Abstract
Mutations in the FLT3 gene represent one of the most common genetic alterations in acute myeloid leukemia (AML) and are associated with an increased risk of relapse and poor prognosis (Port, Böttcher et al. 2014). FLT3 inhibitors have significantly improved clinical outcomes in AML patients with FLT3 internal tandem duplication (ITD) mutations. However, currently available FLT3 inhibitors often exhibit off-target effects, leading to numerous adverse reactions (Majothi, Adams et al. 2020). Therefore, the identification of more specific FLT3 inhibitors has great clinical significance.
We subsequently conducted high-throughput (HT) virtual screening for small molecules that bind to autoinhibited conformation of the FLT3 protein in the National Cancer Institute Developmental Therapeutics Program (NCI DTP) library. 159 small-molecule compounds were selected. Following in vitro screening in 32D and 32D-ITD cells, the most promising lead compound TIP-20 was identified. The predicted minimum binding capacity of TIP-20 for the FLT3 protein was −9.1. The docking results showed that TIP-20 can occupy the ATP pocket of FLT3 well. The terminal pyridine ring penetrates into the DFG-out hydrophobic pocket, triazole ring forms one key hydrogen bond with the amino acid residue Glu-661 in the hinge region, with an interaction distance of 2.0 Å. After aniline benzene ring bridging, the terminal imidazole ring forms a hydrogen bond with Cys-694. Cellular thermal shift assay (CETSA) confirmed the direct binding of TIP-20 to the FLT3 protein. TIP-20 inhibited FLT3 kinase activity detected by homogeneous time-resolved fluorescence. TIP-20 was further evaluated in FLT3-ITD and FLT3 wild-type AML cell lines to assess its selective antileukemia activity. IC50 values varied significantly across cell lines, with MV4-11 and MOLM-13 (FLT3-ITD positive) being the most sensitive (1.68 μM and 5.78 μM, respectively), whereas K562, THP-1, and Kasumi-1 exhibited reduced sensitivity or resistance, with IC50 values ranging from 2.7× 104 μM to >5.1 × 109 μM. TIP-20 inhibited proliferation, induced apoptosis, and caused G0/G1 cell cycle arrest in FLT3-ITD AML cell lines. Furthermore, TIP-20 suppressed the phosphorylation of FLT3 and its downstream signaling components, including AKT, STAT5, and ERK.
TIP-20 is a novel FLT3 inhibitor with potent anti-leukemic activity. Importantly, it demonstrates high specificity toward FLT3-ITD AML cells and possesses a favorable therapeutic window. These findings suggest that TIP-20 may represent a promising candidate for targeted therapy in FLT3-ITD AML.
