Molecular Design, Synthesis And Evaluation Of The Bioactivity Of New Antibacterial Sensitizer

Objective：The infection caused by multi-drug resistance gram-negative bacteria is one of themain death cause of patients. The common multi-drug resistance G-bacteria strains includeEscherichia coli (E. coli), Acinetobacter baumannii, Klebsiella pneumoniae andPseudomonas aeruginosa. A serious phenomenon is34%of clinical isolates are E. coli inintensive care units, and44%of those are multi-drug resistance. In view of this, it seemsmore important to discover new efficient anti-E. coli drugs or antibacterial sensitizer.AcrAB-TolC, a resistance-nodulation-cell-division (RND) superfamily derived effluxpump system, plays a very important role for multi-drug resistance in E. coli. Previously,we demonstrated that artesunate (AS), an artemisinin (ART) derivant, significantlyincreased the antibacterial effect of β-lactam antibiotics against E. coli via decreasing AcrBmRNA expression. The aim of the present study plan to obtain new antibacterial sensitizerwith better bioactivities targeting AcrB, using ART and its derivatives AS anddihydroartemisinin (DHA) as direct chemicals to design and synthesize new derivants. Andthe bioactivity of new ART derivants will be also studied.Methods：1. The molecular interactions between AcrB, ART or its derivants were computermodelled by molecular docking method. The structures of new molecules were designedbased on the interaction details.2. Twenty-one ART derivants were synthesized by side chain modifications.3. Construction of recombinant E. coli AG100A-AcrB which used as in vitrobioactivity screening system targeting AcrB.4. Two-fold diluted method was used to measure the susceptibility of E. coliAG100A-AcrB to various β-lactam antibiotics ampicillin or cefuroxime in the absence orpresence of new ART derivants. 5. Dynamic growth assays of effects of ampicillin or cefuroxime alone or combinedwith new ART derivants on E. coli AG100A-AcrB.6. Dynamic growth assays of effects of ampicillin or cefuroxime alone or combinedwith DHA-7on E. coli ATCC35218.7. Semi-quantitative RT-PCR analysis of the mRNA levels of AcrB and upstreamregulators within ATCC35218after the treatment of DHA-7combined with ampicillin.8．Realtime PCR analysis of the mRNA levels of AcrB and upstream regulators withinATCC35218after the treatment of the treatment of DHA-7combined with ampicillin.Results：1. The molecular docking results suggested the three candidate ligands could dock intoAcrB very well by immobilizing the parent sesquiterpene lactone nuclear to Gln176andSer46. In the most favored docking mode, although three ligands had the same parentsesquiterpene lactone structure, only DHA and AS had the same docking poses, while ARTwas different from the two derivants. The succinic acid ester tail of AS could extended inthe drug transport tunnel of the AcrB very well. It was pretended to prevent the drug bind tothe active center and then block the drug efflux tunnel.2. The sesquiterpene lactone nuclear was used as the pharmacophore based on themolecular docking results, and the side chain was modified to obtain twenty-one ARTderivants, seventeen of which were new structures.3. DHA-7was considered to be the best antibacterial sensitizer according to the invitro bioavtivity evaluations.4. Semi-quantitative RT-PCR and Realtime PCR results demonstrated that althoughDHA-7itselves could not change the mRNA expressions of AcrB and its upstreamregulators of ATCC35218, while DHA-7combined with ampicillin could significantlydecrease the mRNA expressions of AcrB and its upstream positive regulators, MarA, Rob,FIS, etc. Meanwhile, the results also suggested that DHA-7combined with ampicillin didnot decrease AcrB expression by change the negative regulators, AcrR, MppA, etc.Conclusions：New ART derivants targeting multi-drug efflux pump transportor AcrB of E. coli wasdesigned using molecular docking method based on computer modeling. Twenty-one newART derivants were synthesized, and seventeen of which were new structures. DHA-7was considered to be the best antibacterial sensitizer according to the in vitro evaluations.DHA-7could increase the sensibility of E. coli to ampicillin or cefuroxime throughsignificantly decreased mRNA expression of multi-drug efflux pump transporter AcrBcombined with antibiotics. The results also suggested the inhibition of AcrB might beassociated with the decreased mRNA expressions of AcrB positive regulators, MarA, Roband FIS, etc.