| The overuse of antibiotics has resulted in a large number of bacteria becoming resistant. Metallo-β-lactamase (MpLs)-mediated resistance to β-lactam antibiotics emerged as the most fearful threat to these lifesaving drugs. Such situation has caused great concern from governments, pharmaceutical industry and academia. In an effort to hold back the attack of antibiotic resistant pathogens, the key proteins in the resistance process were systematacially studied. Beside the development of MβLs inhibitors, alternative therapeutics such as photodynamic antimicrobial chemotherapy (PACT) and other materials that can effectively inactivate the growth of antibiotic resistant bacteria have been explored. As the results, series of highly promising options have been put forward to response the emergency. The following specific points show the main research contents of this work.In chapter 2, the purification processes of CcrA, ImiS and L1, as the representatives of MβLs B1-B3 subgroups, were described in details. A rapid detection reagent of such hydrolase was synthesized in a convenient way. We first reported two carbon monoxide-releasing molecules with efficient antibacterial effects, which provided a good option for future development of antibacterial agents.In chapter 3, we first reported the thermokinetic parameters of penicillin G, cephalothin V and imipenem hydrolysis catalyzed with metallo-β-lactamase CcrA from Bacteroides fragilis by microcalorimetrey, which presented the activation free energy, activation enthalpy, activation entropy and apparent activation energy. We also systematically studied the corresponding steady-state kinetic parameters of the above three reactions. The thermokinetic and dynamic parameters could be useful to deep understand the reaction and mechanism of antibiotic hydrolysis catalyzed by metallo-β-lactamases.In chapter 4, we have synthesized and characterized 6 basic porphyrin photosensitizers. Through photodynamic inactivation evaluation, H2(p-NH2)TPP was chosen to react with vancomycin moiety to obtain a novel phototherapeutic conjugate. The conjugate was isolated using a gel molecular sieve purification technique and confirmed by MALDI-TOF mass spectrum. Surface plasmon resonance analysis with living bacterial cells demonstrated that the selectivity of porphyrin was successfully improved by linking to the vancomycin ligand. Moreover, the conjugate displayed an overall photodynamic inactivation activity towards 6 Gram-positive bacterial strains upon white light exposure, with a killing efficiency from 70% to 95%. The constructed conjugation can be used to identify Gram-positive bacteria and generate singlet oxygen effectively under white light irradiation, which overcomes the disadvantage of common photosensitizers. The skeleton and data gained in this chapter provided a starting point for the exploration of alternative options to fighting against antibiotic resistant bacterial infections.In the last chapter, based on the thermokinetic and dynamic parameters which obtained in chapter 3, a new scaffold, azolylthioacetamide, was designed and constructed.19 correlative compounds were synthesized and isolated in total. IC50 values against metallo-β-lactamase NDM-1, ImiS and L1 were evaluated respectively. The obtained molecules specifically inhibited NDM-lwith IC50 values distributed from 0.1-1.7 micromole using cephalothin V as substrate. Structure-activity relationships revealed that the electron-donating/hydrophobic group on the benzene ring effective improves inhibitory activity of the inhibitors. Besides, the internuclear distance between the aromatic oxygen/nitrogen atom and the key heteroatom (azole nitrogen and the sulphur atom) played significant role in the bioactivity.
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