Design,Synthesis And Effect Of New AcrB Inhibitors Based On Artesunate's Antibacterial Emhancement Action

Objective:Under the selective pressure of antibiotics,bacteria multidrug resistance(MDR)almost is produced for all antibiotics in clinical.After the 1970 s,MDR increases every year,how to deal with drug-resistant infection has become a serious problem.In 2013,clinical strains of bacteria collected in China's main regional hospitals,the Gram-positive bacteria accounted for 27%,Gram-negative bacteria accounted for 73%.Among Gram-negative bacteria Escherichia coli(E.coli),Klebsiella and Proteus mirabilis in ESBLs producing strains rates were 54.0%,31.8% and 16.5%,respectively [1].Thus,E.coli are in first place in the clinical detection of resistant pathogens in hospitals.So,to research an effective and difficult to produce drug-resistant for E.coli is very necessary.Therefore,it is of great significance to search for new antibiotics or other antibacterial agents.Our group has searched for pre-drug which had antibacterial sensitization activity for a long time.It was found that artesunate(AS),one of the important derivative of artemisinin,had no antibacterial activity in itself but has the activity of enhancing ?-lactam antibiotics against Escherichia coli,which is called as antibacterial sensitizing activity.At same time,the molecular mechanism of action of AS was explored and it was found that it could inhibit antibiotic efflux by inhibiting the transporter AcrB's mRNA expression of AcrAB-TolC system,leading to increased antibiotics aggregation within the bacteria.Because of the poor activity of AS on clinical strains,other artemisinin derivatives with the similar activity were investigated in our lab.Based on the other important derivatives of artemisinin had no such an activity,via comparison we found that they share a common nucleus structure but different branched chains.So the branching structure of artesunate was suspected to play an important role in the docking of artesunate.Therefore,the computer molecular simulation docking method was used,and the results showed artesunate branch played an important role.For this reason,its branched structure of artesunate was modified to obtain a series of new artemisinin derivatives.Among these derivatives,the compounds named as DHA7 has better antimicrobial sensitization activity than AS,and the mechanism is related to the inhibition of AcrB,which is similar to AS.The activity of DHA7 confirmed our hypothesis that the relationship between the branching and activity of artemisinin,but the disadvantages were that DHA7 were also less active on the clinical strains and poor solubility.Therefore,new derivatives of artemisinin were investigated to to obtain better agents.In this study,the multi-drug efflux pump transporter AcrB of E.coli is used as the target,too.Based on the previous results,the structure-activity relationship analysis is investigated and molecular docking method was used to simulate the interaction of the target protein with artemisinin and its derivatives.And then,the chemical structure of the new artemisinin derivatives was synthesized in our lab.Herein,a novel artemisinin derivative,named as DHA27 with strong antibacterial sensitization activity in vitro was obtained and its solubility was improved via chemical salt formation.The mechanism of its antimicrobial sensitization was related to AcrB,too.Methods:1.The structure-activity relationship of 18 compounds is investigated by computer-aided design method.The chemical structure of artemisinin derivatives is determined and the design principles of artemisinin derivatives are established.2.Molecular docking method is used to simulate the artesunate and DHA7 and E.coli multi-drug efflux pump transporter AcrB binding,based on the basis for the establishment of artemisinin derivatives molecular screening principle;3.Four supposed and representative artemisinin derivatives are synthesized in our lab.4.DHA27 is prepared as hydrochloride by chemical method to increase the water solubility of the compound.5.In vitro antibacterial activity of ?-lactam antibiotic ampicillin against E.coli ATCC35218 is screened by two-fold microplate dilution of artemisinin derivative.6.The growth inhibition effect of ampicillin on the growth of E.coli ATCC35218 is investigated by the dynamic growth curve method and coating plate method.7.The antimicrobial activity of DHA27 hydrochloride combined with ?