Structure-based virtual screening was performed to investigate the mechanism of action of dual-target inhibitors against FMS-like tyrosine kinase 3 (FLT3) and histone deacetylase 8 (HDAC8) in leukemia Free

ObjectiveThe aim of this study was to identify dual-target inhibitors of FMS-like tyrosine kinase 3 (FLT3) and histone deacetylase 8 (HDAC8) using structure-based virtual screening technology, and to systematically investigate their mechanisms of action in the context of anti-acute myeloid leukemia (AML) therapy.

MethodsVirtual Screening and Molecular Docking:The crystal structures of FLT3 (PDB ID: 6IL3) and HDAC8 (PDB ID: 1T69) were obtained from the Protein Data Bank (PDB) and the conformation was optimized for energy minimization by Molecular Operating Environment (MOE) software.After screening candidate molecules from an internal database of 35,000 compounds and converting their two-dimensional structure into three-dimensional conformation, MOE's Dock tool was used to attach the target protein active site, and the binding affinity was evaluated by dG scoring algorithm.In Vitro Inhibition Assays:A standardized kinase response and HDAC8 fluorescence assay kit (Fluor de Lys®-HDAC8) was used to determine the compound's half-inhibitory concentration (IC₅₀) for FLT3 and HDAC8.Molecular Dynamics (MD) Simulations：GROMACS software (version 2021.5) and AMBER99SB-ILDN force field were used to simulate FH-5 and the target protein complex for 50 ns, and root mean square deviation (RMSD), root mean square fluctuation (RMSF) and secondary structure stability were analyzed.In Vitro Antiproliferation Assay：MTT assay was used to evaluate the inhibitory activity of FH-5 on the proliferation of KG-1 leukemia cells.In Vivo Antitumor Testing：A xenograft model of KG-1 cells (nu/nu mice) was established, and FH-5 (2.5, 5, 10 mg/kg) was administered in groups to monitor the changes in tumor volume and body weight, and the dose-dependent effects were analyzed.

Results1. Six potential dual-target inhibitors (FHs 1-6) were virtually screened out, and the docking scores of FH-5 against FLT3 and HDAC8 were significantly better than those of the positive controls quinzatinib (−12.61 kcal/mol) and volinota (−12.65 kcal/mol). 2. In vitro enzyme inhibition experiments showed that the IC₅₀ value of FH-5 against FLT3 and HDAC8 was 0.31 ± 0.02 nM and 2.91 ± 0.79 nM, respectively, and the activity was 32 times that of quinzatinib and 3 times that of vorinoat. 3. Molecular dynamics simulation showed that the main chain RMSD of FH-5 and FLT3 and HDAC8 complexes stabilized below 0.7-0.9 nm and 0.2 nm, respectively, and the fluctuation value of key residues (such as Arg834 of FLT3 and Asn178 of HDAC8) was lower than 0.4 nm, which verified the binding stability. 4. The proliferation inhibition rate of FH-5 on KG-1 cells exceeded 95%, and the IC₅₀ value was 23.2 nM. 5. In vivo experiments, the tumor inhibition rate of 10 mg/kg FH-5 group reached 78%, and the weight of mice was not significantly affected, showing a dose-dependent anti-tumor effect (P< 0.001).

ConclusionIn this study, we successfully obtained the FLT3/HDAC8 dual-target inhibitor FH-5 through structure-based virtual screening technology. It stably binds to the active site of the target protein through hydrogen bonding, hydrophobic interaction and ionic bonding, and shows strong anti-AML activity in vitro and in vivo, providing a novel candidate drug and theoretical basis for the treatment of AML.

Key words Fms-like tyrosine kinase 3; histone deacetylase 8; acute myeloid leukemia; dual-targeting inhibitors; structure-based virtual screening