Durlobactam, a Diazabicyclooctane ß-Lactamase Inhibitor, Inhibits BlaC and Peptidoglycan Transpeptidases of Mycobacterium tuberculosis.

Peptidoglycan synthesis is an underutilized drug target in Mycobacterium tuberculosis (Mtb). Diazabicyclooctanes (DBOs) are a class of broad-spectrum ß-lactamase inhibitors that also inhibit certain peptidoglycan transpeptidases that are important in mycobacterial cell wall synthesis. We evaluated the DBO durlobactam as an inhibitor of BlaC, the Mtb ß-lactamase, and multiple Mtb peptidoglycan transpeptidases (PonA1, LdtMt1, LdtMt2, LdtMt3, and LdtMt5). Timed electrospray ionization mass spectrometry (ESI-MS) captured acyl-enzyme complexes with BlaC and all transpeptidases except LdtMt5. Inhibition kinetics demonstrated durlobactam was a potent and efficient DBO inhibitor of BlaC (KI app 9.2 ± 0.9 µM, k2/K 5600 ± 560 M-1 s-1) and similar to clavulanate (KI app 3.3 ± 0.6 µM, k2/K 8400 ± 840 M-1 s-1); however, durlobactam had a lower turnover number (tn = kcat/kinact) than clavulanate (1 and 8, respectively). KI app values with durlobactam and clavulanate were similar for peptidoglycan transpeptidases, but ESI-MS captured durlobactam complexes at more time points. Molecular docking and simulation demonstrated several productive interactions of durlobactam in the active sites of BlaC, PonA1, and LdtMt2. Antibiotic susceptibility testing was conducted on 11 Mtb isolates with amoxicillin, ceftriaxone, meropenem, imipenem, clavulanate, and durlobactam. Durlobactam had a minimum inhibitory concentration (MIC) range of 0.5-16 µg/mL, similar to the ranges for meropenem (1-32 µg/mL) and imipenem (0.5-64 µg/mL). In ß-lactam + durlobactam combinations (1:1 mass/volume), MICs were lowered 4- to 64-fold for all isolates except one with meropenem-durlobactam. This work supports further exploration of novel ß-lactamase inhibitors that target BlaC and Mtb peptidoglycan transpeptidases.

3-O-Substituted Quercetin: an Antibiotic-Potentiating Agent against Multidrug-Resistant Gram-Negative Enterobacteriaceae through Simultaneous Inhibition of Efflux Pump and Broad-Spectrum Carbapenemases.

The discovery of safe and efficient inhibitors against efflux pumps as well as metallo-ß-lactamases (MBL) is one of the main challenges in the development of multidrug-resistant (MDR) reversal agents which can be utilized in the treatment of carbapenem-resistant Gram-negative bacteria. In this study, we have identified that introduction of an ethylene-linked sterically demanding group at the 3-OH position of the previously reported MDR reversal agent di-F-Q endows the resulting compounds with hereto unknown multitarget inhibitory activity against both efflux pumps and broad-spectrum ß-lactamases including difficult-to-inhibit MBLs. A molecular docking study of the multitarget inhibitors against efflux pump, as well as various classes of ß-lactamases, revealed that the 3-O-alkyl substituents occupy the novel binding sites in efflux pumps as well as carbapenemases. Not surprisingly, the multitarget inhibitors rescued the antibiotic activity of a carbapenem antibiotic, meropenem (MEM), in NDM-1 (New Delhi Metallo-ß-lactamase-1)-producing carbapenem-resistant Enterobacteriaceae (CRE), and they reduced MICs of MEM more than four-fold (synergistic effect) in 8-9 out of 14 clinical strains. The antibiotic-potentiating activity of the multitarget inhibitors was also demonstrated in CRE-infected mouse model. Taken together, these results suggest that combining inhibitory activity against two critical targets in MDR Gram-negative bacteria, efflux pumps, and ß-lactamases, in one molecule is possible, and the multitarget inhibitors may provide new avenues for the discovery of safe and efficient MDR reversal agents.

Metallo-ß-lactamase inhibitors: A continuing challenge for combating antibiotic resistance.

