Metallo-?-Lactamases and Aptamer-Based Inhibition
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ABSTRACT:
SUBMITTER: Schlesinger S
PROVIDER: S-EPMC4053962 | biostudies-literature | 2011 Feb
REPOSITORIES: biostudies-literature
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Project description:The hydrolysis of β-lactam antibiotics by β-lactamase enzymes is the most prominent antibiotic resistance mechanism for many pathogenic bacteria. Out of this broad class of enzymes, metallo-β-lactamases are of special clinical interest because of their broad substrate specificities. Several in vitro inhibitors for various metallo-β-lactamases have been reported with no clinical efficacy. Previously, we described a 10-nucleotide single stranded DNA aptamer (10-mer) that inhibits Bacillus cereus 5/B/6 metallo-β-lactamase very effectively. Here, we find that the aptamer shows uncompetitive inhibition of Bacillus cereus 5/B/6 metallo-β-lactamase during cefuroxime hydrolysis. To understand the mechanism of inhibition, we report a 2.5 Å resolution X-ray crystal structure and solution-state NMR analysis of the free enzyme. Chemical shift perturbations were observed in the HSQC spectra for several residues upon titrating with increasing concentrations of the 10-mer. In the X-ray crystal structure, these residues are distal to the active site, suggesting an allosteric mechanism for the aptamer inhibition of the enzyme. HADDOCK molecular docking simulations suggest that the 10-mer docks 26 Å from the active site. We then mutated the three lysine residues in the basic binding patch to glutamine and measured the catalytic activity and inhibition by the 10-mer. No significant inhibition of these mutants was observed by the 10-mer as compared to wild type. Interestingly, mutation of Lys50 (Lys78; according to standard MBL numbering system) resulted in reduced enzymatic activity relative to wild type in the absence of inhibitor, further highlighting an allosteric mechanism for inhibition.
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Project description:The metallo-beta-lactamases fall into two groups: Ambler class B subgroups B1 and B2 and Ambler class B subgroup B3. The two groups are so distantly related that there is no detectable sequence homology between members of the two different groups, but homology is clearly detectable at the protein structure level. The multiple structure alignment program MAPS has been used to align the structures of eight metallo-beta-lactamases and five structurally homologous proteins from the metallo-beta-lactamase superfamily, and that alignment has been used to construct a phylogenetic tree of the metallo-beta-lactamases. The presence of genes from Eubacteria, Archaebacteria, and Eukaryota on that tree is consistent with a very ancient origin of the metallo-beta-lactamase family.
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Project description:New Delhi metallo-β-lactamase-1 (NDM-1) poses an immediate threat to our most effective and widely prescribed drugs, the β-lactam-containing class of antibiotics. There are no clinically relevant inhibitors to combat NDM-1, despite significant efforts toward their development. Inhibitors that use a carboxylic acid motif for binding the ZnII ions in the active site of NDM-1 make up a large portion of the >500 inhibitors reported to date. New and structurally diverse scaffolds for inhibitor development are needed urgently. Herein we report the isosteric replacement of one carboxylate group of dipicolinic acid (DPA) to obtain DPA isosteres with good inhibitory activity against NDM-1 (and related metallo-β-lactamases, IMP-1 and VIM-2). It was determined that the choice of carboxylate isostere influences both the potency of NDM-1 inhibition and the mechanism of action. Additionally, we show that an isostere with a metal-stripping mechanism can be re-engineered into an inhibitor that favors ternary complex formation. This work provides a roadmap for future isosteric replacement of routinely used metal binding motifs (i.e., carboxylic acids) for the generation of new entities in NDM-1 inhibitor design and development.
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Project description:In an attempt to exploit the hydrolytic mechanism by which β-lactamases degrade cephalosporins, we designed and synthesized a series of novel cephalosporin prodrugs aimed at delivering thiol-based inhibitors of metallo-β-lactamases (MBLs) in a spatiotemporally controlled fashion. While enzymatic hydrolysis of the β-lactam ring was observed, it was not accompanied by inhibitor release. Nonetheless, the cephalosporin prodrugs, especially thiomandelic acid conjugate (8), demonstrated potent inhibition of IMP-type MBLs. In addition, conjugate 8 was also found to greatly reduce the minimum inhibitory concentration of meropenem against IMP-producing bacteria. The results of kinetic experiments indicate that these prodrugs inhibit IMP-type MBLs by acting as slowly turned-over substrates. Structure-activity relationship studies revealed that both phenyl and carboxyl moieties of 8 are crucial for its potency. Furthermore, modeling studies indicate that productive interactions of the thiomandelic acid moiety of 8 with Trp28 within the IMP active site may contribute to its potency and selectivity.
