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Background and purpose: The production of metallo‐β‐lactamases is a major mechanisms adopted by bacterial pathogens to resist carbapenems. Repurposing approved drugs to restore the efficacy of carbapenems represents an efficient and cost‐effective approach to fight infections caused by carbapenem resistant pathogens. Experimental approach: The nitrocefin hydrolysis assay was employed to screen potential New Delhi metallo‐lactamase‐1 (NDM‐1) inhibitors from a commercially available U.S. Food and Drug Administration (FDA) approved drug library. The mechanism of inhibition was clarified by metal restoration, inductively coupled plasma mass spectrometry (ICP‐MS) and molecular dynamics simulation. The in vitro synergistic antibacterial effect of the identified inhibitors with meropenem was determined by the checkerboard minimum inhibitory concentration (MIC) assay, time‐dependent killing assay and combined disc test. Three mouse infection models were used to further evaluate the in vivo therapeutic efficacy of combined therapy. Key results: Twelve FDA‐approved compounds were initially screened to inhibit the ability of NDM‐1 to hydrolyse nitrocefin. Among these compounds, dexrazoxane, embelin, candesartan cilexetil and nordihydroguaiaretic acid were demonstrated to inhibit all tested metallo‐β‐lactamases and showed an in vitro synergistic bactericidal effect with meropenem against metallo‐β‐lactamases‐producing bacteria. Dexrazoxane, embelin and candesartan cilexetil are metal ion chelating agents, while the inhibition of NDM‐1 by nordihydroguaiaretic acid involves its direct binding to the active region of NDM‐1. Furthermore, these four drugs dramatically rescued the treatment efficacy of meropenem in three infection models. Conclusions and implications: Our observations indicated that dexrazoxane, embelin, candesartan cilexetil and nordihydroguaiaretic acid are promising carbapenem adjuvants against metallo‐β‐lactamases‐positive carbapenem resistant bacterial pathogens. [ABSTRACT FROM AUTHOR]

At present, the resistance of New Delhi metallo-β-lactamase-1 (NDM-1) to carbapenems and cephalosporins, one of the mechanisms of bacterial resistance against β-lactam antibiotics, poses a threat to human health. In this work, based on the virtual ligand screen method, we found that carnosic acid1 (CA), a natural compound, exhibited a significant inhibitory effect against NDM-1 (IC50 = 27.07 μM). Although carnosic acid did not display direct antibacterial activity, the combination of carnosic acid and meropenem still showed bactericidal activity after the loss of bactericidal effect of meropenem. The experimental results showed that carnosic acid can enhance the antibacterial activity of meropenem against Escherichia coli ZC-YN3. To explore the inhibitory mechanism of carnosic acid against NDM-1, we performed the molecular dynamics simulation and binding energy calculation for the NDM-1-CA complex system. Notably, the 3D structure of the complex obtained from molecular modeling indicates that the binding region of carnosic acid with NDM-1 was not situated in the active region of protein. Due to binding to the allosteric pocket of carnosic acid, the active region conformation of NDM-1 was observed to have been altered. The distance from the active center of the NDM-1-CA complex was larger than that of the free protein, leading to loss of activity. Then, the mutation experiments showed that carnosic acid had lower inhibitory activity against mutated protein than wild-type proteins. Fluorescence experiments verified the results reported above. Thus, our data indicate that carnosic acid is a potential NDM-1 inhibitor and is a promising drug for the treatment of NDM-1 producing pathogens. [ABSTRACT FROM AUTHOR]

Background and Purpose: Bacteria producing New Delhi metallo-β-lactamase-1 (NDM-1) are an increasing clinical threat. NDM-1 can inactivate almost all β-lactams and is not sensitive to any existing β-lactamase inhibitors. To identify effective inhibitors of the NDM-1 enzyme and clarify the mechanism of action, a "lead compound" for developing more potent NDM-1 inhibitors needs to be provided.Experimental Approach: Natural compounds were tested by enzyme inhibition screening to find potential inhibitors. MIC assays, growth curve assays, and time-kill assays were conducted to evaluate the in vitro antibacterial activity of pterostilbene and the combination of pterostilbene and meropenem. A murine thigh model and a mouse pneumonia model were used to evaluate the in vivo efficacy of combined therapy. Molecular modelling and a mutational analysis were used to clarify the mechanism of action.Key Results: Pterostilbene significantly inhibited NDM-1 hydrolysis activity in enzyme inhibition screening assays and effectively restored the effectiveness of meropenem in vitro with NDM-expressing isolates in antibacterial activity assays. In addition, the combined therapy effectively reduced the bacterial burden in a murine thigh model and protected mice from pneumonia caused by Klebsiella pneumoniae. By means of molecular dynamics simulation, we observed that pterostilbene localized to the catalytic pocket of NDM-1, hindering substrate binding to NDM-1 and reducing NDM-1 activity.Conclusions and Implications: These findings indicated that pterostilbene combined with meropenem may offer a new safe and potential "lead compound" for the further development of NDM-1 inhibitors. [ABSTRACT FROM AUTHOR]