ß-lactam antibiotics are the most successful and commonly used antibacterial agents, but the emergence of resistance to these drugs has become a global health threat. The expression of ß-lactamase enzymes produced by pathogens, which hydrolyze the amide bond of the ß-lactam ring, is the major mechanism for bacterial resistance to ß-lactams. In particular, among class A, B, C and D ß-lactamases, metallo-ß-lactamases (MBLs, class B ß-lactamases) are considered crucial contributors to resistance in gram-negative bacteria. To combat ß-lactamase-mediated resistance, great efforts have been made to develop ß-lactamase inhibitors that restore the activity of ß-lactams. Some ß-lactamase inhibitors, such as diazabicyclooctanes (DBOs) and boronic acid derivatives, have also been approved by the FDA. Inhibitors used in the clinic can inactivate mostly serine-ß-lactamases (SBLs, class A, C, and D ß-lactamases) but have not been effective against MBLs until now. In order to develop new inhibitors particularly for MBLs, various attempts have been suggested. Based on structural and mechanical studies of MBL enzymes, several MBL inhibitor candidates, including taniborbactam in phase 3 and xeruborbactam in phase 1, have been introduced in recent years. However, designing potent inhibitors that are effective against all subclasses of MBLs is still extremely challenging. This review summarizes not only the types of ß-lactamase and mechanisms by which ß-lactam antibiotics are inactivated, but also the research finding on ß-lactamase inhibitors targeting these enzymes. These detailed information on ß-lactamases and their inhibitors could give valuable information for novel ß-lactamase inhibitors design.

Rational Design of Benzobisheterocycle Metallo-ß-Lactamase Inhibitors: A Tricyclic Scaffold Enhances Potency against Target Enzymes.

Antimicrobial resistance is a global public health threat. Metallo-ß-lactamases (MBLs) inactivate ß-lactam antibiotics, including carbapenems, are disseminating among Gram-negative bacteria, and lack clinically useful inhibitors. The evolving bisthiazolidine (BTZ) scaffold inhibits all three MBL subclasses (B1-B3). We report design, synthesis, and evaluation of BTZ analogues. Structure-activity relationships identified the BTZ thiol as essential, while carboxylate is replaceable, with its removal enhancing potency by facilitating hydrophobic interactions within the MBL active site. While the introduction of a flexible aromatic ring is neutral or detrimental for inhibition, a rigid (fused) ring generated nM benzobisheterocycle (BBH) inhibitors that potentiated carbapenems against MBL-producing strains. Crystallography of BBH:MBL complexes identified hydrophobic interactions as the basis of potency toward B1 MBLs. These data underscore BTZs as versatile, potent broad-spectrum MBL inhibitors (with activity extending to enzymes refractory to other inhibitors) and provide a rational approach to further improve the tricyclic BBH scaffold.

Structure Guided Discovery of Novel Pan Metallo-ß-Lactamase Inhibitors with Improved Gram-Negative Bacterial Cell Penetration.

The use of ß-lactam (BL) and ß-lactamase inhibitor combination to overcome BL antibiotic resistance has been validated through clinically approved drug products. However, unmet medical needs still exist for the treatment of infections caused by Gram-negative (GN) bacteria expressing metallo-ß-lactamases. Previously, we reported our effort to discover pan inhibitors of three main families in this class: IMP, VIM, and NDM. Herein, we describe our work to improve the GN coverage spectrum in combination with imipenem and relebactam. This was achieved through structure- and property-based optimization to tackle the GN cell penetration and efflux challenges. A significant discovery was made that inhibition of both VIM alleles, VIM-1 and VIM-2, is essential for broad GN coverage, especially against VIM-producing P. aeruginosa. In addition, pharmacokinetics and nonclinical safety profiles were investigated for select compounds. Key findings from this drug discovery campaign laid the foundation for further lead optimization toward identification of preclinical candidates.

Fluorinated captopril analogues inhibit metallo-ß-lactamases and facilitate structure determination of NDM-1 binding pose.

Bacterial resistance to the majority of clinically used ß-lactam antibiotics is a global health threat and, consequently, the driving force for the development of metallo-ß-lactamase (MBL) inhibitors. The rapid evolution of new MBLs calls for new strategies and tools for inhibitor development. In this study, we designed and developed a series of trifluoromethylated captopril analogues as probes for structural studies of enzyme-inhibitor binding. The new compounds showed activity comparable to the non-fluorinated inhibitors against the New Delhi Metallo-ß-lactamase-1 (NDM-1). The most active compound, a derivative of D-captopril, exhibited an IC50 value of 0.3 µM. Several compounds demonstrated synergistic effects, restoring the effect of meropenem and reducing the minimum inhibitory concentration (MIC) values in NDM-1 (up to 64-fold), VIM-2 (up to 8-fold) and IMP-26 (up to 8-fold) harbouring Escherichia coli. NMR spectroscopy and molecular docking of one representative inhibitor determined the binding pose in NDM-1, demonstrating that fluorinated analogues of inhibitors are a valuable tool for structural studies of MBL-inhibitor complexes.

