NDM-1 Inhibitors Research Articles

New derivatives of dipicolinic acid as metallo-β-lactamase NDM-1 inhibitors

New derivatives of dipicolinic acid as metallo-β-lactamase NDM-1 inhibitors

Binding selectivity analysis of new delhi metallo-beta-lactamase-1 inhibitors using molecular dynamics simulations: Exploring possibilities for decoding antimicrobial drug resistance

BackgroundNew Delhi metallo-beta-lactamase-1 (NDM1) confers resistance to several bacterial species against a broad range of beta-lactam antibiotics and turning them into superbugs that pose a significant threat to healthcare systems worldwide. As such, it is a potentially relevant biological target for counteracting bacterial infections. Given the lack of effective treatment options against NDM1 producing bacteria, finding a reliable inhibitor for the NDM1 enzyme is crucial. MethodsUsing molecular dynamics simulations, the binding selectivities and affinities of three ligands, viz. PNK, 3S0, and N1G were investigated against NDM1. ResultsThe results indicate that N1G binds with more affinity to NDM1 than PNK and 3S0. The binding energy decomposition analysis revealed that residues I35, W93, H189, K211, and N220 showed significant binding energies with PNK, 3S0, and N1G, and hence are crucially involved in the binding of the ligands to NDM1. Molecular dynamics trajectory analysis further elicited that the ligands influence dynamic flexibility of NDM1 morphology, which contributes to the partial selectivities of PNK, 3S0, and N1G. ConclusionsThis in silico study offers a vital information for developing potential NDM1 inhibitors with high selectivity. Nevertheless, in vitro and in vivo experimental validation is mandated to extend the possible applications of these ligands as NDM1 inhibitors that succor in combating antimicrobial resistance.

Exploiting the Carboxylate-Binding Pocket of β-Lactamase Enzymes Using a Focused DNA-Encoded Chemical Library.

β-Lactamase enzymes hydrolyze and thereby provide bacterial resistance to the important β-lactam class of antibiotics. The OXA-48 and NDM-1 β-lactamases cause resistance to the last-resort β-lactams, carbapenems, leading to a serious public health threat. Here, we utilized DNA-encoded chemical library (DECL) technology to discover novel β-lactamase inhibitors. We exploited the β-lactamase enzyme-substrate binding interactions and created a DECL targeting the carboxylate-binding pocket present in all β-lactamases. A library of 106 compounds, each containing a carboxylic acid or a tetrazole as an enzyme recognition element, was designed, constructed, and used to identify OXA-48 and NDM-1 inhibitors with micromolar to nanomolar potency. Further optimization led to NDM-1 inhibitors with increased potencies and biological activities. This work demonstrates that the carboxylate-binding pocket-targeting DECL, designed based on substrate binding information, aids in inhibitor identification and led to the discovery of novel non-β-lactam pharmacophores for the development of β-lactamase inhibitors for enzymes of different structural and mechanistic classes.

Prediction of Phytochemicals for Their Potential to Inhibit New Delhi Metallo β-Lactamase (NDM-1).

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.

Rational design of novel compounds to serve as potential NDM-1 inhibitors using molecular docking, molecular dynamics simulation, and physicochemical studies

ABSTRACT New Delhi Metallo-β-lactamase (NDM-1) is an enzyme that hydrolyses a wide range of β-lactam antibiotics, including most carbapenems, leading to antimicrobial resistance. The development of a novel NDM-1 inhibitor for use in combination with carbapenems may help to combat drug-resistant pathogens. Twenty compounds were designed based on the structural features of the NDM-1 active site to inhibit bacterial NDM-1 and protect β-lactam antibiotics from enzyme attack. The designed molecules were naphthalene, thiazole, and sulfone derivatives because they could coordinate with the zinc ions and form hydrophobic contracts with the enzyme’s active site. A molecular docking protocol was used to identify potential inhibitor(s) of the NDM-1 target protein. Furthermore, drug-likeness and pharmacokinetic properties of the designed molecules were predicted. Three compounds with the more negative ΔGbinding results were selected for further investigation using molecular dynamic (MD) simulations. T016 had a significantly more negative binding free energy than the positive control and other designed molecules, had stable MD simulations (Root-mean-square deviation < 0.5 Å), passed Lipinski’s rule of five, and had favourable physicochemical and pharmacokinetic properties. The findings can be used to inform the synthesis and in vitro testing of the selected molecules.

