Characterization And Inhibition Of Metal Inside The β- Lactamase

Most infections are caused by bacteria. Antibiotics, as a powerful weapon, are used to combate these infections for a long time. Most of the antibiotics are β-lactams. However, most of the bacteria are ever-evolving and resistant to nearly all available β-lactams in the fight against human. Bacterial resistance to β-lactams is most often the result of the production of metallo-β-lactamases (MβLs), which are enzymes that hydrolyze and inactivate the antibiotic. Therefore, it is particularly important of overexpression, characterization and design and synthesis of inhibitors of MβLs. Based on this, we carried out our research as follows:1. MβL L1was over-expressed in BL21(DE3)pLysS E. coli, purified by FPLC, and identified by SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis). Then L1was characterized by UV-visible spectroscopy, fluorescence spectroscopy and kinetic. In additional, the metal free L1(Apo-L1) and recombinant L1were also prepared and their effective diameters were measured. The inhibition study indicated that the cefazolin combination with N-Heterocyclic dicarboxylic acids lc against E.coli-Ll exhibited very effective inhibitory activity, making the sterilization efficacy of antibiotics increased16times.2. We firstly determined the thermokinetic parameters of cefalexin hydrolysis with MβL L1in the absence and presence of inhibitor. The presence of inhibitor caused a value decrease of the rate constant k, and a value increase of the apparent activation energy E, activation enthalpy ΔHθ≠, activation free energy ΔGθ≠and activation entropy ΔSθ≠in comparison with the absence of inhibitor, proposing that the changes of k, E, ΔHθ≠and ΔSθ≠values can be employed to identify the inhibitors of MβLs. The information gained in this work will be valuable for the screening of enzyme inhibitors.3. Eight new compounds were synthesized and characterized by1H NMR,13C NMR and MS. Then the inhibition studies against MβLs and antibacterial activities of the compounds in combination with existing P-lactam antibiotics were evaluated. Biological activity assays demonstrated that the compound1is a broad-spectrum inhibitor of MβLs and three subgroup MβLs all can be inhibited by it. Especially against MβL ImiS, the value of IC50is0.03μM. Seven compounds2-8all exhibited inhibitory activity against MβL L1, Compound6 exhibited the most effective inhibitory activity and the Ki values displaying0.11μM. Compounds6combination with cefazolin against E. coli BL21(DE3) that produce MβLs showed a value of MIC is2μg/mL (the control is8μg/mL) and it meant that antimicrobial effect of antibiotics was increased4times. These seven compounds2-8exhibited selective inhibitory activity against MβL L1can be used for further inhibition mechanism research.