Inhibition Studies Of Metallo-β-Lactamases And Drug-resistant Bacteria By Hydroxamate,sulfonamide And Sulfonate

Drug resistance is a major public health safety issue.The containment of bacterial resistance mediated byβ-lactamases has become the global action plan announced at the G20 Summit（2016）and China’s“National Action Plan for Containment of Bacterial Drug Resistance”.β-Lactamases include the serineβ-lactamases（SβLs）and metallo-β-lactamases（MβLs）.However,the drug-resistant bacteria carrying MβLs（also known as superbugs）are resistant to almost allβ-lactam antibiotics,and there are no MβLs inhibitors for clinical purpose to date.To this end,hydroxamates,sulfonamides and sulfonates were designed and synthesized,and their inhibitory activities against MβLs and drug-resistant bacteria with MβLs were evaluated.This thesis is divided into the following two parts:1.Targeted at the lysine residues in the active site of MβLs,13 hydroxamate inhibitors were designed and synthesized,which were characterized by ¹H and ¹³C NMR and confirmed by HRMS.Bioactivity evaluation showed that these synthetic hydroxamate derivatives inhibited both MβLs IMP-1 and NDM-1 well,with an IC₅₀ value as low as 0.10and 0.23μM,respectively.Multiplex dilution and equilibrium dialysis experiments showed that the hydroxamate irreversibly inhibited IMP-1,which means that where the enzyme,when treated by an inhibitor,cannot restore activity by multidilution or dialysis.The ICP-MS analysis indicated that the inhibition of IMP-1 by the hydroxamate is different from that of EDTA,where the Zn（II）of the enzyme active center is not stripped by the inhibitor.Thermal shift and MALDI-TOF mass spectrometry evaluation showed that hydroxamate forms a covalent complex after binding to IMP-1.2.Thirty-one quinoline sulfonamide and sulfonate derivatives were synthesized.The enzyme kinetic assays showed that these derivatives have good inhibitory activity against NDM-1,with an IC₅₀ value as low as 0.02μM.The structure-activity relationship showed that the substituents of phenyl ring or phenyl groups with halogen atoms more significantly improved inhibitory effect of quinoline derivatives on NDM-1 than the aliphatic alkyl substituents.The MIC evaluation revealed that a dose of 16μg/m L quinoline sulfonamide derivatives increased inhibitory efficacy of meropenem on clinically resistant bacteria with NDM-1,resulting in a 64 fold decrease in MIC.Most importantly,inhibitor 1e reduced the bacterial load of EC08 in the spleen and liver of mice in combination with meropenem after a single intraperitoneal administration.Docking studies revealed that the inner ring nitrogen atom of quinoline ring and the outer ring nitrogen atom of sulfonamide coordinated to the Zn（II）ion at active site of NDM-1.This work provides potential lead compounds for further the development of inhibitors targeting NDM-1.