Ki23819 (KRN383•HCl) Inhibits Kinase Activity of Wild Type and Mutant FLT3 Receptor Tyrosine Kinase In Vitro.

[Background] FLT3 is a class III receptor tyrosine kinase which is widely expressed on hematopoietic stem/progenitor cells. Two types of constitutively active FLT3 mutations have been reported to be expressed on a subset of leukemic cells; internal tandem duplications (ITD) and kinase domain mutations. The former are associated with poor prognosis in acute myeloid leukemia (AML) patients. Although several inhibitors targeting FLT3-ITD are tested in clinical trials, their cytotoxic effects are still unsatisfactory. Innate and acquired resistance is also a problem to be solved. [Purpose] To screen a novel potent FLT3 inhibitor and characterize its in vitro activity. [Materials and Methods] MOLM13 and MV4-11 cells, human leukemia cell lines expressing FLT3-ITD, were exposed to candidate compounds for 48 hours, and cytotoxic effect was assessed by colorimetric assay. Inhibitory effect on autophosphorylation was evaluated by immunoprecipitation and Western blotting. These effects were also tested in 32D cells engineered to express wild type FLT3 (FLT3-WT) or FLT3-ITD. FLT3-WT was activated with 50 ng/ml FLT3 ligand for 15 min. Proapoptotic effect was confirmed by flow cytometry with Annexin V staining. In vitro kinase assay was performed to demonstrate direct inhibition of tyrosine kinase activity of FLT3-ITD. Inhibitory effects on downstream signaling molecules, ERK and STAT5, were assessed by Western blotting. [Results] Among candidates for VEGFR inhibitors from a library, a quinoline-urea derivative Ki23819 (KRN383•HCl) was identified to specifically inhibit proliferation and induce apoptosis to MOLM13 and MV4-11 cells. Ki23819 inhibited proliferation of MV4-11 cells more effectively than SU11248, a precedent FLT3 inhibitor (IC50 <1 nM vs 3~10 nM). Similar results were obtained when MOLM13 cells were used. Ki23819 inhibited autophosphorylation of both ligand-activated FLT3-WT and FLT3-ITD (IC50 30 nM and 3 nM, respectively), and abrogated IL-3-independent proliferation of 32D cells expressing FLT3-ITD (IC50 3~10 nM). In vitro kinase assay demonstrated direct inhibition of kinase activity of FLT3-ITD (IC50 7.8 nM). This compound also inhibited ERK and STAT5 constitutively activated by FLT3-ITD. The IC50 for inhibition of phosphorylation in 32D FLT3-ITD cells was 3 nM for both proteins, which is equivalent to that for inhibition of FLT3-ITD autophosphorylation. [Conclusion] Ki23819 is a novel and potent candidate for antileukemic agents against FLT3-ITD positive AML. In vivo activity of KRN383, the free base of Ki23819, is also to be reported in this ASH meeting (Nishiyama et al.).