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A Lysine-Targeted Affinity Label for Serine-?-Lactamase Also Covalently Modifies New Delhi Metallo-?-lactamase-1 (NDM-1).

ABSTRACT: The divergent sequences, protein structures, and catalytic mechanisms of serine- and metallo-?-lactamases hamper the development of wide-spectrum ?-lactamase inhibitors that can block both types of enzymes. The O-aryloxycarbonyl hydroxamate inactivators of Enterobacter cloacae P99 class C serine-?-lactamase are unusual covalent inhibitors in that they target both active-site Ser and Lys residues, resulting in a cross-link consisting of only two atoms. Many clinically relevant metallo-?-lactamases have an analogous active-site Lys residue used to bind ?-lactam substrates, suggesting a common site to target with covalent inhibitors. Here, we demonstrate that an O-aryloxycarbonyl hydroxamate inactivator of serine-?-lactamases can also serve as a classical affinity label for New Delhi metallo-?-lactamase-1 (NDM-1). Rapid dilution assays, site-directed mutagenesis, and global kinetic fitting are used to map covalent modification at Lys211 and determine KI (140 ?M) and kinact (0.045 min-1) values. Mass spectrometry of the intact protein and the use of ultraviolet photodissociation for extensive fragmentation confirm stoichiometric covalent labeling that occurs specifically at Lys211. A 2.0 Å resolution X-ray crystal structure of inactivated NDM-1 reveals that the covalent adduct is bound at the substrate-binding site but is not directly coordinated to the active-site zinc cluster. These results indicate that Lys-targeted affinity labels might be a successful strategy for developing compounds that can inactivate both serine- and metallo-?-lactamases.

SUBMITTER: Thomas PW

PROVIDER: S-EPMC6597302 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Project description:Bacteria expressing New Delhi metallo-β-lactamase-1 (NDM-1) can hydrolyze β-lactam antibiotics (penicillins, cephalosporins, and carbapenems) and, thus, mediate multidrug resistance. The worldwide dissemination of NDM-1 poses a serious threat to public health, imposing a huge economic burden in the development of new antibiotics. Thus, there is an urgent need for the identification of novel NDM-1 inhibitors from a pool of already-known drug molecules. Here, we screened a library of FDA-approved drugs to identify novel non-β-lactam ring-containing inhibitors of NDM-1 by applying computational as well as in vitro experimental approaches. Different steps of high-throughput virtual screening, molecular docking, molecular dynamics simulation, and enzyme kinetics were performed to identify risedronate and methotrexate as the inhibitors with the most potential. The molecular mechanics/generalized Born surface area (MM/GBSA) and molecular dynamics (MD) simulations showed that both of the compounds (risedronate and methotrexate) formed a stable complex with NDM-1. Furthermore, analyses of the binding pose revealed that risedronate formed two hydrogen bonds and three electrostatic interactions with the catalytic residues of NDM-1. Similarly, methotrexate formed four hydrogen bonds and one electrostatic interaction with NDM-1's active site residues. The docking scores of risedronate and methotrexate for NDM-1 were -10.543 kcal mol-1 and -10.189 kcal mol-1, respectively. Steady-state enzyme kinetics in the presence of risedronate and methotrexate showed a decreased catalytic efficiency (i.e., kcat/Km) of NDM-1 on various antibiotics, owing to poor catalytic proficiency and affinity. The results were further validated by determining the MICs of imipenem and meropenem in the presence of risedronate and methotrexate. The IC50 values of the identified inhibitors were in the micromolar range. The findings of this study should be helpful in further characterizing the potential of risedronate and methotrexate to treat bacterial infections.

