Synthesis and biological characterization of dual-specific proteolysis target chimeras (PROTACs) for the kinases FLT3-ITD and MAPK14 with antileukemic activity

Acute myeloid leukemia (AML) is a hematological malignancy frequently driven by mutations in the FLT3 gene, particularly internal tandem duplications (FLT3-ITD), which contribute to aberrant cell proliferation and resistance to tyrosine kinase inhibitors (FLT3i). The limitations of current FLT3i therapies, including drug resistance, off-target effects, and poor selectivity, necessitate the development of novel therapeutic strategies. Proteolysis-targeting chimeras (PROTACs) represent a promising approach to achieve selective degradation of oncogenic proteins. We developed and characterized an optimized series of FLT3-targeting PROTACs based on our previously described compound MA49, with a focus on linker modifications and pharmacophore optimization to improve degradation efficiency and pharmacokinetic properties. Among these, compounds MA190 and MA191,containing rigid cyclohexyl-piperidine/piperazine linkers, demonstrate superior degradation of FLT3-ITD in MV4-11 AML cells at nanomolar concentrations, achieving >95% reduction in FLT3-ITD levels, outperforming MA49. These compounds exhibit good solubility, high chemical and plasma stability, and improved kinase selectivity. In cellular assays, MA190 and MA191 induce potent apoptosis in FLT3-ITD+ AML cells but have minimal effects on cells with wild-type FLT3. Proteomic analysis reveal that MA191 also degrades MAPK14 (p38α), a kinase upregulated in leukemia, in addition to FLT3. Dual targeting of FLT3-ITD and MAPK14 enhances pro-apoptotic signaling. The co-inhibition using MA191 or a combination of doramapimod (a MAPK14 inhibitor) with a non-degrading FLT3 inhibitor results in greater caspase-3 activation than either treatment alone. This synergistic effect underscores the therapeutic advantage of degrading multiple oncogenic drivers simultaneously.