Novel Cephalosporin Conjugates Display Potent and Selective Inhibition of Imipenemase-Type Metallo-β-Lactamases.
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ABSTRACT:
SUBMITTER: Tehrani KHME
PROVIDER: S-EPMC8273888 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:An evolution of antibiotic-resistant bacteria has resulted in the need for new antibiotics. β-Lactam based drugs are the most predominantly prescribed antibiotics to combat bacterial infections; however, production of β-lactamases, which catalyze the hydrolysis of the β-lactam bond of this class of antibiotics, by pathogenic bacteria such as Bacillus cereus, are rendering them useless. Some inhibitors of β-lactamases have been found, but there are no inhibitors against a class of β-lactamases known as metallo-β-lactamases, and it has been reported that the number of bacteria that produce metallo-β-lactamases is on the rise. Finding inhibitors of metallo-β-lactamases is thus an urgent necessity. One way to approach the problem is by employing the combinatorial method SELEX. The SELEX method is significant in discovering and producing new classes of inhibitors, as well as providing insight into the development of these inhibitors and paves the way for future aptamer applications that further novel drug discovery.
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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: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:There are currently no clinically available inhibitors of metallo-?-lactamases (MBLs), enzymes that hydrolyze ?-lactam antibiotics and confer resistance to Gram-negative bacteria. Here we present 6-phosphonomethylpyridine-2-carboxylates (PMPCs) as potent inhibitors of subclass B1 (IMP-1, VIM-2, and NDM-1) and B3 (L1) MBLs. Inhibition followed a competitive, slow-binding model without an isomerization step (IC50 values of 0.3-7.2 ?M; Ki values of 0.03-1.5 ?M). Minimum inhibitory concentration assays demonstrated potentiation of ?-lactam (Meropenem) activity against MBL-producing bacteria, including clinical isolates, at concentrations at which eukaryotic cells remain viable. Crystal structures revealed unprecedented modes of binding of inhibitor to B1 (IMP-1) and B3 (L1) MBLs. In IMP-1, binding does not replace the nucleophilic hydroxide, and the PMPC carboxylate and pyridine nitrogen interact closely (2.3 and 2.7 Å, respectively) with the Zn2 ion of the binuclear metal site. The phosphonate group makes limited interactions but is 2.6 Å from the nucleophilic hydroxide. Furthermore, the presence of a water molecule interacting with the PMPC phosphonate and pyridine N-C2 ?-bond, as well as the nucleophilic hydroxide, suggests that the PMPC binds to the MBL active site as its hydrate. Binding is markedly different in L1, with the phosphonate displacing both Zn2, forming a monozinc enzyme, and the nucleophilic hydroxide, while also making multiple interactions with the protein main chain and Zn1. The carboxylate and pyridine nitrogen interact with Ser221 and -223, respectively (3 Å distance). The potency, low toxicity, cellular activity, and amenability to further modification of PMPCs indicate these and similar phosphonate compounds can be further considered for future MBL inhibitor development.
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Project description:Multidrug-resistant bacterial infections mediated by metallo-β-lactamases (MβLs) have grown into an emergent health threat, and development of novel antimicrobials is an ideal strategy to combat the infections. Herein, a novel vancomycin derivative Vb was constructed by conjugation of triazolylthioacetamide and vancomycin molecules, characterized by reverse-phase high performance liquid chromatography (HPLC) and confirmed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). The biological assays revealed that Vb effectively inhibited S. aureus and methicillin-resistant S. aureus (MRSA), gradually increased the antimicrobial effect of β-lactam antibiotics (cefazolin, meropenem and penicillin G) and exhibited a dose-dependent synergistic antibacterial effect against eight resistant strains tested, which was confirmed by the time-kill curves determination. Most importantly, Vb increased the antimicrobial effect of meropenem against the clinical isolates EC08 and EC10 and E. coli producing ImiS and CcrA, resulting in a 4- and 8-fold reduction in MIC values, respectively, at a dose up to 32 μg/mL. This work offers a promising scaffold for the development of MβLs inhibitors, specifically antimicrobials for clinically drug-resistant isolates.
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Project description:Antibiotic resistance, particularly beta-lactam resistance, is a major problem worldwide. Imipenemase or IMP-type metallo-β-lactamase (MBL) has become a more prominent enzyme, especially in Asia, since it was discovered in the 1990s in Japan. There are currently 88 variants of IMP-type enzymes. The most commonly identified variant of IMP-type enzymes is IMP-1 variant. IMP-type MBLs have been detected in more than ten species in Enterobacterales. Pseudomonas aeruginosa is the most frequent carrier of IMP-type enzymes worldwide. In Asia, IMP-type MBLs have been distributed in many countries. This work investigated a variety of currently available IMP-type MBLs at both a global level and a regional level. Out of 88 variants of IMP-type MBLs reported worldwide, only 32 variants were found to have susceptibility profiles. Most of the bacterial isolates carrying IMP-type MBLs were resistant to Carbapenems, especially Imipenem and Meropenem, followed by the 3rd-generation cephalosporins, and interestingly, monobactams. Our results comprehensively indicated the distribution of IMP-type MBLs in Asia and raised the awareness of the situation of antimicrobial resistance in the region.
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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.
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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: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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