Inhibition Studies Of Metallo-?-Lactamase And Antibiotic-Resistant Bacteria

The excessive use of?-lactam antibiotics,including penicillins,cephalosporins,carbapenems,and monobactams,has resulted in the emergence and dissemination of highly efficient resistance bacteria,which poses a serious threat to human health and economic and social development.An important mechanism of resistance to?-lactam antibiotics involves?-lactamase catalysis.Metallo-?-lactamases?M?Ls?can hydrolyse almost all?-lactam antibiotics and their plasmids are easily transferred horizontally between different pathogens,making its monitoring and inhibition a great challenge.Obviously,the monitoring and labeling studies of M?Ls have extremely important value in academic and clinical application for human health.In this work,in order to develop the M?Ls inhibitor and cooperate with the existing antibiotics to deal with bacterial resistance,we carried out the following research work.1.To probe a truth that mercaptoacetate thioester inhibiting L1 is contribution of the thioester itself or its hydrolysate,ten mercaptoacetate thioesters were synthesized,which specifically inhibited L1,exhibiting an IC₅₀ ranging from 0.17 to 1.2?M.These thioesters restored 2-4-fold antimicrobial activity of cefazolin against E.coli expressing L1.UV-Vis monitoring shown that 9 was unhydrolyzed in Tris buffer?pH 6.0-8.5?,but hydrolyzed by L1,further HPLC monitoring indicated that 1/3 of the thioester were converted to mercaptoacetic acid.STD-NMR monitoring suggested that both thioester and its hydrolysate mercaptoacetic acid jointly inhibited L1.Docking studies suggested that the carboxyl oxygen of 4 and 9 interacts Zn²⁺of L1,and bond tightly at active sites by forming hydrogen bond with residual Asp120,and hydrophobic interaction with residual Phe156,Pro226 and Trp36.2.Twenty ebselen derivatives were designed and synthesized,which is characterized by ¹H NMR,¹³C NMR and MS.The biological activity evaluation showed that all compounds tested effectively inhibit NDM-1,IMP-1 and ImiS,but no inhibition on L1.By chemical coupling of ebselen,cephalosporins core and azolyl thioacetamide,a bifunctional broad-spectrum M?Ls inhibitor GAE was synthesized,which can effectively inhibit NDM-1?B1?,ImiS?B2?and L1?B3?,and produce antibacterial activity against E.coli expressing M?Ls in combination with antibiotics.In addition,by modifying the physicochemical properties of ebselen,the broad-spectrum antimicrobial agents 3f and 3s were synthesized.The compounds 3f and 3s showed potential broad-spectrum antibacterial activity against 6 Gram-positive bacteria and 12 Gram-negative strains in the range of MIC0.25-128?g/m L and 2-64?g/m L,respectively.3.Based on the structural characteristics of the active sites of M?Ls and ebselen core,we designed and developed a selective inhibition and labeling agent RB and the dual covalent NDM-1 inhibitors SB and CB.RB can selectively inhibit and label different subclasses M?Ls using SDS-PAGE.3D-superresolution fluorescence imaging was first used to observe the real-time distribution change process of intracellular protein NDM-1 in vivo and the target initially associate on the surface of bacteria cells,a high density of uniform localization of NDM-1 throughout the cytosol of cells,and the protein accumulated in formation of inclusion bodies at the cell poles.Flow cytometry assays showed that 73.2%of RB entered the E.coli cells with NDM-1 at a inhibitor concentration of 20?M where the ebselen bound to NDM-1.The UV real-time activity monitoring showed that the RB effectively inhibit the hydrolysis of antibiotic by NDM-1 resistant strain E.coli?BL21?and clinical strain EC08;It is worth pointing out that SB and CB is the first dual covalent inhibitor of NDM-1,which provides a new method to fight the infection of drug-resistant bacteria.