Design,Synthesis And Reversal Of Bacterial Resistance Of Small Molecular Inhibitors Based On AcrB

Antibacterial drugs play not only a key role in the prevention and treatment of bacterial infections,but also a supporting role in the successful application of new technologies such as as organ transplantation,tumor chemotherapy and radiotherapy.However,the widespread prevalence and spread of bacterial resistance in the world pose a serious threat to human health.In particular,infections caused by Gram-negative bacteria such as Enterobacteriaceae,Pseudomonas aeruginosa,Klebsiella pneumoniae,and Acinetobacter baumanmii have led to the reduced or completely lost efficacy of many effective antibacterial agents clinically.More seriously,only a few new structural types of antibacterial drugs have been listed worldwide in this new century.In view of this situation,the World Health Organization(WHO)warns that in the near future,there will be no drugs available for the treatment of the serious infections caused by multidrug-resistant Gram-negative bacteria.Therefore,bacterial multidrug resistance and the infection caused by these bacteria with the multidrug resistance have become an increasingly serious global public health problem.It is urgent to develop new antibacterial drugs with new mechanisms of action to meet the challenge of bacterial resistance.AcrB is a transport protein widely existing on the cell membrane of Gram-negative bacteria.It is highly conserved,and its function is to pump harmful substances out of the bacterial cells.The bacteria overexpressing of AcrB could pump out various drug molecules,which would lead to too low drug concentration to inhibit or kill the bacteria,resulting in the emergence of multidrug-resistant phenotypes.If AcrB efflux is inhibited or interfered,the drug concentration in the bacteria will increase,thereby to restore antibacterial potency.Therefore,AcrB is a promising target for the development of novel antibacterial agents.We selected the AcrB as the new target in this dissertation,and chose AcrB inhibitors NDGA and A3 reproted previously by our group as the lead compounds.Based on the structural properties of the AcrB,three different structural skeleton types of novel compournds(toally 10 series)were designed and synthesized via using the principles of medicinal chemistry,computer-aided drug design and biochemistry methods.Th:rough the retrosynthetic analysis,the simple and efficent synthetic routes were established,and 106 target compounds were synthesized using these synthetic routes and their structures were characterized.In the aspect of reversal activity evaluation,we first measured the inherent antibacterial activity of the target compounds to determine their concentration range when combination with antimicrobial agents,so as to eliminate the effect of the target compounds itself on the antibacterial activity.Then the target compounds were used in combination with the antimicrobial agents such as chloramphenicol,erythromycin,tetraphenylphosphonium chloride and levofloxacin to determine their antibacterial sensitizing activity.Finally,the compounds with potent sensitizing activity were selected to further determine their efflux inhibition ability by Nile Red efflux assay and effect on outer or inner membrane permeabilization by nitrocefin uptake assay and DiOC2(3)fluorescence assay,which further confirmed their target-activity in the cell level.The study of catechol derivetives as AcrB inhibitors.Based on the structure of NDGA found by our group previously and properties of the AcrB binding sites,four novel series of catechol derivatives(WA,WB,WC and WD)were designed and synthesized through the diversified modification of the phenyl ring and linker,and evaluated for their antibacterial sensitizing activity.The results showed that compounds WA5,WA7,WB11,WD5 and WD6 exhibited good antibacterial sensitizing activity.In particular,compound WD6 at a low concentration of 8 ?g/mL potentiated the activity of of chloramphenicol and erythromycin against E.coli BW25113 by 2-fold;at a concentration of 32 ?g/mL,it showed synergism with all the tested antibiotics,indicating its broad spectrum;when deployed at 128 ?g/mL,its antibacterial sensitizing activity was further improved,which was significantly effective as potentiator of chloramphenicol,erythromycin,tetraphenylphosphonium chloride and levofloxacin with a 4-,4-,4-and 4-fold MIC-decrease in