Discovery of thiosemicarbazone derivatives as effective New Delhi metallo-β-lactamase-1(NDM-1) inhibitors against NDM-1 producing clinical isolates

New Delhi metallo-b-lactamase-1(NDM-1)is capable of hydrolyzing nearly allβ-lactam antibiotics,posing an emerging threat to public health.There are currently less effective treatment options for treating NDM-1 positive"superbug",and no promising NDM-1 inhibitors were used in clinical practice.In this study,structure eactivity relationship based on thiosemicarbazone derivatives wassystematically characterized and their potential activities combined with meropenem(MEM)were evaluated.Compounds 19 bg and 19 bh exhibited excellent activity against 10 NDM-positive isolate clinical isolates in reversing MEM resistance.Further studies demonstrated compounds 19 bg and 19 bh were uncompetitive NDM-1 inhibitors with Ki Z 0.63 and 0.44 mmol/L,respectively.Molecular docking speculated that compounds 19 bg and 19 bh were most likely to bind in the allosteric pocket which would affect the catalytic effect of NDM-1 on the substrate meropenem.Toxicity evaluation experiment showed that no hemolysis activities even at concentrations of 1000 mg/m L against red blood cells.In vivo experimental results showed combination of MEM and compound 19 bh was markedly effective in treating infections caused by NDM-1 positive strain and prolonging the survival time of sepsis mice.Our finding showed that compound 19 bh might be a promising lead in developing new inhibitor to treat NDM-1 producing superbug.

First identification of isatin-β-thiosemicarbazones as novel inhibitors of New Delhi metallo-β-lactamase-1： Chemical synthesis, biological evaluation and molecular simulation

New Delhi metallo-b-lactmase-1（NDM-1） catalyzes the hydrolysis of b-lactam antibiotics and cleaves the b-lactam ring of the molecule, conferring bacterial resistance against these medicines. In an effort to discover novel agents to treat this superbug, an old drug methisazone was found to be a weak NDM-1 inhibitor, with an IC50 of 297.6 mmol/L. Based on this result, a series of isatin-β-thiosemicarbazones（IBTs）were synthesized and biologically evaluated as novel NDM-1 inhibitors. Nine of the IBT compounds showed IC50 values of 〈10 mmol/L, the best of which was 2.72 mmol/L. Comparative field analysis（Co MFA） contour maps were generated to depict the structural features and molecular docking was performed to understand the possible binding mode of these inhibitors. The present research hereby has provided valuable information for further discovery of NDM-1 inhibitors.

Identification and Distribution of the Clinical Isolates of Imipenem-resistant Pseudomonas aeruginosa Carrying Metallo-β-lactamase and/or Class 1 Integron Genes

To investigate the distribution of the genes of two major metallo-β-1actamases （MBL;i.e.,IMP and VIM） and class 1 integrons （intI） in the clinical imipenem-resistant Pseudomonas aeruginosa, a total of 65 isolates, from a university hospital in Sichuan between December 2004 and April 2005 were screened for MBL genes by PCR using primers specific for blaIMP-1, blaVIM and blaVIM-2 genes. The MBL-positive isolates were further assessed for class 1 integrons by PCRusing specific primers. The nucleotide sequences of several PCR products were also determined. The results revealed that the blaVIM gene was found in 81.5% （53/65） of all isolates, blaVIM-2 gene was found in only 1 isolate and the intl gene was observed in 45.3% （24/53） of blaVIM-positive isolates. One isolate carried simultaneously both blaIMP-1 and intl genes, and to the best of our knowledge this is the first report of such isolate in southwest China. These observations highlight that the genes for VIM β-1actamase and class 1 integrons were predominantly present among the imipenem-resistant P. aeruginosa tested, confirming the current widespread threat of imipenem-resistant, integron-borne P.aeruginosa.

Insights on the structural characteristics of NDM-1: The journey so far

New Delhi metallo-β-lactamase (NDM-1) has created a medical storm ever since it was first reported;as it is active on virtually all clinically used β-lactam antibiotics. NDM-1 rampancy worldwide is now considered a nightmare scenario, particularly due to its rapid dissemination. An underlying theme in the majority of recent studies is structural characterization as knowledge of the three-dimensional structure of NDM-1 shall help find connections between its structure and function. Moreover, structural details are even critical in order to reveal the resistance mecha- nism to β-lactam antibiotics. In this perspective, we review structural characteristics of NDM-1 that have been delineated since its first report. We anticipate that these structure-function connections made by its characterization shall further serve as future guidelines for elucidating pathways towards de novo design of functional inhibitors.

