Mechanism And Bioactivity Evaluation Of Thiosemicarbazone Inhibitors On NDM-1 Enzyme

Objective:1.Screening of target compounds with the best activity and strong water solubility against New Delhi metallo-β-Lactamase-1(NDM-1)produced by meropenem(MEM)from a series of newly synthesized thiosemicarbazones.2.The pharmacodynamic activity of the compound in vivo and in vitro was evaluated and its inhibitory mechanism on NDM-1 enzyme was elucidated,which provides theoretical basis and data support for the development of NDM enzyme inhibitors.Methods:1.The antibacterial activity of a series of thiosemicarbazone compounds combined with MEM against NDM-1-producing strains was detected by K-B disk method,and the target compounds with good bacteriostatic effect and strong water solubility were screened.2.The in vitro activity of the target compound combined with MEM was further evaluated by microbroth dilution method and time-germicidal curve.3.The potential toxicity of the target compound was evaluated by erythrocyte hemolysis test,blood routine and blood biochemical test,cytotoxicity test and HE staining test.4.The in vivo pharmacodynamic activity of the target compound combined with MEM was investigated based on the mouse sepsis model infected by NDM-1 positive strains.5.By measuring the IC50 and Ki values of the target compound to NDM-1 enzyme,the interaction mode between the compound and NDM-1 enzyme was analyzed,and then the molecular docking simulation was used to further analyze the inhibition mechanism of the compound.Results:1.Fifty-four thiosemicarbazones with different structures were evaluated by K-B disk method.(E)-N’-(2-hydroxybenzylidene)piperazine-1-carthiohy drazide thiosemicarbazone(No.48)and(E)-N’-(2-hydroxybenzylidene)-4-methylpiperazine-1-carthiohydrazide thiosemicar-bazone(No.51)with good bacteriostatic effect and strong water solubility were selected for further study.2.When compound 48 or 51 was combined with MEM,it had good antibacterial activity against 30 producing NDM-1 clinical isolates,and had real-time bactericidal efficacy under the condition of bacterial growth.3.Target compounds 48 and 51 have low toxicity to erythrocytes and Hela cells.4.The target compound 48 or 51 combined with MEM had obvious therapeutic effect on septic model mice infected by NDM-1 enzyme producing strain,and could significantly improve the survival rate of infected mice(P<0.01).The mortality of compound 51 combined with MEM group was 0.5.Compounds 48 and 51 acted on NDM-1 enzyme by anti-competitive inhibition,and molecular docking analysis speculates that compounds 48 and 51 are most likely to bind to the allosteric pocket adjacent to the substrate pocket,thus affecting the hydrolysis of MEM by NDM-1.Conclusion:Thiosemicarbazone compounds 48 or 51 combined with MEM had good antibacterial activity and low toxicity to NDM-1 positive strains in vivo and in vitro.Mechanism analysis showed that 48 and 51 exhibited inhibitory activity on NDM-1 through anti-competitive inhibition.