Discovery of FLC-8 as the First Covalent FLT3 Inhibitor Targeting Cys807 for FLT3 Mutant Acute Myeloid Leukemia
Zi-Xuan Wang1, Xiao-Long Jing1, Qian Liu1
1National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine, Nanjing University of Chinese Medicine, 138 Xianlin Road, Nanjing, Jiangsu 210023, China.
Abstract:
FLT3 is a validated therapeutic target in acute myeloid leukemia (AML), yet resistance mutations frequently limit current inhibitors. Here, we report a series of 6-methylisoxazolo[5,4-b]pyridin-3-amines that covalently target Cys807, a previously unexploited nucleophilic residue within the FLT3 kinase domain. Compound 18 (FLC-8) potently inhibited FLT3-WT (IC50 = 10.2 nM) and clinically relevant mutants G697R (IC50 = 11.6 nM) and N676D (IC50 = 24.1 nM). Covalent engagement of Cys807 was confirmed by mass spectrometry, peptide mapping, and loss of activity upon C807S mutation. FLC-8 suppressed FLT3-mediated STAT5, AKT, and ERK signaling and induced apoptosis in AML cells while maintaining low-nanomolar potency over 72 h. Kinome profiling revealed a narrow inhibition spectrum. In vivo, FLC-8 inhibited MV4-11 xenograft growth (TGI: 136-178% at 10-50 mg/kg) without overt toxicity. These findings identify Cys807 as a covalent binding hotspot in FLT3 and establish FLC-8 as a promising scaffold for next-generation FLT3 inhibitor development.
Insights
Researchers developed novel covalent inhibitors targeting Cys807 in FMS-like tyrosine kinase 3 (FLT3) to overcome resistance in acute myeloid leukemia (AML). Compound FLC-8 shows potent inhibition of FLT3 and its mutants, demonstrating promise for next-generation AML therapies.
Area of Science:
- Medicinal Chemistry
- Oncology
- Molecular Biology
Background:
- FMS-like tyrosine kinase 3 (FLT3) is a key therapeutic target in acute myeloid leukemia (AML).
- Resistance mutations frequently emerge, limiting the efficacy of current FLT3 inhibitors.
- Cys807 in the FLT3 kinase domain represents a novel, unexploited target for covalent inhibition.
Purpose of the Study:
- To design and synthesize novel 6-methylisoxazolo[5,4-b]pyridin-3-amine derivatives as covalent FLT3 inhibitors.
- To evaluate the inhibitory activity of these compounds against wild-type FLT3 and clinically relevant mutants.
- To assess the in vitro and in vivo efficacy and safety of the lead compound.
Main Methods:
- Synthesis of a series of 6-methylisoxazolo[5,4-b]pyridin-3-amine compounds.
- Biochemical assays to determine IC50 values against FLT3-WT and mutants.
- Mass spectrometry and peptide mapping to confirm covalent adduct formation at Cys807.
- Cellular signaling pathway analysis (STAT5, AKT, ERK) and apoptosis assays.
- In vivo studies using MV4-11 xenograft models in mice.
Main Results:
- Compound 18 (FLC-8) demonstrated potent inhibition of FLT3-WT (IC50 = 10.2 nM) and mutants G697R (IC50 = 11.6 nM) and N676D (IC50 = 24.1 nM).
- Mass spectrometry confirmed covalent binding to Cys807.
- FLC-8 suppressed downstream signaling, induced apoptosis in AML cells, and maintained potency over 72 hours.
- FLC-8 exhibited a narrow kinome inhibition profile.
- In vivo, FLC-8 significantly inhibited tumor growth in MV4-11 xenografts without apparent toxicity.
Conclusions:
- Cys807 is identified as a covalent binding hotspot on FLT3.
- FLC-8 is a potent and selective covalent FLT3 inhibitor with promising anti-leukemic activity.
- FLC-8 represents a promising scaffold for developing next-generation FLT3 inhibitors for AML treatment.