IMB-XH1作为新型新德里金属-β-内酰胺酶-1(NDM-1)抑制剂的研究（英文）

革兰氏阴性菌的耐药问题日益严峻,已经引起了公众的广泛关注。产新德里金属β-内酰胺酶(NDM-1)的"超级细菌"对几乎所有的β-内酰胺类抗生素耐药。然而临床现有的β-内酰胺酶抑制剂对NDM-1等金属β-内酰胺酶无效,因此,临床上迫切需要有效NDM-1抑制剂。在这项研究中,优化了NDM-1抑制剂的高通量筛选模型。应用该模型筛选NDM-1抑制剂,并从不同来源的化合物样品中筛选得到一种NDM-1的抑制剂IMB-XH1。研究发现IMB-XH1与β-内酰胺类抗生素联合使用可以提高表达NDM-1的大肠埃希菌E.coliBL21(DE3)(pET-30a(+)-NDM-1)对β-内酰胺类抗生素的敏感性。酶促动力学研究表明IMB-XH1是NDM-1的非竞争性抑制剂。IMB-XH1还具有对其他金属β-内酰胺酶的抑制活性,如IMP-4,ImiS和L1。IMB-XH1作为一种新型的NDM-1抑制剂,值得进一步对其活性评价和作用机制进行深入研究。

A structural view of the antibiotic degradation enzyme NDM-1 from a superbug

Gram-negative Enterobacteriaceae with resistance to carbapenem conferred by New Delhi metallo-β-lactamase 1(NDM-1)are a type of newly discovered antibioticresistant bacteria.The rapid pandemic spread of NDM-1 bacteria worldwide(spreading to India,Pakistan,Europe,America,and Chinese Taiwan)in less than 2 months characterizes these microbes as a potentially major global health problem.The drug resistance of NDM-1 bacteria is largely due to plasmids containing the blaNDM-1 gene shuttling through bacterial populations.The NDM-1 enzyme encoded by the blaNDM-1 gene hydrolyzes β-lactam antibiotics,allowing the bacteria to escape the action of antibiotics.Although the biological functions and structural features of NDM-1 have been proposed according to results from functional and structural investigation of its homologues,the precise molecular characteristics and mechanism of action of NDM-1 have not been clarified.Here,we report the threedimensional structure of NDM-1 with two catalytic zinc ions in its active site.Biological and mass spectroscopy results revealed that D-captopril can effectively inhibit the enzymatic activity of NDM-1 by binding to its active site with high binding affinity.The unique features concerning the primary sequence and structural conformation of the active site distinguish NDM-1 from other reported metallo-β-lactamases(MBLs)and implicate its role in wide spectrum drug resistance.We also discuss the molecular mechanism of NDM-1 action and its essential role in the pandemic of drug-resistant NDM-1 bacteria.Our results will provide helpful information for future drug discovery targeting drug resistance caused by NDM-1 and related metallo-β-lactamases.

An unexpected similarity between antibiotic-resistant NDM-1 and beta-lactamaseⅡfrom Erythrobacter litoralis

NDM-1(New Delhi metallo-beta-lactamase)gene encodes a metallo-beta-lactamase(MBL)with high carbapenemase activity,which makes the host bacterial strain easily dispatch the last-resort antibiotics known as carbapenems and cause global concern.Here we present the bioinformatics data showing an unexpected similarity between NDM-1 and beta-lactamase II from Erythrobacter litoralis,a marine microbial isolate.We have further expressed these two mature proteins in E.coli cells,both of which present as a monomer with a molecular mass of 25 kDa.Antimicrobial susceptibility assay reveals that they share similar substrate specificities and are sensitive to aztreonam and tigecycline.The conformational change accompanied with the zinc binding visualized by nuclear magnetic resonance,Zn2+-bound NDM-1,adopts at least some stable tertiary structure in contrast to the metal-free protein.Our work implies a close evolutionary relationship between antibiotic resistance genes in environmental reservoir and in the clinic,challenging the antimicrobial resistance monitoring.