the antibacterial sensizing activity respectively,better than NDGA.The structure-activity relationships(SARs)were summarized as follows:(1)the sensitizing activity of the catechol derivetives was closely related to the the substituents on the benzene ring in their structure,and among various substituents,the hydrophilic groups was perferred group;(2)the introduction of aromatic rings with large steric h:indrance such as biphenyl,naphthalene and anthracene groups at the end of side chain were unfavorable for retaining potency;(3)The intermediate linker was best with a triatomic chain,and the introduction of an amide group intruducted in the linker could significantly improve the antibacterial sensitizing activity.In order to confirm the efflux inhibition activity,we selected the compounds with antibacterial sensitizing activity to determine their efflux inhibition ability,and further selected the most active and representative compounds WA5,WA7,WA9,WB11,WC1,WC2,WD5 and WD6 to determine their effect on outer and inner membrane permeabilization.The results showed that compounds WA7,WA9,WA10,WB11,WC3 and WC4 were able to completely inhibit the efflux of Nile Red at concentrations of 100 pM and 200 ?M,compounds WA5,WA7,WA9,WB11 and WC2 had no influence on the permeability of the outer membrane of E.coli at concentrations of 128 and 256 ?g/mL,and only compounds WA7,WD5 and WD6 at 32 ?g/mL did not dissipate the proton motive force(pmf).In summary,compounds WA7 and WD6 were ideal AcrB inhibitors,which meeted all the characteristics of ideal efflux pump inhibitors(EPIs):(1)it potentiated the antibacterial activity of antibiotics against bacteria strains expressing AcrB efflux pump;(2)it did not exhibit antibacterial sensitizing activity against sensitive bacteria strains lacking the AcrB efflux pump;(3)it inhibited extrusion of AcrB substrates(e.g.,Nile Red);(4)it had no influence on the permeability of the outer membrane;(5)it did not affect the energy source of AcrB(proton gradient).The study of 2-naphthamide derivatives as AcrB inhibitors.Based on the structural properties of a hydrophilic,water-filled cavity in the exit region of the AcrB binding pocket and took compound A3 as a lead compound,two novel series of 2-naphthamide derivatives E and F(28 compounds)were designed and synthesized as AcrB inhibitors via introducing the structurally diverse polar groups such as fatty amines,aromatic rings with polar substituents and nitrogen containing heterocyclic rings,etc.as hydrogen bond receptors or donors at the C-4 side chain of compound A3,to explore their interactions with the surrounding amino acid residues and their accommodation with the volume in the cavity.The biological activity results indicated that compounds E3,E9,E10,F1,F7,F9 and F10 showed synergism with the antibiotics tested,exhibiting improved antibacterial sensitizing activity,better than compound A3.Among them,compounds E3 and E10 reduced the MIC of erythromycin by 8-fold at a lower concentration of 128 ?g/mL than that of the reference PA?N(200 ?g/mL).Furthermore,compound E10 reduced the MIC of chloramphenicol by 4-fold(128 ?g/mL)while compound E9(256 ?g/mL)was able to potentiate the activity of chloramphenicol by 8-fold against the wild type E.coli BW25113.Subsequently,the compounds with potent antibacterial sensitizing activity were selected to determine their efflux inhibition ability and effect on outer or inner membrane permeabilization.The results showed that to a certain degree,all of the tested compounds exhibited Nile Red efflux inhibitory activity at different concentrations.Among them,compound E10 exhibited the best efflux inhibition activity,which were able to completely inhibit the efflux of Nile Red at concentrations of 100?M,compounds E3,E9,F7 and F9 had no influence on the permeability of the outer membrane while compounds E10,F1 and F10 significantly accelerated the rate of nitrocefin hydrolysis.Compounds E3,E9,E10,F7,F9 and F10 except F1 did not dissipate the proton motive force(pmf).Thus,compounds E3,WE9 and F7 were ideal AcrB inhibitors.The study of benzochromene derivatives as AcrB inhibitors.On the basis of crystal research and molecular modeling,the novel benzo[h]chromene structure was designed by combining the effective pharmacophore naphthalene ring of 2-naphthamide A crB inhibitors with pyranopyridine ring using the fragment splicing and scaffold hopping strategies.Based on this skeleton