Discovery of environment-sensitive fluorescent probes for detecting and inhibiting metallo-β-lactamase

Metallo-β-lactamases (MβLs) hydrolyze almost all β-lactam antibiotics, including last-resort carbapenems, and is emerging as a global antibiotic resistance threat. Discovering novel fluorescent molecules for visualizing MβLs has proved challenging. Herein, based on covalent and Zn(II)-binding scaffold of MβLs inhibitor, we designed and synthesized a novel series of environment-sensitive fluorescent probes ESA, DHA and DHS, to detect and inhibit the enzymatic activity of MβLs. Of these probes, ESA is a highly active NDM-1 inhibitor (IC50 = 81 nM), which exhibited excellent turn-on fluorescent properties to effectively distinguish NDM-1 (B1), ImiS (B2) and L1 (B3) in vitro. Cell imaging indicated that ESA can label and track the distribution process of the intracellular protein NDM-1 in living cells. Molecular docking further elucidated the environment-sensitive fluorescent response nature of ESA to the NDM-1. Significantly, ESA showed excellent synergistic antibacterial effect, combined with meropenem, to overcome NDM-1-mediated drug-resistant pathogens.

New N-Alkylated Heterocyclic Compounds as Prospective NDM1 Inhibitors: Investigation of In Vitro and In Silico Properties.

A new family of pyrazole-based compounds (1–15) was synthesized and characterized using different physicochemical analyses, such as FTIR, UV-Visible, 1H, 13C NMR, and ESI/LC-MS. The compounds were evaluated for their in vitro antifungal and antibacterial activities against several fungal and bacterial strains. The results indicate that some compounds showed excellent antibacterial activity against E. coli, S. aureus, C. freundii, and L. monocytogenes strains. In contrast, none of the compounds had antifungal activity. Molecular electrostatic potential (MEP) map analyses and inductive and mesomeric effect studies were performed to study the relationship between the chemical structure of our compounds and the biological activity. In addition, molecular docking and virtual screening studies were carried out to rationalize the antibacterial findings to characterize the modes of binding of the most active compounds to the active pockets of NDM1 proteins.

1,2,4-Triazole-3-Thione Analogues with a 2-Ethylbenzoic Acid at Position 4 as VIM-type Metallo-β-Lactamase Inhibitors.

Metallo-β-lactamases (MBLs) are increasingly involved as a major mechanism of resistance to carbapenems in relevant opportunistic Gram-negative pathogens. Unfortunately, clinically efficient MBL inhibitors still represent an unmet medical need. We previously reported several series of compounds based on the 1,2,4-triazole-3-thione scaffold. In particular, Schiff bases formed between diversely 5-substituted-4-amino compounds and 2-carboxybenzaldehyde were broad-spectrum inhibitors of VIM-type, NDM-1 and IMP-1 MBLs. Unfortunately, these compounds were unable to restore antibiotic susceptibility of MBL-producing bacteria, probably because of poor penetration and/or susceptibility to hydrolysis. To improve their microbiological activity, we synthesized and characterized compounds where the hydrazone-like bond of the Schiff base analogues was replaced by a stable ethyl link. This small change resulted in a narrower inhibition spectrum, as all compounds were poorly or not inhibiting NDM-1 and IMP-1, but showed a significantly better activity on VIM-type enzymes, with Ki values in the μM to sub-μM range. The resolution of the crystallographic structure of VIM-2 in complex with one of the best inhibitors yielded valuable information about their binding mode. Interestingly, several compounds were shown to restore the β-lactam susceptibility of VIM-type-producing E. coli laboratory strains and also of K. pneumoniae clinical isolates. In addition, selected compounds were found to be devoid of toxicity toward human cancer cells at high concentration, thus showing promising safety.

Metallo-β-Lactamase Inhibitor Phosphonamidate Monoesters.

Being the second leading cause of death and the leading cause of disability-adjusted life years worldwide, infectious diseases remain—contrary to earlier predictions—a major consideration for the public health of the 21st century. Resistance development of microbes to antimicrobial drugs constitutes a large part of this devastating problem. The most widely spread mechanism of bacterial resistance operates through the degradation of existing β-lactam antibiotics. Inhibition of metallo-β-lactamases is expected to allow the continued use of existing antibiotics, whose applicability is becoming ever more limited. Herein, we describe the synthesis, the metallo-β-lactamase inhibition activity, the cytotoxicity studies, and the NMR spectroscopic determination of the protein binding site of phosphonamidate monoesters. The expression of single- and double-labeled NDM-1 and its backbone NMR assignment are also disclosed, providing helpful information for future development of NDM-1 inhibitors. We show phosphonamidates to have the potential to become a new generation of antibiotic therapeutics to combat metallo-β-lactamase-resistant bacteria.