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

ß-Lactamase enzymes hydrolyze and thereby provide bacterial resistance to the important ß-lactam class of antibiotics. The OXA-48 and NDM-1 ß-lactamases cause resistance to the last-resort ß-lactams, carbapenems, leading to a serious public health threat. Here, we utilized DNA-encoded chemical library (DECL) technology to discover novel ß-lactamase inhibitors. We exploited the ß-lactamase enzyme-substrate binding interactions and created a DECL targeting the carboxylate-binding pocket present in all ß-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-ß-lactam pharmacophores for the development of ß-lactamase inhibitors for enzymes of different structural and mechanistic classes.

Aurones and derivatives as promising New Delhi metallo-ß-lactamase (NDM-1) inhibitors.

Bacterial resistance is undoubtedly one of the main public health concerns especially with the emergence of metallo-ß-lactamases (MBLs) able to hydrolytically inactivate ß-lactam antibiotics. Currently, there are no inhibitors of MBLs in clinical use to rescue antibiotic action and the New Delhi metallo-ß-lactamase-1 (NDM-1) is still considered as one of the most relevant targets for inhibitor development. Following a fragment-based strategy to find new NDM-1 inhibitors, we identified aurone as a promising scaffold. A series of 60 derivatives were then evaluated and two of them were identified as promising inhibitors with Ki values as low as 1.7 and 2.5 µM. Moreover, these two most active compounds were able to potentiate meropenem in in vitro antimicrobial susceptibility assays. The molecular modelling provided insights about their likely interactions with the active site of NDM-1, thus enabling further improvement in the structure of this new inhibitor family.

Discovery of hydroxamate as a promising scaffold dually inhibiting metallo- and serine-ß-lactamases.

The bacterial infection mediated by ß-lactamases MßLs and SßLs has grown into an emergent health threat, however, development of a molecule that dual inhibits both MßLs and SßLs is challenging. In this work, a series of hydroxamates 1a-g, 2a-e, 3a-c, 4a-c were synthesized, characterized by 1H and 13C NMR and confirmed by HRMS. Biochemical assays revealed that these molecules dually inhibited MßLs (NDM-1, IMP-1) and SßLs (KPC-2, OXA-48), with an IC50 value in the range of 0.64-41.08 and 1.01-41.91 µM (except 1a and 1d on SßLs, IC50 > 50 µM), and 1f was found to be the best inhibitor with an IC50 value in the range of 0.64-1.32 and 0.57-1.01 µM, respectively. Mechanism evaluation indicated that 1f noncompetitively and irreversibly inhibited NDM-1 and KPC-2, with Ki value of 2.5 and 0.55 µM, is a time- and dose-dependent inhibitor of both MßLs and SßLs. MIC tests shown that all hydroxamates increased the antimicrobial effect of MER on E. coli-NDM-1 and E. coli-IMP-1 (expect 1b, 1d, 1g and 2d), resulting in a 2-8-fold reduction in MICs of MER, 1e-g, 2b-d, 3a-c and 4b-c decreased 2-4-fold MICs of MER on E. coli-KPC-2, and 1c, 1f-g, 2a-c, 3b, 4a and 4c decreased 2-16-fold MICs of MER on E. coli-OXA-48. Most importantly, 1f-g, 2b-c, 3b and 4c exhibited the dual synergizing inhibition against both E. coli-MßLs and E. coli-SßLs tested, resulting in a 2-8-fold reduction in MICs of MER, and 1f was found to have the best effect on the drug-resistant bacteria tested. Also, 1f shown synergizing antimicrobial effect on five clinical isolates EC04, EC06, EC08, EC10 and EC24 that produce NDM-1, resulting in a 2-8-fold reduction in MIC of MER, but its effect on E. coli and K. pneumonia-KPC-NDM was not to be observed using the same dose of inhibitor. Mice tests shown that the monotherapy of 1f or 4a in combination with MER significantly reduced the bacterial load of E. coli-NDM-1 and E. coli-OXA-48 cells in liver and spleen, respectively. The discovery in this work offered a promising bifunctional scaffold for creating the specific molecules that dually inhibit MßLs and MßLs, in combating antibiotic-resistant bacteria.

Aromatic Diboronic Acids as Effective KPC/AmpC Inhibitors.