Project description:The effectiveness of all antibiotics in the β-lactam group to cure bacterial infections has been impaired by the introduction of the New Delhi Metallo-β-lactamase (NDM-1) enzyme. Attempts have been made to discover a potent chemical as an inhibitor to this enzyme in order to restore the efficacy of antibiotics. However, it has been a challenging task to develop broad-spectrum inhibitors of metallo-β-lactamases. Lack of sequence homology across metallo-β-lactamases (MBLs), the rapidly evolving active site of the enzyme, and structural similarities between human enzymes and metallo-β-lactamases, are the primary causes for the difficulty in the development of these inhibitors. Therefore, it is imperative to concentrate on the discovery of an effective NDM-1 inhibitor. This study used various in silico approaches, including molecular docking and molecular dynamics simulations, to investigate the potential of phytochemicals to inhibit the NDM-1 enzyme. For this purpose, a library of about 59,000 phytochemicals was created from the literature and other databases, including FoodB, IMPPAT, and Phenol-Explorer. A physiochemical and pharmacokinetics analysis was performed to determine possible toxicity and mutagenicity of the ligands. Following the virtual screening, phytochemicals were assessed for their binding with NDM-1using docking scores, RMSD values, and other critical parameters. The docking score was determined by selecting the best conformation of the protein-ligand complex. Three phytochemicals, i.e., butein (polyphenol), monodemethylcurcumin (polyphenol), and rosmarinic acid (polyphenol) were identified as result of pharmacokinetics and molecular docking studies. Furthermore, molecular dynamics simulations were performed to determine structural stabilities of the protein-ligand complexes. Monodemethylcurcumin, butein, and rosmarinic acid were identified as potential inhibitors of NDM-1 based on their low RMSD, RMSF, hydrogen bond count, average Coulomb-Schrödinger interaction energy, and Lennard-Jones-Schrödinger interaction energy. The present investigation suggested that these phytochemicals might be promising candidates for future NDM-1 medication development to respond to antibiotic resistance.

Project description:BACKGROUND:New Delhi metallo-?-lactamase (bla NDM), a plasmid-borne carbapenemase gene associated with significant mortality and severely limited treatment options, is of global public health concern as it is found in extremely diverse Gram-negative bacterial strains. This study thus aims to genetically characterize local and global spread of bla NDM. METHODS:To investigate local transmission patterns in the context of a single hospital, whole genome sequencing data of the first 11 bla NDM-positive bacteria isolated in a local hospital were analyzed to: (1) identify and compare bla NDM-positive plasmids; and (2) study the phylogenetic relationship of the bacteria chromosomes. The global analysis was conducted by analyzing 2749 complete plasmid sequences (including 39 bla NDM-positive plasmids) in the NCBI database, where: (1) the plasmids were clustered based on their gene composition similarity; (2) phylogenetic study was conducted for each bla NDM-positive plasmid cluster to infer the phylogenetic relationship within each cluster; (3) gene transposition events introducing bla NDM into different plasmid backbones were identified; and (4) clustering pattern was correlated with the plasmids' incompatibility group and geographical distribution. RESULTS:Analysis of the first 11 bla NDM-positive isolates from a single hospital revealed very low bla NDM-positive plasmid diversity. Local transmission was characterized by clonal spread of a predominant plasmid with 2 sporadic instances of plasmid introduction. In contrast to the low diversity locally, global bla NDM spread involved marked plasmid diversity with no predominant bacterial clone. Thirty-nine (1.4 %) out of the 2749 complete plasmid sequences were bla NDM-positive, and could be resolved into 7 clusters, which were associated with plasmid incompatibility group and geographical distribution. The bla NDM gene module was witnessed to mobilize between different plasmid backbones on at least 6 independent occasions. CONCLUSIONS:Our analysis revealed the complex genetic pathways of bla NDM spread, with global dissemination characterized mainly by transposition of the bla NDM gene cassette into varied plasmids. Early local transmission following plasmid introduction is characterized by plasmid conjugation and bacterial spread. Our findings emphasize the importance of plasmid molecular epidemiology in understanding bla NDM spread.

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A Lysine-Targeted Affinity Label for Serine-?-Lactamase Also Covalently Modifies New Delhi Metallo-?-lactamase-1 (NDM-1).

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