Abyssomicin W and Neoabyssomicin B are potential inhibitors of New Delhi Metallo-β-Lactamase-1 (NDM -1): A computational approach

Background: Antibiotic resistance in bacteria mediated by New Delhi Metallo-β-lactamase (NDM) is a global threat to human health with an enormous economic burden. NDM can hydrolyze all the β-lactam core-containing antibiotics including carbapenems, which are regarded as last resort antibiotics. Materials and Methods: A library of Abyssomicins was virtually screened to identify novel non-β-lactam ring-containing inhibitors of NDM-1. Different computational approaches such as molecular modeling, virtual screening, molecular docking, molecular dynamics simulation, ADMET profiling, and free energy calculations were utilized for this purpose. Results: Virtual screening and ADMET profiling shortlisted Abyssomicin W and Neoabyssomicin B as the most promising candidate molecules. An in-depth analysis of protein-ligand interactions by molecular docking revealed that both ligands bind the active site of NDM-1. The identified inhibitors interacted with key catalytic residues as well as other residues around the active site of NDM-1. Hydrogen bonding and hydrophobic interactions played a significant role in stabilizing the protein-inhibitor complexes. The docking energy of NDM-1-Abyssomicin W, and NDM-1-Neoabyssomicin B complexes were − 9.6 kcal/mol and − 9.5 kcal/mol, respectively, which were higher than NDM-1-Methicillin (control) complex (−7.3 kcal/mol). Molecular dynamics simulation and free energy calculations by MM-PBSA also confirmed the stability of NDM-1-Abyssomicin W, and NDM-1-Neoabyssomicin B complexes. Conclusion: The findings of this study suggest that Abyssomicins serve as potential inhibitors of NDM-1. However, these results need to be validated in vitro and in vivo. This study may serve as a basis for further developing Abyssomicins as novel inhibitors of β-lactamases.

Imitation of β-lactam binding enables broad-spectrum metallo-β-lactamase inhibitors.

Carbapenems are vital antibiotics, but their efficacy is increasingly compromised by metallo-β-lactamases (MBLs). Here we report the discovery and optimization of potent broad-spectrum MBL inhibitors. A high-throughput screen for NDM-1 inhibitors identified indole-2-carboxylates (InCs) as potential β-lactamase stable β-lactam mimics. Subsequent structure-activity relationship studies revealed InCs as a new class of potent MBL inhibitor, active against all MBL classes of major clinical relevance. Crystallographic studies revealed a binding mode of the InCs to MBLs that, in some regards, mimics that predicted for intact carbapenems, including with respect to maintenance of the Zn(II)-bound hydroxyl, and in other regards mimics binding observed in MBL-carbapenem product complexes. InCs restore carbapenem activity against multiple drug-resistant Gram-negative bacteria and have a low frequency of resistance. InCs also have a good in vivo safety profile, and when combined with meropenem show a strong in vivo efficacy in peritonitis and thigh mouse infection models.

Efforts towards the inhibitor design for New Delhi metallo-beta-lactamase (NDM-1).

Antimicrobial stewardship is imperative when treating bacterial infections because the misuse and overuse of antibiotics have caused pathogens to develop life-threatening resistance mechanisms. The New Delhi metallo-beta-lactamase (NDM-1) is one of many enzymes that enable bacterial resistance. NDM-1 is a more recently discovered beta-lactamase with the ability to inactivate a wide range of beta-lactam antibiotics. Multiple NDM-1 inhibitors have been designed and tested; however, due to the complexity of the NDM-1 active site, there is currently no inhibitor on the market. Consequently, an infection caused by bacteria possessing the gene for the NDM-1 enzyme is a serious and potentially fatal complication. An abundance of research has been invested over the past decade in search of an NDM-1 inhibitor. This review aims to summarize various NDM-1 inhibitor designs that have been developed in recent years.

Dithiocarbamates combined with copper for revitalizing meropenem efficacy against NDM-1-producing Carbapenem-resistant Enterobacteriaceae

The worldwide prevalence of NDM-1-producing Gram-negative pathogens has drastically undermined the clinical efficacy of carbapenems, prompting a need to devise an effective strategy to preserve their clinical value. Here we constructed a focused compound library of dithiocarbamates and systematically evaluated their potential synergistic antibacterial activities combined with copper. SA09-Cu exhibited excellent inhibition against a series of clinical NDM-1-producing carbapenem-resistant Enterobacteriaceae (CRE) in restoring meropenem effect, and slowed down the development of carbapenem resistance. Enzymatic kinetic and isothermal titration calorimetry studies demonstrated that SA09-Cu was a noncompetitive NDM-1 inhibitor. The electron paramagnetic resonance (EPR) and X-ray photoelectron spectroscopy (XPS) revealed a novel inhibition mechanism, which is that SA09-Cu could convert NDM-1 into an inactive state by oxidizing the Zn(II)-thiolate site of the enzyme. Importantly, SA09-Cu showed a unique redox tuning ability, and avoided to be reduced by intracellular thiols of bacteria. In vivo experiments indicated that SA09 combined with CuGlu could effectively potentiate MER’s effect against NDM-1-producing E. coli (EC23) in the murine infection model. This study provides a highly promising scaffold in developing novel inhibitors to combat NDM-1-producing CREs.