Over 30 compounds, including para-, meta-, and ortho-phenylenediboronic acids, ortho-substituted phenylboronic acids, benzenetriboronic acids, di- and triboronated thiophenes, and pyridine derivatives were investigated as potential ß-lactamase inhibitors. The highest activity against KPC-type carbapenemases was found for ortho-phenylenediboronic acid 3a, which at the concentration of 8/4 mg/L reduced carbapenems' MICs up to 16/8-fold, respectively. Checkerboard assays revealed strong synergy between carbapenems and 3a with the fractional inhibitory concentrations indices of 0.1-0.32. The nitrocefin hydrolysis test and the whole cell assay with E. coli DH5α transformant carrying blaKPC-3 proved KPC enzyme being its molecular target. para-Phenylenediboronic acids efficiently potentiated carbapenems against KPC-producers and ceftazidime against AmpC-producers, whereas meta-phenylenediboronic acids enhanced only ceftazidime activity against the latter ones. Finally, the statistical analysis confirmed that ortho-phenylenediboronic acids act synergistically with carbapenems significantly stronger than other groups. Since the obtained phenylenediboronic compounds are not toxic to MRC-5 human fibroblasts at the tested concentrations, they can be considered promising scaffolds for the future development of novel KPC/AmpC inhibitors. The complexation of KPC-2 with the most representative isomeric phenylenediboronic acids 1a, 2a, and 3a was modeled by quantum mechanics/molecular mechanics calculations. Compound 3a reached the most effective configuration enabling covalent binding to the catalytic Ser70 residue.

Non-ß Lactam Inhibitors of the Serine ß-Lactamase blaCTX-M15 in Drug-Resistant Salmonella typhi.

Antibiotic resistance by bacterial pathogens against widely used ß-lactam drugs is a major concern to public health worldwide, resulting in high healthcare cost. The present study aimed to extend previous research by investigating the potential activity of reported compounds against the S. typhi ß-lactamase protein. 74 compounds from computational screening reported in our previous study against ß-lactamase CMY-10 were subjected to docking studies against blaCTX-M15. Site-Identification by Ligand Competitive Saturation (SILCS)-Monte Carlo (SILCS-MC) was applied to the top two ligands selected from molecular docking studies to predict and refine their conformations for binding conformations against blaCTX-M15. The SILCS-MC method predicted affinities of -8.6 and -10.7 kcal/mol for Top1 and Top2, respectively, indicating low micromolar binding to the blaCTX-M15 active site. MD simulations initiated from SILCS-MC docked orientations were carried out to better characterize the dynamics and stability of the complexes. Important interactions anchoring the ligand within the active site include pi-pi stacked, amide-pi, and pi-alkyl interactions. Simulations of the Top2-blaCTX-M15 complex exhibited stability associated with a wide range of hydrogen-bond and aromatic interactions between the protein and the ligand. Experimental ß-lactamase (BL) activity assays showed that Top1 has 0.1 u/mg BL activity, and Top2 has a BL activity of 0.038 u/mg with a minimum inhibitory concentration of 1 mg/mL. The inhibitors proposed in this study are non-ß-lactam-based ß-lactamase inhibitors that exhibit the potential to be used in combination with ß-lactam antibiotics against multidrug-resistant clinical isolates. Thus, Top1 and Top2 represent lead compounds that increase the efficacy of ß-lactam antibiotics with a low dose concentration.

Discovery of 2-Aminothiazole-4-carboxylic Acids as Broad-Spectrum Metallo-ß-lactamase Inhibitors by Mimicking Carbapenem Hydrolysate Binding.

Metallo-ß-lactamases (MBLs) are zinc-dependent enzymes capable of hydrolyzing all bicyclic ß-lactam antibiotics, posing a great threat to public health. However, there are currently no clinically approved MBL inhibitors. Despite variations in their active sites, MBLs share a common catalytic mechanism with carbapenems, forming similar reaction species and hydrolysates. We here report the development of 2-aminothiazole-4-carboxylic acids (AtCs) as broad-spectrum MBL inhibitors by mimicking the anchor pharmacophore features of carbapenem hydrolysate binding. Several AtCs manifested potent activity against B1, B2, and B3 MBLs. Crystallographic analyses revealed a common binding mode of AtCs with B1, B2, and B3 MBLs, resembling binding observed in the MBL-carbapenem product complexes. AtCs restored Meropenem activity against MBL-producing isolates. In the murine sepsis model, AtCs exhibited favorable synergistic efficacy with Meropenem, along with acceptable pharmacokinetics and safety profiles. This work offers promising lead compounds and a structural basis for the development of potential drug candidates to combat MBL-mediated antimicrobial resistance.

