Crystal Structure of the FLT3 Kinase Domain Bound to the Inhibitor Quizartinib (AC220)

Abstract

More than 30% of acute myeloid leukemia (AML) patients possess activating mutations in the receptor tyrosine kinase FMS-like tyrosine kinase 3 or FLT3. A small-molecule inhibitor of FLT3 (known as quizartinib or AC220) that is currently in clinical trials appears promising for the treatment of AML. Here, we report the co-crystal structure of the kinase domain of FLT3 in complex with quizartinib. FLT3 with quizartinib bound adopts an “Abl-like” inactive conformation with the activation loop stabilized in the “DFG-out” orientation and folded back onto the kinase domain. This conformation is similar to that observed for the uncomplexed intracellular domain of FLT3 as well as for related receptor tyrosine kinases, except for a localized induced fit in the activation loop. The co-crystal structure reveals the interactions between quizartinib and the active site of FLT3 that are key for achieving its high potency against both wild-type FLT3 as well as a FLT3 variant observed in many AML patients. This co-complex further provides a structural rationale for quizartinib-resistance mutations.

High-throughput identification of FLT3 wild-type and mutant kinase substrate preferences and application to design of sensitive in vitro kinase assay substrates

“…Bulky, hydrophobic mutations at position 835 in FLT3 are thought to confer resistance to quizartinib, due to the effects of the side chains on the structure and dynamics of the DFG loop in the kinase domain, with the extra steric bulk disrupting the stability of the inactive conformation. 44,45 The FAStide-E quizartinib dose response results for the D835Y mutant were consistent with this model, with essentially no significant inhibition even at concentrations as high as 100 nM (>10,000-fold higher than the IC50 for quizartinib against the WT FLT3). However using FAStide-F as the substrate, inhibition was observed in the same IC50 range as for sorafenib.…”

The MERTK/FLT3 inhibitor MRX-2843 overcomes resistance-conferring FLT3 mutations in acute myeloid leukemia

“…For instance, the flexible cyclopropylethyl group in MRX-2843 forms favorable van der Waals interactions equally well with either the leucine or phenylalanine side chains, while quizartinib binding is dependent on a stacking interaction with the F691 side chain. Moreover, phenylalanine is also likely to favor a metastable conformation required for quizartinib engagement (24). …”

“…In addition, MRX-2843 is an ATP-competitive type 1 inhibitor, and, unlike quizartinib and other type 2 inhibitors that bind target proteins in their inactive conformation (24), acquisition of mutations that stabilize the active form of the target protein is unlikely to be a mechanism of acquired resistance. Indeed, MRX-2843 retains activity against FLT3-mutant proteins containing activating aa changes at D835.…”

Phase 1 study of quizartinib in combination with induction and consolidation chemotherapy in patients with newly diagnosed acute myeloid leukemia

“…An important limitation of first generation, less potent, FLT3 inhibitors is that while they are able to clear blasts from peripheral blood when used at their MTD, they often have little effect on bone marrow blasts . Quizartinib is the first member of a next‐generation class of FLT3 inhibitors that demonstrates more potent binding to FLT3, more selective inhibition of FLT3 kinase activity, and more potent growth inhibitory activity in AML cells harboring FLT3‐ITD mutation vs multikinase inhibitors including midostaurin and lestaurtinib . Importantly, quizartinib has demonstrated very high single agent activity in patients with FLT3‐ITD mutated AML, with reported composite complete remission rates between 46% and 57%, overall response rates between 65% and 78%, and high rates (35%‐42%) of successful bridging to allogeneic hematopoietic stem cell transplantation (allo‐HSCT) .…”