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Project description:The ascendancy of metallo-beta-lactamases within the clinical sector, while not ubiquitous, has nonetheless been dramatic; some reports indicate that nearly 30% of imipenem-resistant Pseudomonas aeruginosa strains possess a metallo-beta-lactamase. Acquisition of a metallo-beta-lactamase gene will invariably mediate broad-spectrum beta-lactam resistance in P. aeruginosa, but the level of in vitro resistance in Acinetobacter spp. and Enterobacteriaceae is less dependable. Their clinical significance is further embellished by their ability to hydrolyze all beta-lactams and by the fact that there is currently no clinical inhibitor, nor is there likely to be for the foreseeable future. The genes encoding metallo-beta-lactamases are often procured by class 1 (sometimes class 3) integrons, which, in turn, are embedded in transposons, resulting in a highly transmissible genetic apparatus. Moreover, other gene cassettes within the integrons often confer resistance to aminoglycosides, precluding their use as an alternative treatment. Thus far, the metallo-beta-lactamases encoded on transferable genes include IMP, VIM, SPM, and GIM and have been reported from 28 countries. Their rapid dissemination is worrisome and necessitates the implementation of not just surveillance studies but also metallo-beta-lactamase inhibitor studies securing the longevity of important anti-infectives.
| S-EPMC1082798 | biostudies-literature
Project description:Metallo-β-lactamases (MBLs) catalyse the hydrolysis of almost all β-lactam antibacterials including the latest generation carbapenems and are a growing worldwide clinical problem. It is proposed that MBLs employ one or two zinc ion cofactors in vivo. Isolated MBLs are reported to use transition metal ions other than zinc, including copper, cadmium and manganese, with iron ions being a notable exception. We report kinetic and biophysical studies with the di-iron(II)-substituted metallo-β-lactamase II from Bacillus cereus (di-Fe(II) BcII) and the clinically relevant B1 subclass Verona integron-encoded metallo-β-lactamase 2 (di-Fe(II) VIM-2). The results reveal that MBLs can employ ferrous iron in catalysis, but with altered kinetic and inhibition profiles compared to the zinc enzymes. A crystal structure of di-Fe(II) BcII reveals only small overall changes in the active site compared to the di-Zn(II) enzyme including retention of the di-metal bridging water; however, the positions of the metal ions are altered in the di-Fe(II) compared to the di-Zn(II) structure. Stopped-flow analyses reveal that the mechanism of nitrocefin hydrolysis by both di-Fe(II) BcII and di-Fe(II) VIM-2 is altered compared to the di-Zn(II) enzymes. Notably, given that the MBLs are the subject of current medicinal chemistry efforts, the results raise the possibility the Fe(II)-substituted MBLs may be of clinical relevance under conditions of low zinc availability, and reveal potential variation in inhibitor activity against the differently metallated MBLs.
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Project description:β-Lactams are the most successful antibacterials, yet their use is threatened by resistance, importantly as caused by β-lactamases. β-Lactamases fall into two mechanistic groups: the serine β-lactamases that utilise a covalent acyl-enzyme mechanism and the metallo β-lactamases that utilise a zinc-bound water nucleophile. Achieving simultaneous inhibition of both β-lactamase classes remains a challenge in the field. Vaborbactam is a boronate-based inhibitor that reacts with serine-β-lactamases to form covalent complexes that mimic tetrahedral intermediates in catalysis. Vaborbactam has recently been approved for clinical use in combination with the carbapenem meropenem. Here we show that vaborbactam moderately inhibits metallo-β-lactamases from all 3 subclasses (B1, B2 and B3), with a potency of around 20-100 fold below that by which it inhibits its current clinical targets, the Class A serine β-lactamases. This result contrasts with recent investigations of bicyclic boronate inhibitors, which potently inhibit subclass B1 MBLs but which presently lack activity against B2 and B3 enzymes. These findings indicate that cyclic boronate scaffolds have the potential to inhibit the full range of β-lactamases and justify further work on the development of boronates as broad-spectrum β-lactamase inhibitors.