The directed evolution of NDM-1.

ß-Lactam antibiotics are among the most frequently prescribed therapeutic agents. A common mechanism of resistance toward ß-lactam antibiotics is the production of ß-lactamases. These enzymes are capable of hydrolyzing the ß-lactam bond, rendering the drug inactive. Among the four described classes, the metallo- ß-lactamases (MBLs, class B) employ one or two zinc ions in the active site for catalysis. One of the three most clinically relevant MBLs is New Delhi Metallo- ß-Lactamase (NDM-1). The current study sought to investigate the in vitro protein evolution of NDM-1 ß-lactamase using error-prone polymerase chain reaction. Evaluation revealed that variants were not found to confer higher levels of resistance toward meropenem based on amino acid substitutions. Thus, we postulate that increases in transcription or changes in zinc transport may be clinically more relevant to meropenem resistance than amino acid substitutions.

A novel aromatic compound from the fungus Synnemellisia sp. FKR-0921.

The filamentous fungus Synnemellisia sp. strain FKR-0921 was obtained from soil collected on Kume Island, Okinawa. The MeOH extract of FKR-0921 cultured on a solid rice medium yielded a new aromatic compound, synnemellisitriol A (1). The structure, including the absolute configuration, was elucidated by spectroscopic analysis (FT-IR, NMR, and HR-ESI-MS), and the absolute configuration at C-9 of 1 was determined using the modified Mosher's method. Additionally, 1 was evaluated for its biological activities, including metallo-ß-lactamase inhibitory activity, type III secretion system inhibitory activity, antimicrobial activity, antimalarial activity, and cytotoxicity.

Kinetics, Thermodynamics, and Structural Effects of Quinoline-2-Carboxylates, Zinc-Binding Inhibitors of New Delhi Metallo-ß-lactamase-1 Re-sensitizing Multidrug-Resistant Bacteria for Carbapenems.

Carbapenem resistance mediated by metallo-ß-lactamases (MBL) such as New Delhi metallo-ß-lactamase-1 (NDM-1) has become a major factor threatening the efficacy of essential ß-lactam antibiotics. Starting from hit fragment dipicolinic acid (DPA), 8-hydroxy- and 8-sulfonamido-quinoline-2-carboxylic acids were developed as inhibitors of NDM-1 with highly improved inhibitory activity and binding affinity. The most active compounds formed reversibly inactive ternary protein-inhibitor complexes with two zinc ions as proven by native protein mass spectrometry and bio-layer interferometry. Modification of the NDM-1 structure with remarkable entropic gain was shown by isothermal titration calorimetry and NMR spectroscopy of isotopically labeled protein. The best compounds were potent inhibitors of NDM-1 and other representative MBL with no or little inhibition of human zinc-binding enzymes. These inhibitors significantly reduced the minimum inhibitory concentrations (MIC) of meropenem for multidrug-resistant bacteria recombinantly expressing blaNDM-1 as well as for several multidrug-resistant clinical strains at concentrations non-toxic to human cells.

Metal binding pharmacophore click-derived discovery of new broad-spectrum metallo-ß-lactamase inhibitors.

The emergence of metallo-ß-lactamases (MBLs) confers resistance to nearly all the ß-lactam antibiotics, including carbapenems. Currently, there is a lack of clinically useful MBL inhibitors, making it crucial to discover new inhibitor chemotypes that can potently target multiple clinically relevant MBLs. Herein we report a strategy that utilizes a metal binding pharmacophore (MBP) click approach to identify new broad-spectrum MBL inhibitors. Our initial investigation identified several MBPs including phthalic acid, phenylboronic acid and benzyl phosphoric acid, which were subjected to structural transformations using azide-alkyne click reactions. Subsequent structure-activity relationship analyses led to the identification of several potent broad-spectrum MBL inhibitors, including 73 that manifested IC50 values ranging from 0.00012 µM to 0.64 µM against multiple MBLs. Co-crystallographic studies demonstrated the importance of MBPs in engaging with the MBL active site anchor pharmacophore features, and revealed the unusual two-molecule binding modes with IMP-1, highlighting the critical role of flexible active site loops in recognizing structurally diverse substrates/inhibitors. Our work provides new chemotypes for MBL inhibition and establishes a MBP click-derived paradigm for inhibitor discovery targeting MBLs as well as other metalloenzymes.

