| Mixed lineage leukemia(MLL),which is named as the fusion protein of 11q23 MLL gene translocation.Patients with this disease generally have poor prognoses and often suffer from early relapse after treatment with current therapies.DOT1 L is the only known enzyme that can catalyze histone H3 methylation of seventy-ninth lysine(H3K79),and the high methylation of H3K79 can abnomally activate HOXA9,MEISI and other genes to promote the growth of leukemia.DOT1 L is so important target of MLL leukemia that the development of DOT1 L inhibitors has become a new way to treat MLL leukemia.At present,the design of DOT1 L inhibitors is mainly polyamine derivatives,and polyamine compounds have become the hot spot of anticancer drugs due to their multiple targets.Based on the study of a large number of polyamine derivatives,established a virtual screening method for histone methyltransferase DOT1 L inhibitors,and screened compounds with higher affinity for the DOT1 L receptor;Designed a synthetic route?separation and purification method of target compounds,and confirmed the structure,Studied the in vitro activity of target compounds against mixed lineage leukemia cells.The results are as follows:(1)The compound library was composed of a series of polyamine derivatives which had been designed based on the previous research and related literature,and a positive compound EPZ-5676 in pHase I clinical trials.Receptor model 4HRA selected from the protein crystal database PDB.The receptor and ligand were subjected to molecular docking test by Sybyl-X2.0 software,and the highest fraction of compound DUOA003 was screened as the target compound.(2)The synthesis of the target compound was completed according to the synthetic route and the structure of the target compound determined by LC-MS,1HNMR,13 CNMR.The structures of the compounds were as follows: N,N'-(propane-1,3-diyl)bis(2,5-dihydroxybenzamide).(3)The mixed lineage leukemia cell line MV4-11 and normal cell line as the research object,the inhibition rate of two cells had been co-incubated with the different concentrations(120,100,50,25,10,1,0.1?M)of the target compound and positive drug ARA-C after 24 h were determined by CCK-8.Results showed DUOA003 and positive drug ARA-C had obviously inhibitory effect on MV4-11 cells,that IC50 were 25 and 28 ?M.IC50 on macrophage were 71 ?M and 11 ?M.The effects of 24 h,48h and 72 h on the inhibition of cell proliferation were investigated in the safe concentration range for normal cells.Results showed there was time and dose dependence in the inhibition rate of DUOA003 on MV4-11.(4)MV4-11 cells which had been induced by high?medium and low concentration of drugs in 24 h and 48 h,stained by Annexin V-FITC/PI method.The state of cells after induction of 48 h was observed by fluorescence microscope,and the apoptosis rate of MV4-11 cells was detected by flow cytometry.Results found the negative control group(17.3±5%)compared to the low concentration group of DUOA003(53.9±8%),middle concentration group(80.9±9%),high concentration group(88.5 + 6%)and ARA-C high concentration group(82.6±9%)showed significant differences(P < 0.001).The apoptosis of MV4-11 cells had been induced by DUOA003 after 48 h was mainly in the late stage of apoptosis,and the apoptosis had been induced by ARA-C was mainly in the early stage of apoptosis.(5)Finally,Caspase-3 activity assay kit was used to detect the changes of the activity of Caspase-3 in the cells had been induced by high,medium and low concentrations of target compounds after 24 h.Caspase-3 activity detection displayed compared with negative control group(0.146±0.002),DUOA003 low concentration group(0.227±0.003),middle concentration group(0.367±0.004)?high concentration group(0.554±0.005),and ARA-C high concentration group(0.477±0.006),there were significant differences(P < 0.001).The inhibition rate and apoptosis rate were time-dependent and dose-dependent.There is Caspase-3-mediated apoptosis during the apoptosis of leukemia cells MV4-11. |
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