Study On Characterization And Inhibition Of Antibiotics Resistant Target Protein M?Ls And Their Drug-Resistant Bacteria

Antibiotics are one of the most effective drug to treat bacterial infection.However,the overuse of antibiotics aggravated the emerging of antibiotic-resistant pathogens,making the original effective antibiotics lose their effect.One of the most important mechanisms of antibiotic-resistance is the production of?-lactamases,which can hydrolyze almost all?-lactam antibiotics,such as penicillins,cephalosporins and carbapenems.To date,serine?-lactamases?S?Ls?inhibitors have been used in clinic,such as clavulanic acid,sulbactam,and tazobactam.The fast evolution of metallo?-lactamases?M?Ls?makes it difficult to develop M?Ls inhibitors.Therefore,developing effective M?Ls inhibitors,real-time monitoring of M?Ls activity and its inhibition are of theoretical significance and application values.The main contents of this study are as following:Obtaining high purity of M?Ls,exploring M?Ls inhibitors and their inhibitory mechanism are effective strategies to overcome?-lactamase-mediated resistance to?-lactams.In this study,six M?Ls belonging to three subclasses of class B,NDM-1,VIM-2,IMP-1,Bla2,ImiS and L1,were successfully expressed and purified.Their purity were over95%.Nine amino acid thioesters 1-9 were evaluated against ImiS,IMP-1,NDM-1,and L1,with an IC₅₀ value ranging from 0.02 to 54.9?M.MIC assays showed that thioesters 1,5and 9 restored 2-32-fold antibacterial activity of cefazolin and/or imipenem against both E.coli BL21 and DH10B strain expressing ImiS,L1,IMP-1 and NDM-1.The study of its mechanism indicated that thioester 1 was partially hydrolyzed by M?Ls to be converted into the mercaptoacetic acid,revealing that the thioester and its hydrolysate mercaptoacetic acid jointly inhibit M?Ls.Docking studies suggested that 1 bound to Zn?II?ion?s?preferentially via its carboxylate group,while other moieties interacted mostly with the conserved active site residues.Based on the principle of heat absorption or release during the specific reaction between enzyme and substrate in enzyme kinetics.Isothermal titration calorimetry?ITC?monitored the heat flow curves of NDM-1,L1 and antibiotic-resistant pathogen producing M?Ls,indicating that ITC can be used to monitor antibiotic-resistant pathogen expressing M?Ls and screening its substrate spectrum by the amount of heat release.Then,cell-based ITC demonstrated that the NDM-1 expressed in bacterial cells was inhibited by four known NDM-1 inhibitors EDTA,D-captopril,ebselen and azolylthioacetamide with IC₅₀ values of3.8,48,0.55,and 17.5?M,respectively,which are in good agreement with the data from inhibition kinetics using UV-Vis spectroscopy or NMR in vivo.Meanwhile,in order to further verify the universality of ITC in evaluating the inhibitory activity of M?Ls in bacterial living cells,the inhibitory activity of L1 inhibitors?mercaptoacetic acid,amino acid thioester and rhodamine?was determined.The results also showed that the IC₅₀ values are consistent with the values determined by UV-Vis spectroscopy.In addition,the method can distinguish reversible and irreversible inhibitor by the determination of Q_total.Finally,the cell-based ITC was used to monitor the heat during antibiotic hydrolysis by clinical antibiotic-resistant pathogen,which were consistent with the values of MIC assay.The above results showed that we established an ITC method for real-time monitoring activity and inhibition of M?Ls in living bacterial cells.Antisense oligodeoxynucleotides?ASO?has been used in drug development by silencing the expression of antibiotic resistance genes with the characteristics of rapid design and synthesis.In this study,PS-ODN targeting NDM-1 was designed and synthesized.The PLGA-PEI nanoparticles were prepared by one-step method.The results of DLS showed that the particles were uniform in size,and the morphology of the nanoparticles was spherical by scanning electron microscope?SEM?.The Zeta potential was positive charge,indicating PEI bind to the surface of nanoparticles effectively and resulting in good binding ability to PS-ODN.The optimal drug loading conditions were obtained by adjusting the ratio of PLGA-PEI to PS-ODN,and the loading efficiency was above 95%.PLGA-PEI/PS-ODN and imipenem showed good bacteriostatic effect on drug-resistant bacteria expressing NDM-1.When the concentration of PS-ODN nanoparticles was above 7?g/mL,PS-ODN nanoparticles increased 4-8-fold antimicrobial activity of imipenem with dose dependent.The results of Blue-Carba and realtime PCR revealed that the mechanism of antisense nanoparticles is to inhibit the expression of NDM-1 and antisense nanoparticles had no antibacterial activity.When the concentration of antisense nanoparticles was 28?g/mL,the expression percentage of NDM-1 was 15%of that of normal expression.The results of this study provide a new idea for the treatment of antibiotic resistant bacterial infection with antisense oligodeoxynucleotides.