Inhibition Studies Of Antibiotics Resistant Target Protein Metallo-?-lactamase And Drug-resistant Bacteria

Metallo-?-lactamase?M?Ls?is an important factor of bacterial resistance to ?-lactams.The studies on M?Ls and bacteria which produce M?Ls is of great academic significance and application value for human health and economic development.In order to develop M?Ls inhibitors and cooperate with existing antibiotics to resist to antibiotic-resistant,this thesis has carried out the following three parts of work.A total of 28 azolylthioacetamides has been designed,synthesized,and characterized by melting point,¹H and ¹³C NMR and HRMS.The inhibition study against M?Ls were evaluated,and the results showed that the obtained benzimidazolyl and benzioxazolyl substituted 1-19 specifically inhibited Imi S,and 10 was found to be the most potent inhibitor of Imi S with an IC₅₀ value of 15 n M,and the nitrobenzimidazolyl substituted 20-28 specifically inhibited NDM-1,with 27 being the most potent inhibitor with an IC50 value of 170 n M.Further studies with 10,11,and 27 revealed a mixed inhibition mode.These inhibitors resulted in a 2-4-fold decrease in imipenem MIC values using E.coli cells producing Imi S or NDM-1.Docking studies indicated that 10 and 11 may interact orthosterically with Zn in the active site of Cph A,while 27 could bridge the two Zn??? ions in the active site of NDM-1 via its nitro group.Moreover,the inhibitory activity of twenty carboxyl substituted triazolyl thioacetamides against VIM-2 was evaluated,with IC₅₀ range of 20.6-58.59 ?M.The structure-activity relationship?SAR?indicated that the aromatic carboxyl group at the R₁ position increased the inhibitory activity of inhibitors.The affinity ability of inhibitors binding to VIM-2 was tested using Isothermal Titration calorimetry?ITC?assays,and the results implyied that the initial binding of 1b or 1h to VIM-2 was together with entropy and enthalpy contribution.Inhibitors 1a-j resulted in a 2–4-fold reduction in MIC of cefzolin against cells expressing VIM-2,and the antimicrobial effect of cefazolin increased gradually with dose increasing of 1b,1c,1g,and 1h.A series of rhodanine derivatives?1a-p,2a-p,3a-f,and 4a-p?and a thioenolate 5a was constructed and characterized by melting pointing,¹H and ¹³C NMR and HRMS,and their Z-conformation was confirmed by small molecule X-ray crystal structures.The percentage inhibition of all obtained rhodanines at the concentration of 50 ?M against M?Ls NDM-1,VIM-2,Imi S and L1 indicated that the compounds 1a-m,2a-m and 5a showed various degree of inhibitory ativity.In order to further clarify the inhibitory activity of 1a-m,2a-m,and 5a against the four enzymes,the IC₅₀ evaluations were carried out,and the results showed that inhibitor 1a-p,2a-p and 5a inhibited M?L L1 with an IC50 range of 0.02-1.7 ?M and 2h-m exhibited broad-spectrum inhibition of M?Ls NDM-1,VIM-2,Imi S,and L1 with IC50 values < 16 ?M.Isothermal titration calorimetry assays indicated that the activity of M?Ls was inhibited by 2l in a dose-dependent manner.All inhibitors increased the antimicrobial effect of cefazolin against E.coli cells expressing L1,resulting in a 2-8-fold reduction in MICs.Docking studies suggested that the nitro?NDM-1,Cph A and L1?or carboxyl group?VIM-2?of 2l coordinate one or two Zn?II?ions,while the N-phenyl group of the inhibitor enhances its hydrophobic interaction with M?Ls.These studies demonstrate that the diaryl-substituted rhodanines are good scaffolds for future broad-spectrum inhibitors of M?Ls.M?Ls VIM-2 and L1 were overexpressed,purified,characterized and obtained in M9 medium with purity of more than 95%.After about 500 crystallographic conditions and manual optimization,the final crystallographic conditions of the VIM-2 and L1 were obtained.The complex crystal of VIM-2 and azolazolylthioacetyl 1b were cultured by cocrystallization method,and the crystal structure of the complex was elucidated by collecting and analysis the X-ray diffraction data.The compelx structure revealed that the N atom on the triazole ring and the carboxyl O of 1b coordinated with Zn?II?ion of the enzyme active site,and the carboxyl O formed H-bound with Asn233.The phenyl at R1 and benzimidazole moieties located at both terminal of 1b formed ?-? stacking with His263 and Phe61,respectively.This important structural information provide a new idea for the development of broad-spectrum and tight bonding M?Ls inhibitors.