Research On The Pharmacodynamic Material Basis And Mechanism Of Alternative Antibiotics From Tradition Chinese Medicine Based On Virtual Screening

The infectious diseases are caused through the invasion and rapid reproduction of external bacterial when the epidemic prevention function of the human body is reduced,and even threaten human life in serious cases.It is urgent that develop new anti-bacterial drugs especially the emergence of drug-resistant Pseudomonas aeruginosa and Escherichia coli.As a valuable medical and health resource in China,Traditional Chinese Medicine（TCM）has a long history in the treatment of infectious diseases,which provides the possibility for the discovery of new drugs to treat bacterial infection and prevent or reduce bacterial resistance.In this paper,the research theory and methods of computer-aided drug design were comprehensively applied to study the relevant targets against Pseudomonas aeruginosa and Escherichia coli based on pharmacophore model and molecular docking.Ingredients with potential antibacterial activity were selected from the self-built database of TCM ingredients and the database of common TCM.Then,the drug molecules with obvious antibacterial effect were obtained through in vitro antibacterial experiment.Secondly,the research strategy of network pharmacology was used to explore the material basis and mechanism of active ingredients from TCM against bacterial infection.At the same time,Quantitative Structure Activity Relationships（QSAR）research was carried out to establish QSAR equations with significance linear relationship,and the main factors affecting the activity of inhibitors were screened out.This study provides a theoretical basis for the discovery of new anti-bacterial drugs that can replace antibiotics in TCM.1.Research based on pharmacophore model and molecular docking:According to the pharmacophore model constructed,target proteins with important pharmacodynamic characteristic elements were selected.RMSD was calculated by redocking the eutectic structures of target proteins.Auto Dock Vina program was used to conduct molecular docking of active molecules（Active）and inactive molecules（Decoy）,respectively.The binding energy values were used to draw Receiver Operating Characteristic（ROC）curve and compound histogram.Meanwhile,the Enrichment Factor（EF）and active molecule hit ratio were calculated.The docking models with strong enrichment ability for active molecules were selected to conduct large-scale virtual screening of the self-built database of TCM ingredients and the database of common TCM.The results showed that the molecular docking models of3IX3,3SZT,3G7E and 1S16 target protein had strong enrichment ability of active molecules.A total of 35 TCM ingredients with binding energy below the threshold,molecular structure similar to the original ligand and meeting the five principles of drug-like were obtained by the above docking models.Among them,4,20,3 and 8 TCM ingredients with potential antibacterial activity were screened by 3IX3,3SZT,3G7E and 1S16 target protein docking models,respectively.2.In vitro antibacterial experiment:The minimum inhibitory concentration（MIC）of 35TCM ingredients were determined by microbroth dilution method.The MIC of TCM ingredients with obvious antibacterial effect and ciprofloxacin were determined by microchessboard dilution method,and the diameter size of inhibition zone of TCM ingredients were determined by agar plate punching method.The results showed that 20 of the35 TCM ingredients with potential antibacterial activity had obvious antibacterial effect,indicating that molecular docking models could screen antibacterial active compounds.Among them,17 ingredients had anti-Pseudomonas aeruginosa activity and 3 ingredients had anti-Escherichia coli activity.The strongest antibacterial effects were Phenethyl caffeate and Phloretin,with MIC values of 31.25μg/m L.Piperine followed with 62.5μg/m L.The MIC value of N,N’-bis（salicylidene）-1,3-propanediamino was 125μg/m L and the MIC values of other TCM ingredients were all 500μg/m L.In addition,it was found that the antibacterial activity of 20 TCM ingredients combined with ciprofloxacin was significantly greater than the sum of their individual antibacterial activities by calculating the fractional inhibitory concentration（FIC）index,and the diameter of inhibition zone in vitro of combination drug was increased to varying degrees compared with single drug,indicating that combination drug could enhance the antibacterial effect.3.Study on the material basis and mechanism of TCM anti-infective based on network pharmacology:The potential targets of active ingredients from TCM against bacterial infection were obtained by mapping active ingredient targets with anti-infection disease targets.The key targets were screened by constructing active ingredients-targets-action pathways network and protein-protein interaction network.At the same time,GO function and KEGG pathway enrichment analysis were performed to explore signaling pathways related to bacterial infection,and molecular docking was used for verification.The results showed that 6active ingredients（Puerarin,Methyl vanillate,Phloretin,Chlorogenic acid,Fargesin and Guajavarin）acted together on 22 potential targets,and key targets of CASP3 and SRC mainly through regulating Pathogenic Escherichia coli infection,TNF,Natural killer cell mediated cytotoxicity and other signal pathways to achieve the purpose of anti-bacterial infection.In addition,the results of molecular docking showed that the 6 active ingredients of TCM had good binding effect with 2 key targets,which further clarified the material basis and mechanism of anti-bacterial infection of TCM from the molecular level.4.Quantitative Structure Activity Relationships study:The structures of active molecules belonged to Las R and Rhl R signal receptor proteins,DNA gyrase and DNA topoisomerase IV target were optimized and some microchemical parameters included the highest occupied orbital energy level(E_HOMO),the lowest empty orbital energy level(E_LUMO),orbital energy level difference（ΔE）between HOMO and LUMO,dipole moment（μ）,and lipid-water partition coefficient（log P）were calculated by Gaussian 09 quantum chemistry software with B3LYP/6-31G（d）level.The QSAR equations were established by multiple linear regression method to screen out the main factors affecting the activity of inhibitors,and the linear significance of QSAR equations were reflected by multiple linear fitting.The results showed that E_LUMO,ΔE andμwere the main factors affecting the activity of Las R signal receptor protein,μand log P were the main factors affecting the activity of Rhl R signal receptor protein,E_HOMO,ΔE andμwere the main factors affecting the activity of DNA gyrase,E_LUMO andΔE were the main factors affecting the activity of DNA topoisomerase IV.The square of complex correlation coefficient（R²）of QSAR equations was close to 1,and the statistical significance level（p）was less than 0.05,indicating that the linear relationship of QSAR equations was significant and the statistical significance of QSAR models was good.To sum up,the target proteins screened by pharmacophore model were used for virtual screening based on molecular docking,and a batch of TCM ingredients with potential antibacterial activity were found.The drug molecules with obvious antibacterial effect were obtained through in vitro antibacterial experiment.Secondly,the research strategy of network pharmacology was used to explore the material basis and mechanism of the active ingredients from TCM against bacterial infection.At the same time,QSAR research was carried out to establish QSAR equations with significance linear relationship,and the main factors affecting the activity of inhibitors were screened out.This study provides a new idea and direction to find the active ingredients that can replace the efficacy of antibiotics in TCM.