Inhibition And Activity Assay Studies Of Bacterial Drug-resistance Target Enzymes VanX And M?Ls

Antibiotics are widely used to treat bacterial infections.However,the overuse and abuse of antibiotics has exacerbated the rate at which bacteria develop resistance,leading to high morbidity and mortality in humans and animals.Drug-resistant bacteria resist almost all antibiotic,including penicillin,cephalosporins,carbapenems and glycopeptide antibiotics known as the?last line of defense?.In particular,superbugs mediated by D-D dipeptidase VanX and metal-?-lactamase?M?Ls?render almost all antibiotics ineffective,leading to clinical medical crisis.In view of the importance of bacterial resisitence research,this paper has carried out the following work:The D-D dipeptidase enzyme VanX is the target enzyme of vancomycin resistance in gram-positive bacteria.Continuous activity assaying of VanX has become a challenging task due to lack of chromophoric substrate.Here,an isothermal titration calorimetry?ITC?method was developed to monitor and inhibit the real-time activity of VanX and its drug-resistence bacteria.Furthermore,with the ITC approach,determination of thermodynamic parameters K_m?0.29 mM?and k_cat(37.3 s^-1)of VanX hydrolysis of D-Ala-D-Ala and inhibition constant of D-cysteine inhibitor K_i?20.8?M?,which are consistent with the data from ninhydrin/Cd?II?assays,and also reported in the literature.For the first time,characterization of VanX activity and its inhibition by ITC based on drug-resistence bacteria?including clinical bacteria?in vivo.The results showed that the VanX expressed in E.coli and in vancomycin-resistant enterococcus faecalis?VRE?was effectively inhibited by D-cysteine with IC₅₀ values of 29.8 and 28.6?M,respectively.Also,with ITC,the determined apparent enthalpy change?H_app values?-10.5 and-0.017kJ/mol?of VRE and E.faecalis hydrolyzing D-Ala-D-Ala are correlated well with the MIC results?512 and 4?g/mL?of vancomycin against VRE and E.faecalis.Eight schiff base M?Ls inhibitors were designed and synthesized,which were characterized by ¹H,¹³C NMR and MS.The biological activity assay demonstrated that four of them effectively inhibit M?Ls NDM-1 and VIM-2 with an IC₅₀ range of 0.16-12?M.It is worth pointing out that these four compounds can effectively inhibit drug-resistant bacteria expressing M?Ls in combination with antibiotics,and resulted in a2-128 fold reduction in MIC value of antibiotics.