structure,four series of benzochromene derivatives(WI-WL)were designed,synthesized through the introduction of different polar groups at the C-5 position of the nucleus to investigate whether or not the additional affinity force with surrounding amino acid residues and water molecule formed,and meanwhile,through the introduction of the diverse substituents on the benzene ring at the end of the side chain to explore their SARs.The biological activities of benzo[h]chromenes were summarized as follows.In the series J,compound WJ10 showed better antimicrobial sensitizing activity than the reference PA?N,which was found to potentiate the antibacterial activity of chloramphenicol by 2-fold at a lower concentration of 32 ?g/mL.In the series WK,most of the compounds such as WK1-WK7 exhibited significant antibacterial sensitizing activity,and especially compound WK2 showed significant efficacy,reducing the MIC values of all the antibiotics tested by 4-to 16-fold and showing a broad spectrum feature.In the series WL,compounds WL7 and WL10 owning the morphoyl formyl group at the C-5 position showed the best antibacterial sensitizing activity,which could potentiate the antibacterial activity of all the antibiotics tested in a concentration dependent manner(8 to 128?g/mL),decreasing their MIC values by 2-fold to 16-fold.The SAR study of the benzo[h]chromene derivatives WI-WL indicated that(1)the introduction of different substituents at the C-5 position of benzo[h]chromene nucleus could remarkably affect the antibacterial sensitizing activity,and the relatively small groups with rigid connection with the nucleus were good for retaining or improving the antibacterial sensitizing activity;(2)on the right benzene ring,both a larger hydrophilic group and a smaller lipophilic group could be introduced,which had no influence the antibacterial sensitizing activity.Subsequently,the compounds(WK and WL series)with potent antibacterial sensitizing activity were selected to determine their efflux inhibition ability and effect on outer and inner membrane permeabilization.The results showed that compounds WL8? WL9 exhibited the best efflux inhibition activity,which were able to completely inhibit the efflux of Nile Red at concentrations of 50?M,compounds WK1-WK7 and WL7-WL10 had no influence on the permeability of the outer membrane while compounds WL1 and WL2 had a slight effect on the rate of hydrolysis of nitrocefin at a concentration of 128 ?g/mL.Compounds WK1-WK7?WL2?WL7 and WL8 did not dissipate the proton motive force(pmf).In summary,we chose AcrB inhibitors NDGA and A3 found by our group previously as the lead compounds,based on crystal complex structures of bacterial AcrB protein and respective action modes of catechols,2-naphthalenecarboxamides and benzochromenes,totally 106 compounds that belong to three categories were rationally designed,synthesized and evaluated as AcrB inhibitors via using the principles of medicinal chemistry and computer-aided drug design methods,then we determine their inherent antibacterial activity,antibacterial sensitizing activity,Nile Red efflux inhibition activity,effect on outer-and inner membrane permeabilization,meanwhile,molecular docking was widely used to verify the rationality of the design.After structural modification and optimization based on the SAR information,we got some AcrB inhibitors with high sensitizing activity and selectivity.In particular,compounds WA7 and E9 showed significantly better antibacterial sensitizing activity than the lead compounds NDGA and A3,and they were able to abolish efflux to the level of the pump-deleted strain.Moreover,they did not destroy inner membrane proton gradient and outer membrane permeability,which indicated that WA7 and WE9 were ideal AcrB inhibitors.In addition,benzo[h]chromene AcrB inhibitors were our first discovery of such inhibitors and had not been previously reported in the literature.Especially compounds WK2,WL7 and WL10 exhibited broad-spectrum,high-efficiency antibacterial sensizing activity,better than PA?N,which provided a new insight into discovery of novel AcrB inhibitors.This study not only discovered several novel lead compounds with good sensitizing activity but also deepened our understanding in the spatial structure of the AcrB protein and provided theoretical basis for the rational design of AcrB inhibitors,which provided a new idea for the treatment of multidrug resistant bacteria infection.