-lactam antibiotic ampicillin on clinical isolates of E.coli is investigated using microplate dilution method.8.The antibacterial activity of DHA27 hydrochloride combined with different kinds of antibiotics on E.coli clinical strains is investigated using the microplate dilution method.9.The effect of DHA27 hydrochloride on cell proliferation was studied by MTS method.10.The influence of DHA27 hydrochloride on the drug aggregation in E.coli ATCC35218 isinvestigated using daunorubicin and nile red experimants.11.The effects of DHA27 hydrochloride on the AcrB mRNA expression in E.coli ATCC35218 is detected by Realtime PCR.12.The relationship between the antibacterial activity of DHA27 hydrochloride and ampicillin on E.coli and the AcrB mRNA expression are detected by Realtime PCR.13.The antibacterial activities of DHA27 hydrochloride against E.coli AcrB knockout strain and AcrB covering strain are observed using microplate dilution method.14.The docking method is used to simulate the binding site of AcrB to DHA27 for predicting its docking site of AcrB.15.The effect of DHA27 binding polypeptide on the aggregation of daunorubicin in E.coli ATCC35218 is observed using daunorubicin experimant.Results:1.The structure-activity relationship of the artemisinin-branched structure showed that the spatial and electronic effects of the artemisinin-branched chains played an important role in the antimicrobial-sensitizing activity of the whole compound.Based on the structure-activity relationship,the structural design was carried out,A series of novel artemisinin derivatives were designed and synthesized.2.Molecular docking results showed that artesunate and DHA7 bound AcrB molecules mainly through hydrogen bond.Comparison of the docking results of the two molecule with AcrB showed that the mother of nuclear-binding cavity position of AcrB to artesunate and DHA7 was almost same,but the branch extended to different directions.Above results demonstated that the branched portion of artemisinin played an important role in its binding to the AcrB active center.The branched portion could prevent drug molecules from binding to the active center,and blocked the active center near the drug channel,leading to cut off drug transport.In addition,Gln176,Tyr327,and Phe136 could be bound via hydrogen bond to fix the three molecules of the skeleton structure;3.According to the molecular docking results,the structures of new artemisinin derivatives were screened.The docking cavity core of artemisinin and DHA7 was set as "template",and new artemisinin derivatives were molecularly docked,22 compounds were obtained by screening the compounds which were docked in the "template" as candidate compounds,4.Four compounds with high molecular docking were selected from 22 candidate compounds and synthesized.5.New artemisinin derivatives,named as DHA27 with strong antimicrobial activity,were obtained by screening in vitro antibacterial activity.6.The efficacy of DHA27 combined with ampicillin on the clinical strains of E.coli could reach to 78%(31/40),and DHA27 combined with other ?-lactam antibiotics had good activity against clinical strains,too.However,when it combined with non-?-lactam antibiotics,the activity was poor.7.DHA27 was prepared as a hydrochloride salt to solve its solubility problem.8.DHA27 could increase drug accumulation in the bacteria in vitro.9.DHA27 could significantly down-regulate the AcrB mRNA expression of E.coli,and DHA27 had a positive correlation with the AcrB mRNA expression.10.DHA27 lost antibacterial enhancing activity for AcrB knockout bacterial strain,but restored it activity if AcrB came back to the bacterial strain.11.Molecular docking results showed DHA27 bound AcrB molecule through hydrogen bond;the binding sites of AcrB probably were Ser46 and Ser134.12.Daunorubicin aggregation experiments showed that DHA27 could bind peptide fragments with Ser46 and Ser134,but couldn't bind the peptides when Ser46 and Ser134 were mutated.13.DHA27 had no obvious proliferative toxicity against RAW 264.7 cells.Conclusions:1.AcrB,a multidrug efflux pump transporter of E.coli,was used as a target to study the structure-activity relationship and molecular docking analysis,22 candidate compounds were desinged.2.A new artemisinin derivative named as DHA27 with high biological activity was selected.3.DHA27 had antimicrobial sensitization activity on both standard strains and clinical E.coli strains.4.The mechanism of DHA27 was related to increased drug accumulation via inhibition of AcrB,its binding sites might be Ser46 and Ser134.