatekeeper F691 residue, and F830 within the highly conserved DFG motif in the activation loop. This reliance makes quizartinib highly critically vulnerable to gatekeeper and activation loop mutations while minimizing the impact of other mutations. Moreover, we identify PLX3397, a novel FLT3 inhibitor that retains activity against the F691L mutant due to a binding mode that depends less critically on specific interactions with the gatekeeper position.