Recent research and development of NDM-1 inhibitors.

Bacteria carrying New Delhi metallo-β-lactamase-1 (New Delhi metallo-β-lactamase, NDM-1) resistance gene is a new type of "superbug", which can hydrolyze almost all β-lactam antibiotics, rapidly spread among the same species and even spread among different species. NDM-1 belongs to the class B1 broad-spectrum enzyme of β-lactamase. The two positively charged zinc ions in the active center have electrostatic interaction with the hydroxyl ions in them to seize the hydrogen atom near the water molecule to form a bridging ring water molecule, which strengthens its nucleophilicity and attacks the carbonyl group on the lactam ring; thus, catalyzing the hydrolysis of β-lactam antibiotics. Since NDM-1 has an open active site and unique electrostatic structure, it essentially provides a wider range of substrate specificity. Due to its flexible hydrolysis mechanism and more and more variants also aggravate the threat of drug-resistant bacteria infection, there is still no effective inhibitor in clinic, which is a serious threat to human health and public health safety. The electron-rich substituents of NDM-1 inhibitors coordinate with two positively charged zinc ions in the active center of the enzyme through ion-dipole interaction to produce NDM-1 inhibitory activity. In this review, the research progress of NDM-1 enzyme and its inhibitors in the past 5 years was reviewed. The crystal structure, active center structure, surrounding important amino acid residues, newly discovered inhibitors and their action mechanism are classified and summarized in detail, which can be used as a reference for the development of effective drugs against drug-resistant bacteria targeting NDM-1.

N-acylhydrazones confer inhibitory efficacy against New Delhi metallo-β-lactamase-1

The expression of β-lactamases, especially metallo-β-lactamases (MβLs) in bacteria is one of the main causes of drug resistance. In this work, an effective N-acylhydrazone scaffold as MβL inhibitor was constructed and characterized. The biological activity assays indicated that the synthesized N-acylhydrazones 1–11 preferentially inhibited MβL NDM-1, and 1 was found to be the most effective inhibitor with an IC50 of 1.2 µM. Analysis of IC50 data revealed a structure–activity relationship, which is that the pyridine and hydroxylbenzene substituents at 2-position improved inhibition of the compounds on NDM-1. ITC and enzyme kinetics assays suggested that it reversibly and competitively inhibited NDM-1 (Ki = 0.29 ± 0.05 µM). The synthesized N-acylhydrazones showed synergistic antibacterial activities with meropenem, reduced 4–16-fold MIC of meropenem on NDM-1- producing E. coli BL21 (DE3), while 1 restored 4-fold activity of meropenem on K. pneumonia expressing NDM-1 (NDM-K. pneumoniae). The mice experiments suggested that 1 combined meropenem to fight against NDM-K. pneumoniae infection in the spleen and liver. Cytotoxicity assays showed that 1 and 2 have low cytotoxicity. This study offered a new framework for the development of NDM-1 inhibitors.

Docking and Antibacterial Activity of Novel Nontoxic 5-Arylidenepyrimidine-Triones as Inhibitors of NDM-1 and MetAP-1

Background: Antibiotic resistance, which occurs through the action of metallo-β-lactamases (NDM-1), is a serious problem in the treatment of infectious diseases. Therefore, the discovery of new NDM-1 inhibitors and promising antibacterial agents as inhibitors of alternative targets (MetAP-1) is important. Method & results: In this study, a virtual library of 5-arylidene barbituric acids was created and molecular docking was performed for identification of novel possible inhibitors of NDM-1 and MetAP-1. Antibacterial activity (agar well-diffusion assay) and cytotoxicity (alamarBlue assay) of perspective compounds were evaluated. Pharmacokinetic profiles and molecular properties were predicted. Conclusion: We have identified possible novel inhibitors of NDM-1 and MetAP-1 with bacteriostatic activity, most of which are not cytotoxic and have potential excellent drug-likeness properties.

