| Multidrug-resistant(MDR)Gram-negative bacteria have become a threat to global public health and it is urgent to develop new antibiotics.Auranofin was approved by FDA for the treatment of rheumatoid arthritis,its safety has been well demonstrated.It was reported that auranofin exhibited significant antibacterial activity against Gram-positive bacteria by inhibiting thioredoxin reductase(TrxR)However,it showed poor antibacterial activity against Gram-negative bacteria because gram-negative bacteria contain a glutathione system to compensate for the deficiency of TrxR.In addition,auranofin still has some problems in vivo,such as insufficient activity and poor cell selectivity.Nitrogen heterocyclic carbene(NHC)is a kind of multifunctional ligand,which can combine with transition metal to form more stable complexes.The introduction of selenium on the basis of gold complex can effectively reduce the toxic and side effects of gold complex.It is hoped that the gold complexes with better antibacterial activity can be synthesized by structural modification of auranofin.Objective:To prepare gold complex with better antibacterial activity and lower toxicity by structural modification of auranofin,and to study its antibacterial activity and mechanism.Methods:1.The structure of auranofin was modified to synthesize 17 new gold(Ⅰ)selenium N-heterocyclic carbene complexes.2.The antibacterial activity of 17 Gold(Ⅰ)selenium N-heterocyclic carbene complexes was screened by broth dilution method.The bacteria used for activity screening included:carbapenem-resistant Acinetobacter Baumannii(CRAB),carbapenem-resistant Klebsiella pneumoniae,carbapenem-resistant Pseudomonas aeruinosa,carbapenem-resistant Escherichia coli,vancomycin-resistant Staphylococcus aureus.The gold complexes with the best antibacterial activity were tested for cytotoxicity and in vitro activity.What’s more,mice skin infection model and mice abdominal infection model were prepared to measure antibacterial activity in vivo.3.Study on the antibacterial mechanism of H7,H8:(1)Determination of intracellular TrxR activity of CRAB.The effects of H7,H8 on purified TrxR were determined.The expression of TrxR gene was verified by qPCR.(2)Intracellular reactive oxygen species(ROS)and oxidative stress related indexes(GSH,MDA,SOD)were determined.Intracellular ATP content was determined.(3)Determination of cell membrane integrity and loss of intracellular contents.(4)In order to explore other antimicrobial targets,DNA damage of H7 and H8 was determined by nucleic acid electrophoresis.(5)Method of GC-MS untargeted metabolomics was used to determine the changes of bacterial intracellular metabolites under the action of H7 and H8.Results:1.Seventeen gold(Ⅰ)selenium N-heterocyclic carbene complexes were screened for their antimicrobial activity,among which H7 and H8 had the best antimicrobial activity against CRAB.The minimum inhibitory concentration(MIC)of H7 and H8 was 10 μM.the minimum bactericidal concentration(MBC)was 20 μM.The antibacterial activity of auranofin was stronger than that of positive control.CCK8 assay showed that the cytotoxicity of H7 and H8 was lower than that of auranofin.In vivo experiments on mice,selenium-gold complexes H7 and H8 can significantly promote wound healing and improve the survival rate of mice.2.The mechanism of selenium-gold compounds H7 and H8 was studied.The results showed that:(1)H7 and H8 inhibited the activity of CRAB intracellular and purified TrxR enzyme(rabbit source),and H7 and H8 had stronger TrxR inhibitory activity than auranofin.QPCR results showed that H7 and H8 acted on CRAB to reduce the expression of TrxC.(2)Inhibition of TrxR activity leads to intracellular redox imbalance,resulting in ROS generation.The ROS produced in CRAB by H7 and H8 was significantly higher than that of auranofin.When the bacteria were in the state of oxidative stress,the content of MDA increased,the activity of SOD decreased,and the content of GSH decreased.(3)Excessive ROS results in cell membrane damage and leakage of cell contents.(4)The extracted DNA was treated with H7 and H8 for nucleic acid electrophoresis.The results showed that H7 and H8 could not only lead to intracellular oxidative stress imbalance by inhibiting TrxR,but also achieve multi-target antibacterial effect by damaging bacterial DNA.3.GC-MS untargeted metabolomics results showed that H7 and H8 induced significant changes in 70 metabolites.Compared with the control group,50 metabolites were up-regulated and 20 metabolites were down-regulated in H7 group.Compared with the control group,50 metabolites were up-regulated and 20 metabolites were down-regulated in H8 group.Enrichment analysis showed that arginine biosynthesis metabolism,glutathione metabolism and alanine,aspartate and glutamate metabolism were all significantly perturbed following treatment with H7 and H8.Conclusion:In this study,17 novel Gold(Ⅰ)Selenium N-heterocyclic Carbene Complexes were prepared,and the dominant compounds H7 and H8 were screened by antibacterial activity.H7 and H8 showed significant antibacterial activity for CRAB in vitro and in vivo.Compared with auranofin,H7 and H8 have better antibacterial activity and lower toxicity.Then,auranofin was used as a positive control to study the mechanism of H7 and H8.It was found that H7 and H8 had stronger TrxR inhibitory activity than auranofin.The same concentration can induce more obvious ROS production,with stronger ability to destroy cell membrane.It was further confirmed by GC-MS untargeted metabolomics that H7 and H8 significantly damaged intracellular amino acid metabolism,which was consistent with the result of inducing ROS production.Excessive intracellular ROS significantly damaged amino acid structure.In conclusion,through the structural modification of auranofin,the Gold(Ⅰ)selenium N-heterocyclic carbene complexes H7 and H8 have lower cytotoxicity and stronger antibacterial activity,providing new ideas for the development of antibacterial drugs. |
PDF Full Text Request