Metallo-ß-lactamase-mediated antimicrobial resistance and progress in inhibitor discovery.

Resistance to ß-lactam antibiotics is rapidly growing, substantially due to the spread of serine-ß-lactamases (SBLs) and metallo-ß-lactamases (MBLs), which efficiently catalyse ß-lactam hydrolysis. Combinations of a ß-lactam antibiotic with an SBL inhibitor have been clinically successful; however, no MBL inhibitors have been developed for clinical use. MBLs are a worrying resistance vector because they catalyse hydrolysis of all ß-lactam antibiotic classes, except the monobactams, and they are being disseminated across many bacterial species worldwide. Here we review the classification, structures, substrate profiles, and inhibition mechanisms of MBLs, highlighting current clinical problems due to MBL-mediated resistance and progress in understanding and combating MBL-mediated resistance.

Ruthenium-Catalyzed Cycloaddition for Introducing Chemical Diversity in Second-Generation ß-Lactamase Inhibitors.

Ruthenium(II) alkyne azide cycloaddition (RuAAC) is an attractive reaction to access 1,5-triazole derivatives and is applicable to internal alkynes. Here, we explore RuAAC to introduce molecular diversity on the diazabicyclooctane (DBO) scaffold of ß-lactamase inhibitors. The methodology presented is fully regioselective and enabled synthesis of a series of 1,5-triazole DBOs and trisubstituted analogues. Molecular modelling and biological evaluation revealed that the DBO substituents provided putative stabilizing interactions in the active site of broad-spectrum ß-lactamase KPC-2 and promising activity against a hyperpermeable strain of Escherichia coli producing KPC-2.

A modified bonded model approach for molecular dynamics simulations of New Delhi Metallo-ß-lactamase.

Modelling metalloproteins using the classical force fields is challenging. Several methods have been devised to model metalloproteins in force fields. Of these methods, the bonded model, combined with Restrained Electrostatic Potential (RESP) charge fitting, proved its superiority. The latter method was facilitated by the development of the python-based Metal Centre Parameter Builder (MCPB.py) AmberTool. However, the standard bonded model method offered by the MCPB.py tool may not be appropriate for validating and refining the binding modes predicted by docking when crystal structures are lacking. That is because the representation of coordination interactions between any bound ligand and metal ions by covalent bonds can hinder the flexibility of the ligand. Therefore, a new modification to the standard bonded model approach is proposed here. Molecular dynamics (MD) simulations based on the new modified bonded model (MBM) approach avoid the bias caused by coordination bonds and, unlike hybrid QM/MM MD, allow for sufficient sampling of the binding mode given the currently available computational power. The MBM MD approach reproduced the studied crystal structure conformations of New Delhi Metallo-ß-lactamase 1 (NDM-1). Furthermore, the MBM approach described the binding interactions of intact ß-lactams with NDM-1 reasonably, and predicted a non-productive binding mode for the poor NDM-1 substrate aztreonam whilst predicting productive binding modes for known good substrates. This study presents a useful MD method for metallo-ß-lactamases and provides better understanding of ß-lactam substrates recognition by NDM-1. The proposed MBM approach might also be useful in the investigation of other metal-containing protein targets.

New polyimidazole ligands against subclass B1 metallo-ß-lactamases: Kinetic, microbiological, docking analysis.

Beta-lactam antibiotics are one of the most commonly used drug classes in managing bacterial infections. However, their use is threatened by the alarming phenomenon of antimicrobial resistance, which represents a worldwide health concern. Given the continuous spread of metallo-ß-lactamases (MBLs) producing pathogens, the need to discover broad-spectrum ß-lactamase inhibitors is increasingly growing. A series of zinc chelators have been synthesized and investigated for their ability to hamper the Zn-ion network of interactions in the active site of MBLs. We assessed the inhibitory activity of new polyimidazole ligands N,N'-bis((imidazol-4-yl)methyl)-ethylenediamine, N,N,N'-tris((imidazol-4-yl)methyl)-ethylenediamine, N,N,N,N'-tetra((imidazol-4-yl-methyl)-ethylenediamine toward three different subclasses B1 MBLs: VIM-1, NDM-1 and IMP-1 by in vitro assays. The activity of known zinc chelators such as 1,4,7,10,13-Pentaazacyclopentadecane, 1,4,8,11-Tetraazacyclotetradecane and 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid was also assessed. Moreover, a molecular docking study was carried to gain insight into the interaction mode of the most active ligands.