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Project description:The urgent demand for effective countermeasures against metallo-β-lactamases (MBLs) necessitates development of novel metallo-β-lactamase inhibitors (MBLIs). This study is dedicated to identifying critical chemical moieties within previously developed MBLIs, and critical MBLs should serve as the target in MBLI evaluations. Using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA), a systematic literature analysis was conducted, and the NCBI RefSeq genome database was exploited to access the abundance profile and taxonomic distribution of MBLs and their variant types. Through the implementation of two distinct systematic approaches, we elucidated critical chemical moieties of MBLIs, providing pivotal information for rational drug design. We also prioritised MBLs and their variant types, highlighting the imperative need for comprehensive testing to ensure the potency and efficacy of the newly developed MBLIs. This approach contributes valuable information to advance the field of antimicrobial drug discovery.
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Project description:Infections caused by multidrug resistant (MDR) bacteria are a major public health threat. Carbapenems are among the most potent antimicrobial agents that are commercially available to treat MDR bacteria. Bacterial production of carbapenem-hydrolysing metallo-β-lactamases (MBLs) challenges their safety and efficacy, with subclass B1 MBLs hydrolysing almost all β-lactam antibiotics. MBL inhibitors would fulfil an urgent clinical need by prolonging the lifetime of these life-saving drugs. Here we report the synthesis and activity of a series of 2-mercaptomethyl-thiazolidines (MMTZs), designed to replicate MBL interactions with reaction intermediates or hydrolysis products. MMTZs are potent competitive inhibitors of B1 MBLs in vitro (e.g., K i = 0.44 μM vs. NDM-1). Crystal structures of MMTZ complexes reveal similar binding patterns to the most clinically important B1 MBLs (NDM-1, VIM-2 and IMP-1), contrasting with previously studied thiol-based MBL inhibitors, such as bisthiazolidines (BTZs) or captopril stereoisomers, which exhibit lower, more variable potencies and multiple binding modes. MMTZ binding involves thiol coordination to the Zn(ii) site and extensive hydrophobic interactions, burying the inhibitor more deeply within the active site than d/l-captopril. Unexpectedly, MMTZ binding features a thioether-π interaction with a conserved active-site aromatic residue, consistent with their equipotent inhibition and similar binding to multiple MBLs. MMTZs penetrate multiple Enterobacterales, inhibit NDM-1 in situ, and restore carbapenem potency against clinical isolates expressing B1 MBLs. Based on their inhibitory profile and lack of eukaryotic cell toxicity, MMTZs represent a promising scaffold for MBL inhibitor development. These results also suggest sulphur-π interactions can be exploited for general ligand design in medicinal chemistry.
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Project description:Bulgecin A, a sulphonated N-acetyl-D-glucosamine unit linked to a 4-hydroxy-5-hydroxymethylproline ring by a beta-glycosidic linkage, is a novel type of inhibitor for binuclear metallo-beta-lactamases. Using steady-state kinetic analysis with nitrocefin as the beta-lactam substrate, bulgecin A competitively inhibited the metallo-beta-lactamase BceII from Bacillus cereus in its two-zinc form, but failed to inhibit when the enzyme was in the single-zinc form. The competitive inhibition was restored by restoring the second zinc ion. The single-zinc metallo-beta-lactamase from Aeromonas veronii bv. sobria, ImiS, was not inhibited by bulgecin A. The tetrameric L1 metallo-beta-lactamase from Stenotrophomonas maltophilia was subject to partial non-competitive inhibition, which is consistent with a kinetic model in which the enzyme bound to inhibitor retains catalytic activity. Docking experiments support the conclusion that bulgecin A co-ordinates to the zinc II site in metallo-beta-lactamases via the terminal sulphonate group on the sugar moiety.
| S-EPMC1134987 | biostudies-literature