pexidartinib and quizartinib

FMS-like tyrosine kinase 3 (FLT3) has been found to be mutated in ~ 30% of acute myeloid leukaemia patients. Small-molecule inhibitors targeting FLT3 that are currently approved or still undergoing clinical trials are subject to drug resistance due to FLT3 mutations. How these mutations lead to drug resistance is hitherto poorly understood. Herein, we studied the molecular mechanism of the drug resistance mutations D835N, Y842S and M664I, which confer resistance against the most advanced inhibitors, quizartinib and PLX3397 (pexidartinib), using enzyme kinetics and computer simulations. In vitro kinase assays were performed to measure the comparative catalytic activity of the native protein and the mutants, using a bacterial expression system developed to this aim. Our results reveal that the differential drug sensitivity profiles can be rationalised by the dynamics of the protein-drug interactions and perturbation of the intraprotein contacts upon mutations. Drug binding induced a single conformation in the native protein, whereas multiple conformations were observed otherwise (in the mutants or in the absence of drugs). The end-point kinetics measurements indicated that the three resistant mutants conferred catalytic activity that is at least as high as that of the reference without such mutations. Overall, our calculations and measurements suggest that the structural dynamics of the drug-resistant mutants that affect the active state and the increased conformational freedom of the remaining inactive drug-bound population are the two major factors that contribute to drug resistance in FLT3 harbouring cancer cells. Our results explain the mechanism of drug resistance mutations and can aid to the design of more effective tyrosine kinase inhibitors.

Topics: Aminopyridines; Benzothiazoles; Drug Resistance, Neoplasm; fms-Like Tyrosine Kinase 3; Humans; Mutation; Neoplasms; Phenylurea Compounds; Protein Conformation; Protein Kinase Inhibitors; Pyrroles

Topics: Aminopyridines; Antineoplastic Agents; Benzothiazoles; Biomarkers; Bone Marrow; Cell Line, Tumor; Clinical Trials as Topic; Dasatinib; fms-Like Tyrosine Kinase 3; Gene Expression; Hair; Hematopoietic Stem Cells; Humans; Indazoles; Leukemia, Myeloid, Acute; Niacinamide; Phenylurea Compounds; Pigmentation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-kit; Pyrimidines; Pyrroles; Sorafenib; Sulfonamides; Thiazoles

Tyrosine kinase domain mutations are a common cause of acquired clinical resistance to tyrosine kinase inhibitors (TKI) 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-mediated resistance are incompletely understood. We report the first cocrystal structure of FLT3 with the TKI quizartinib, which demonstrates that quizartinib binding relies on essential edge-to-face aromatic interactions with the gatekeeper F691 residue, and F830 within the highly conserved Asp-Phe-Gly motif in the activation loop. This reliance makes quizartinib critically vulnerable to gatekeeper and activation loop substitutions while minimizing the impact of mutations elsewhere. Moreover, we identify PLX3397, a novel FLT3 inhibitor that retains activity against the F691L mutant due to a binding mode that depends less vitally on specific interactions with the gatekeeper position.. We report the first cocrystal structure of FLT3 with a kinase inhibitor, elucidating the structural mechanism of resistance due to the gatekeeper F691L mutation. PLX3397 is a novel FLT3 inhibitor with in vitro activity against this mutation but is vulnerable to kinase domain mutations in the FLT3 activation loop.

Topics: Aminopyridines; Animals; Antineoplastic Agents; Benzothiazoles; Cell Line, Tumor; Drug Resistance, Neoplasm; Enzyme Activation; fms-Like Tyrosine Kinase 3; Heterografts; Humans; Leukemia, Myeloid, Acute; Mice; Models, Molecular; Molecular Conformation; Mutation; Phenylurea Compounds; Protein Binding; Protein Interaction Domains and Motifs; Protein Kinase Inhibitors; Pyrroles; Recurrence; Structure-Activity Relationship