Discovery And Investigation Of Inhibitors Targeting MLL1-WDR5 Interaction

Mixed-lineage leukemia 1?MLL1?protein is a member of mixed lineage leukemia?MLL?family Histone 4 Lysine 3?H3K4?methyltransferases,whose optimal catalytic activity is determined by its partner proteins including WD Repeat Domain 5?WDR5?,Retinoblastoma-binding Protein 5?RbBP5?,and Absent,Small,or Homeotic 2-like?ASH2?.The protein-protein interaction?PPI?between WDR5 and MLLI is particular important for maintaining optimal H3K4 methyltransferase activity of MLLI.It's reported that the dysregulation of MLL1 catalytic function is relevant to mixed-lineage leukemia and blocking interaction of MLL1-WDR5 can inhibit the development of acute leukemia.Therefore,targeting WDR5-MLL1 interaction could be a promising therapeutic strategy for leukemia harboring MLL1 fusion proteins.To date,several peptidomimetic and non-peptidomimetic small-molecule inhibitors targeting MLL1-WDR5 protein-protein interaction?PPI?have been reported,but no inhibitor has been approved into clinical trial because of bad druggability,such as bad cellular permeability,toxicity and poor selectivity.Consequently,it's still necessary to find other small-molecule WDR5-MLL1 inhibitors with novel scaffolds,good selectivity and cellular permeability.In this study,through fluorescence polarization?FP?-based high-throughput screening,4 hit compounds DC M4,DC M5,DC M33 and DC M39?the IC50 values are 18.53 ± 0.72 ?M,13.62 ± 1.21 ?M,27.79 ± 2.20 ?M and 18.53 ± 1.14 ?M,respectively?,with potent inhibitory activities in vitro against WDR5-MLL1 interaction were discovered.Nuclear Magnetic Resonance?NMR?assays were carried out to confirm the direct binding between hit compounds and WDR5.Subsequent similarity-based analog searching of the 4 hits led to several inhibitors with better activities,among which DC_M4₁ and DC_M5₂ displayed highest inhibitory activity with IC50 values of 13 ± 0.2 ?M and 10 ± 1.5?M,respectively.Furthermore,Surface Plasmon Resonance was carried out to detect the KD values of DC_M4₁ and DC M5 2.Molecular docking study was performed to predict the binding modes between two most potent inhibitors and WDR5.The inhibitory activities against other methyltransferases suggested that DC_M4₁ had good selectivity against MLL1.In addition,the cell viability assay presented that DC M4 1 could inhibit the proliferation of MV4-11 and K562 cell lines,particularly the growth of MV4-11 cell line which possesses MLL fusion protein.In conclusion,based on the combination of high-throughput screening platform,biophysical,biochemical and cell experiments as well as computer aided drug design,a novel,potent,selectivity and cell-permeable inhibitor?DC_M4₁?against MLL1-WDR5 PPI was identified.Our research achievements could offer new inhibitor scaffolds for small-molecule inhibitors against MLL1-WDR5 PPI,which in the future could be structurally modified therefore leading to more potent lead compounds or molecular probes.Moreover,our research results may contribute to accelerating the molecular mechanism research of MLL1 in vivo and in vitro as well as the application of small-molecule inhibitors in the treatment of leukemia.