N6-methyladenosine (m6A) methylation represents a widespread modification of eukaryotic mRNAs. METTL3, an RNA methyltransferase, facilitates the addition of m6A and serves as the sole catalytic subunit utilizing S-adenosylmethionine (SAM) as a methyl donor. STM2457, a predecessor of the compound STC-15, developed by Storm Therapeutics, has demonstrated antiproliferative effect in acute myeloid leukemia (AML). Currently, STC-15 is being evaluated in phase 1 clinical trials. Traditional Chinese Medicine (TCM) holds a significant position in the realm of clinical treatment in China. Consequently, we endeavored to identify a novel METTL3 inhibitor derived from TCM monomers through the application of molecular docking techniques and subsequent biological experimental validation.
Molecular modeling
The crystal structure of METTL3 was obtained from the Protein Data Bank (PDB ID: 5K7U). Then, water molecules and the original ligand were manually removed by using PyMol software[1]. This docking job to predict the binding pose of isoliquiritigenin(provided by Topscience) was carried out by Autodock (version 4.2.6)[2]. In the beginning, prepare_ligand4.py and prepare_recptor4.py scripts from AutoDockTools 1.5.6 were used to prepare the initial files including adding charges and hydrogen atoms. Next, a grid box of 60 × 60 × 60 with a spacing of 0.375Å was set to enclose the whole binding site. The Lamarckian genetic (LGA) was adopted to search the best binding poses. The specific docking settings were as follow: trials of 100 dockings, 300 individuals per population with a crossover rate of 0.8 and the local search rate was set to 0.06. Other parameters were set as default during the docking.
Biological experimental verification
Through molecular docking, we identified isoliquiritigenin interacts with METTL3 in the MTase domain. Subsequent experimental validation was performed as follows: 1) The Cellular Thermal Shift Assay (CETSA) and Drug Affinity Responsive Target Stability (DARTS) assays were subsequently employed to assess the potential binding interaction between isoliquiritigenin and METTL3. These assays demonstrated that isoliquiritigenin conferred protection against high-temperature-induced denaturation and protease- mediated degradation, indicating that METTL3 is a preferential target of isoliquiritigenin. 2) RNA dot plot assays revealed that isoliquiritigenin differentially reduced m6A modification levels in AML cells, exhibiting more potency compared to STM2457. 3) The antiproliferative effects of the compound on various AML cell lines were assessed using the CellTiter-Glo® Luminescent Cell Viability Assay (MOLM13, GI50=7.8μM; THP-1, GI50=9.3μM). 4) Isoliquiritigenin mitigated the expression of target-related proteins (MCL1, c-Myc) and apoptotic proteins (cleaved caspase-3 and cleaved PARP) in MOLM13 and THP-1 cells in a concentration- dependent manner. 5) Flow cytometry analysis demonstrated that isoliquiritigenin exhibited a concentration- dependent cytotoxic effect on primary AML cells, surpassing the pharmacological efficacy of STM2457. 6) In a subsequent study, the combination of isoliquiritigenin with Venetoclax, as opposed to Venetoclax alone, resulted in a more pronounced reduction in MCL1 and c-Myc protein expression.
The results demonstrate that a novel inhibitor of the METTL3, TCM monomer isoliquiritigenin, exhibit potential anti-leukemia activity. Additionally, these findings provide a molecular rationale for the augmented therapeutic efficacy observed when isoliquiritigenin is combined with venetoclax, suggesting a promising potential therapeutic strategy for AML.
[1] Schrodinger, LLC, The PyMOL Molecular Graphics System, Version 1.8, (2015).
[2] G.M. Morris, H. Ruth, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell, A.J. Olson, Software news and updates AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility, J. Comput. Chem. 30 (2009) 2785-2791.
No relevant conflicts of interest to declare.
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