In previous literature, it was found that the activity of New Delhi Metallo-β-lactamase-1 (NDM-1) was inhibited by 2,6-dipicolinic acid (DPA) derivatives. To identify the mechanism of interaction between the inhibitors and NDM-1, molecular dynamics simulations were performed for the complex systems. Via the molecular modelling, inhibitors were found to be able bind to the region of catalytic activity of NDM-1. However, the detailed binding sites of the inhibitors differed with their structures. It was determined that His189, Lys211, Met248, Ser249, His250, and Ser251 are key residues for the binding of inhibitor 36 with NDM-1, and Asp124 is the only critical residue in the NDM-1-DPA complex. Furthermore, because of the interaction of the benzene ring in inhibitor 36 with the side chain of Lys211, inhibitor 36 can form 4 strong hydrogen bonds with protein. For the NDM-1-DPA complex, owing to the absence of the aniline group, DPA can only form a weak interaction with the residues around the binding site of NDM-1, except for Asp124, leading to a weaker inhibitory activity. Therefore, we believe that the strong interaction of the inhibitor with Lys211 results in effective inhibition, and the aniline group is the element required for the inhibitory activity. [ABSTRACT FROM AUTHOR]

New Delhi metallo-β-lactamase (NDM-1), one of the metallo-β-lactamases (MBLs), leads to antibiotic resistance in clinical treatments due to the strong ability of hydrolysis to almost all kinds of β-lactam antibiotics. Therefore, there is the urgent need for the research and development of the novel drug-resistant inhibitors targeting NDM-1. In this study, ZINC05683641 was screened as potential NDM-1 inhibitor by virtual screening and the inhibitor mechanism of this compound was explored based on molecular dynamics simulation. The nitrocefin assay showed that the IC50 value of ZINC05683641 was 13.59 ± 0.52 μM, indicating that the hydrolytic activity of NDM-1 can be obviously suppressed by ZINC05683641. Further, the binding mode of ZINC05683641 with NDM-1 was obtained by molecular modeling, binding free energy calculation, mutagenesis assays and fluorescence-quenching assays. As results, ILE-35, MET-67, VAL-73, TRP-93, CYS-208, ASN-220 and HIS-250 played the key roles in the binding of NDM-1 with ZINC05683641. Interestingly, these key residues were exactly located in the catalytic activity region of NDM-1, implying that the inhibitor mechanism of ZINC05683641 against NDM-1 was the competitive inhibition. These findings will provide an available approach to research and develop new drug against NDM-1 and treatment for bacterial resistance. [ABSTRACT FROM AUTHOR]

Abstract The monobactam antibiotic, aztreonam, can be hydrolyzed by some β-lactamases (NDM-1), whereas other β-lactamases, including VIM-1, have no hydrolytic activity on aztreonam. NDM-1 and VIM-1 are both metallo-β-lactamases (MBLs), but they catalyze hydrolysis at different active site residues, which is reflected in their obvious activity difference with aztreonam. In this work, to explore the catalytic hydrolysis mechanism of MBLs at the atomic level, molecular dynamic simulations were performed for NDM-1-aztreonam and VIM-1-aztreonam complexes based on the molecular docking analysis. Molecular modeling revealed the binding of aztreonam to the active regions of NDM-1 and VIM-1. Residues Met67, Phe70, Asp124, Thr190, and His250 play key roles in the binding of aztreonam with NDM-1. His116, Asp118, Cys198, His201, and His240 are the critical residues for the binding of aztreonam with VIM-1. Interestingly, Asp124 displayed the strongest binding energy (ΔE total = −11.28 kcal/mol), which was nearly 10 times higher than that of the other residues in the NDM-1-aztreonam complex. A similar result was found for the binding of aztreonam with VIM-1, with Asp118 displaying the strongest binding energy (ΔE total = −2.95 kcal/mol). These results implicated aspartic acid as the critical active site in the catalytic hydrolysis pocket of NDM-1 and VIM-1. However, in the VIM-1-aztreonam complex, because of the strong π-π interaction between the thiazol ring group of aztreonam and the imidazole ring groups of His201 and His240, the binding energy obtained from Asp118 become significantly weaker than that of aztreonam with Asp124 of NDM-1. Analysis of the simulation trajectory indicated that the thiazol ring plane of aztreonam is almost parallel to the imidazole ring planes of His201 and His240, implying that they can form a strong π-π interaction, which is consistent with the above results. On the basis of the computational biology results, it was confirmed that aspartic acid in the active pocket of NDM-1 and VIM-1 can effectively promote substrate hydrolysis, while histidine on the other side of the active pocket can block the binding of the substrate with aspartic acid, leading to the loss of hydrolysis. Graphical abstract Unlabelled Image Highlights • The interaction of aztreonam towards the NDM-1 and VIM-1 complexes were explored. • The binding sites of aztreonam in the complexes were identified. • Aspartic acid in the active pocket of NDM-1 and VIM-1 promote substrate hydrolysis. • Histidine in the active pocket block the hydrolysis of the substrate. [ABSTRACT FROM AUTHOR]