Design,Synthesis And Reversal Of Multidrug Resistance Of Quinazoline Derivatives Targeting AcrB Efflux Protein

Antibacterial drugs greatly guarantee the survival of human beings and improve the quality of human life.However,on a global scale,the long-tenn use and abuse of antibacterial drugs have led to the emergence and deterioration of bacterial resistance.The emergence,development and spread of multi-drug resistant bacteria pose a serious threat to human health and safety.In particular,the infectious diseases caused by Gram-negative bacteria such as Enterobact.er,Klebsiella pneumoniae,Acinetobacter baumannii and Pseudomonas aeruginosa,have resulted in antibacterial efficacy of conventional drugs continuously decreasing or even disappearing.The World Health Organization(WHO)reports that multi-drug resistance of Gram-negative bacteria has become a global public health problem.Therefore,it is imperative to develop new drugs with novel mechanisms of action or to increase the antibacterial efficacy of traditional antibiotics.In recent years,the mechanism of bacterial resistance has been continuously studied,and the efflux pump is confirmed to be the important one.The AcrAB-TolC efflux pump is widely distributed on the cell membrane of Gram-negative bacteria,and its overexpression is an important cause of multi-drug resistance of Gram-negative bacteria.In the AcrAB-TolC efflux system,AcrB protein plays a critical role in drug efflux transport.Inhibition of AcrB efflux protein can enhance the sensitivity of drug-resistant bacteria to antibacterial drugs,reverse multi-drug resistance of bacteria,and restore the original antibacterial efficacy of antibacterial drugs.AcrB has become the ideal drug target for combatting bacterial resistance.According to the structure of AcrB and the binding characteristics of the substrate,44 novel quinazoline derivatives were designed and synthesized as AcrB inhibitors by hybridizing the function fragment of the 4-isopentyloxy-2-naphthylbenzamide and MBX-2319 with refering to their binding mode in AcrB.The biological evaluation including intrinsic antibacterial activity assay,antibacterial sensitizing activity assay,Nile Red efflux inhibition assay,outer and inner membrane permeability assays and cytotoxicity test was carreid out.In the intrinsic antibacterial activity assay,the MIC value of the target compound against the wild type E.coli BW25113 strain is greater than 128μg/mL.In the antibacterial sensitizing activity assay,19 compounds show sensitizing activity against chloramphenicol,erythromycin and TPP against wild type E.coli BW25113 strain.In the sernes A,8 compounds exhibite antibacterial sensitizing activities.Among them,A-GXJ-15 is the best potentiator of chloramphenicol,erythromycin and TPP with 8-fold MIC decrase respectively,at 128μg/mL.In the series B,there are 11 compounds exhibiting a broad spectrum of sensitizing activity.B-GXJ-7 has the bestsensitizing activity at 128 μg/mL,it can decrease the MIC of chloramphenicol,erythromycin and TPP by 16,2 and 4 times,respectively.The other compounds possss the sensitizing activity of chloramphenicol,erythromycin and TPP from 2 to 4 times.The results of Nile Red efflux inhibition assay shows that compounds A-GXJ-8,A-GXJ-15,A-GXJ-16 and A-GXJ-21 can completely inhibit the efflux of Nile Red at 50 μM,wheras A-GXJ-10,A-GXJ-14,B-GXJ-5,B-GXJ-6,B-GXJ-7,B-GXJ-11 at 100 μM,which is better than or comparable to the positive control 4-isopentyloxy-2-naphthylbenzamide.The results of the outer and inner membrane permeability assay showed that all the tested compounds have no effect on the outer membrane permeability.The compounds A-GXJ-12 and B-GXJ-13 don’t dissipate the proton motive force at 128μg/mL.Taken together,the compounds A-GXJ-12 and B-GXJ-13 are ideal AcrB inhibitors with the desired charateristics:(1)it can potentiated the sensitivity of AcrB overexpressing strains to antimicrobial agents;(2)it inhibit the efflux of AcrB substrates(e.g.,Nile Red);(3)it has no effect on the bacterial outer membrane permeability;(4)it does not destroy the intimal proton gradient.Compounds A-GXJ-15 and B-GXJ-7 exhibit potent sensitizing activity of antibacterial agents and efflux inhibitory activity of AcrB substrates,and do not destroy the permeability of the outer membrane,but dissipate the proton motive force,indicating that in addition to AcrB inhibition,there are other mechanisms of these two compounds to potentiate the antibacterial activity of the tested antibiotics and TPP.It is highly probable that the compound blocks the energy source of AcrB operation.At the same time,the IC50 values of the compounds A-GXJ-15 and B-GXJ-7 for Hela cells were both more than 128μg/mL,indicating that the compounds were not toxic to mammalian cells.The quinazoline derivatives as AcrB inhibitors discussed in this paper are our first discovery and they have not been reported in the literature before.We were able to design and synthesize a novel chemical compound with great potential to further optimization as AcrB inhibitors.This study provides new directions and ideas for the research and discovery of novel AcrB inhibitors.To a certain extent,it contributes to the treatment of infections caused by AcrB-mediated multi-drug resistant bacteria.