Labeling And Inhibition Studies Of New Delhi Metallo-?-lactamase-1(NDM-1)

?-Lactam antibiotics,including penicillins,cephalosporins and carbapenems,remain the most important and frequently used antimicrobial agents,constituting more than 50%of the antibiotics prescribed worldwide.However,the immoderate use of?-lactams and the lack of supervision have resulted in a large number of bacteria that are resistant to most?-Lactam antibiotics,and antibiotic resistance has seriously threatened human life and health.Most commonly,bacteria become resistant to?-lactams by producing metallo-?-lactamases?M?Ls?,which catalyze the hydrolysis of?-lactam antibiotics.In recent years,the widespread spread of superbacteria carrying New Delhi metal-?-lactamases?NDM-1?has triggered a serious biomedical crisis,and the bacteria can hydrolyze almost all antibiotics used clinically.Monitoring and inhibition of NDM-1 has proven challenging due to its shuttling between pathogenic bacteria and hydrolytic substrate is broad-spectrum.Therefore,the study of monitoring,labeling and inhibition of NDM-1 is of great academic and clinical value to human health.Based on this target,this paper has carried out the following research work.Based on the residue Cys221 at active site of NDM-1,eighteen ebsulfur derivatives were synthesized,characterized by ¹H and ¹³C NMR and confirmed by HRMS.Enzymatic kinetic study indicated that eighteen ebsulfurs gained except 1a–b and 1f inhibited NDM-1with an IC₅₀ value ranging of 0.16–9?M,and these inhibitors was found to be the dose-and time-dependent inhibitor.Also,these ebsulfurs effectively restored the antibacterial activities of cefazolin against E.coli expressing NDM-1,and the best effect was observed to be from 1g,1i and 1n,resulting in an 256-fold reduction in MIC of the antibiotic at a concentration of 16?g/mL.The equilibrium dialysis study implied that the ebsulfur disrupted the coordination of one Zn?II?ion at active site of NDM-1 and released one zinc ion.A fluorescent inhibitor Ebs-R was constructed through attaching rhodamine B to an ebsulfur moiety.Labeling and tracking NDM-1 using Ebs-R in vitro and in vivo further explained that the inhibitor covalently bound to the target through SDS-PAGE analysis and confocal microscopic imaging.Real-time activity monitoring of the NDM-1 inside living E.coli BL21 cells indicated that 1g effectively inhibited the enzyme with an IC₅₀ value of35.1?M.Moreover,these inhibitors also can effectively restore the antibacterial activity of cefazolin against clinical strains E.coli EC08 producing NDM-1.The cytotoxicity of ebsulfurs against L929 mouse fibroblastic cells showed that these compounds were less toxic and could be further developed into clinical potential inhibitors of M?Ls.According to the mechanism of cephalosporin hydrolysis by M?Ls,a method of monitoring M?Ls and screening inhibitors was established based on the combination of thiophenol probe and thiophenolized cephalosporal substrate.A thiophenolized cephalosporal Ce-s and two thiophenol probes F-1 and F-2 were successfully constructed.The selective recognition of eight sulfhydryl compounds showed that the probe F-2exhibited a high specificity and sensitivity toward thiophenol.It was found that the substrate Ce-s was hydrolyzed by NDM-1 and hydrolysate was detected by probe F-2 is a substrate concentration-and time-dependent process.The inhibition and screening of seven NDM-1 inhibitors reported in vivo and in vitro showed that this approach can be used for preliminary evaluation and screening of M?Ls inhibitors.