Synthesis of 1,3,4-trisubstituted pyrrolidines as meropenem adjuvants targeting New Delhi metallo-β-lactamase

A promising NDM-1 inhibitor was discovered by the construction of pyrrolidine library via boric acid-catalyzed 1,3-dipolar cycloaddition and cell-based screens.

Diaryl-substituted thiosemicarbazone: A potent scaffold for the development of New Delhi metallo-β-lactamase-1 inhibitors

The superbug infection caused by New Delhi metallo-β-lactamase (NDM-1) has become an emerging public health threat. Inhibition of NDM-1 has proven challenging due to its shuttling between pathogenic bacteria. A potent scaffold, diaryl-substituted thiosemicarbazone, was constructed and assayed with metallo-β-lactamases (MβLs). The obtained twenty-six molecules specifically inhibited NDM-1 with IC50 0.038–34.7 µM range (except 1e, 2e, and 3d), and 1c is the most potent inhibitor (IC50 = 0.038 µM). The structure-activity relationship of synthetic thiosemicarbazones revealed that the diaryl-substitutes, specifically 2-pyridine and 2-hydroxylbenzene improved inhibitory activities of the inhibitors. The thiosemicarbazones exhibited synergistic antimycobacterial actions against E. coli-NDM-1, resulted a 2–512-fold reduction in MIC of meropenem, while 1c restored 16–256-, 16-, and 2-fold activity of the antibiotic on clinical isolates ECs, K. pneumonia and P. aeruginosa harboring NDM-1, respectively. Also, mice experiments showed that 1c had a synergistic antibacterial ability with meropenem, reduced the bacterial load clinical isolate EC08 in the spleen and liver. This work provided a highly promising scaffold for the development of NDM-1 inhibitors.

Benzimidazole and Benzoxazole Zinc Chelators as Inhibitors of Metallo-β-Lactamase NDM-1.

Bacterial expression of β-lactamases, which hydrolyze β-lactam antibiotics, contributes to the growing threat of antibacterial drug resistance. Metallo-β-lactamases, such as NDM-1, use catalytic zinc ions in their active sites and hydrolyze nearly all clinically available β-lactam antibiotics. Inhibitors of metallo-β-lactamases are urgently needed to overcome this resistance mechanism. Zinc-binding compounds are promising leads for inhibitor development, as many NDM-1 inhibitors contain zinc-binding pharmacophores. Here, we evaluated 13 chelating agents containing benzimidazole and benzoxazole scaffolds as NDM-1 inhibitors. Six of the compounds showed potent inhibitory activity with IC50 values as low as 0.38 μM, and several compounds restored the meropenem susceptibility of NDM-1-expressing E. coli. Spectroscopic and docking studies suggest ternary complex formation as the mechanism of inhibition, making these compounds promising for development as NDM-1 inhibitors.

Discovery and characterization of New Delhi metallo-β-lactamase-1 inhibitor peptides that potentiate meropenem-dependent killing of carbapenemase-producing Enterobacteriaceae.

Metallo-β-lactamases (MBLs) are an emerging class of antimicrobial resistance enzymes that degrade β-lactam antibiotics, including last-resort carbapenems. Infections caused by carbapenemase-producing Enterobacteriaceae (CPE) are increasingly prevalent, but treatment options are limited. While several serine-dependent β-lactamase inhibitors are formulated with commonly prescribed β-lactams, no MBL inhibitors are currently approved for combinatorial therapies. New compounds that target MBLs to restore carbapenem activity against CPE are therefore urgently needed. Herein we identified and characterized novel synthetic peptide inhibitors that bound to and inhibited NDM-1, which is an emerging β-lactam resistance mechanism in CPE. We leveraged Surface Localized Antimicrobial displaY (SLAY) to identify and characterize peptides that inhibit NDM-1, which is a primary carbapenem resistance mechanism in CPE. Lead inhibitor sequences were chemically synthesized and MBCs and MICs were calculated in the presence/absence of carbapenems. Kinetic analysis with recombinant NDM-1 and select peptides tested direct binding and supported NDM-1 inhibitor mechanisms of action. Inhibitors were also tested for cytotoxicity. We identified approximately 1700 sequences that potentiated carbapenem-dependent killing against NDM-1 Escherichia coli. Several also enhanced meropenem-dependent killing of other CPE. Biochemical characterization of a subset indicated the peptides penetrated the bacterial periplasm and directly bound NDM-1 to inhibit enzymatic activity. Additionally, each demonstrated minimal haemolysis and cytotoxicity against mammalian cell lines. Our approach advances a molecular platform for antimicrobial discovery, which complements the growing need for alternative antimicrobials. We also discovered lead NDM-1 inhibitors, which serve as a starting point for further chemical optimization.