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Serine/metallo-carbapenemase-coproducing pathogens, often referred to as "superbugs", are a significant clinical problem. They hydrolyze nearly all available β-lactam antibiotics, especially carbapenems considered as last-resort antibiotics, seriously endangering efficacious antibacterial treatment. Despite the continuous global spread of carbapenem resistance, no dual-action inhibitors are available in therapy. This Perspective is the first systematic investigation of all chemotypes, modes of inhibition, and crystal structures of dual serine/metallo-carbapenemase inhibitors. An overview of the key strategy for designing dual serine/metallo-carbapenemase inhibitors and their mechanism of action is provided, as guiding rules for the development of clinically available dual inhibitors, coadministrated with carbapenems, to overcome the carbapenem resistance issue.

The overuse of antibiotics exacerbates the development of antibiotic-resistant bacteria, threatening global public health, while most traditional antibiotics act on specific targets and sterilize through chemical modes. Therefore, it is a desperate need to design novel therapeutics or extraordinary strategies to overcome resistant bacteria. Herein, we report a positively charged nanocomposite PNs-Cur with a hydrodynamic diameter of 289.6 nm, which was fabricated by ring-opening polymerization of ε-caprolactone and Z-Lys

The drug resistance caused due to the overuse of antibiotics has grown into an emerging threat. It is urgent to develop novel strategies to combat bacterial resistance. In this work, a quinine-based quaternized polymer with amphiphilicity (QMTA) was prepared by free-radical copolymerization of quinine and (2-methacryloyloxyethyl) trimethyl ammonium chloride (MTA). Biological activity assays revealed that the polymer sterilized the drug-resistant Gram-negative E. coli and non-resistant Gram-positive bacteria B. subtilis, exhibiting a MIC value of 2 μg mL−1 and a bactericidal rate of more than 95%. Most importantly, both E. coli and B. subtilis treated with the polymer didn't develop resistance even after their 16th passages. Also, the polymer has low toxicity at a dose of up to 128 μg mL−1. The mechanism studies through SEM and 3D-SIM images, fluorescence staining and membrane potential determination reveal that the positively charged QMTA initially concentrates on the surface of bacteria by electrostatic adsorption, changes the membrane potential, binds to the bacteria by its quinine chain, and destroys the membrane structure of the bacteria. This study provides a potential approach through the existing drug to develop new bactericidal reagent/polymer materials without resistance.

It is imperative to develop innovative and efficient antibacterial agents, on account of the mounting prevalence of complicated infections induced by multidrug-resistant bacteria. In this work, Chol-b-Lys nanoparticles (CL-NPs) with a diameter of 304.9 nm were prepared by ring-opening polymerization of the monomers cholesterol-ethylenediamine conjugate (Chol-NH2) and L-Lys-N-carboxyanhydrides (L-Lys-NCAs) and further self-assembly. The nanoparticles exhibited biological activity against the drug-resistant and non-resistant bacteria, including Gram-negative E. coli and Gram-positive B. subtilis, E. faecalis and S. aureus, with a MIC value in the range of 32–2 μg mL−1 and a more than 95% bactericidal rate. Importantly, both bacteria treated with CL-NPs didn’t develop resistance even after their 16th passages. Also, the CL-NPs have low toxicity (32 μg mL−1). The mechanism characterization shows that the positively charged nanoparticles are initially gathered on the surface of bacteria by electrostatic adsorption, changes the membrane permeability, inserts into the lipid bilayer bacteria membrane with cholesterol groups, destroys the membrane structure and results in bacterial apoptosis. This work provides a potential approach by mimicking the structure of cell membrane to develop new bactericidal reagent/nanoparticle materials which is hard to cause resistance.

The emerging COVID-19 pandemic generated by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has severely threatened human health. The main protease (M

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 (IC

Antibiotic resistance mediated by β-lactamases including metallo-β-lactamases (MβLs) has become an emerging threat. In this study, we employed UV-vis to monitor the activity and inhibition of New Delhi MβL (NDM), K. pneumoniae carbapenemase (KPC) and MβL VIM-2 in live bacterial cells in real time. The monitoring showed that the faropenem was hydrolyzed by six strains which express β-lactamases, K. pneumoniae, P. aeruginosa, E. coli expressing extended-spectrum β-lactamase (ESBL), NDM-1 (EC07), VIM-2, and methicillin-resistant S. aureus (MRSA), but not by E. coli and S. aureus which do not express β-lactamases. Also, four β-lactams cefazolin, meropenem, faropenem and amoxicillin were hydrolyzed by the NDM and KPC expressed in K. pneumoniae. Cell-based studies revealed that the targets NDM and KPC expressed in K. pneumoniae cells were inhibited by three known inhibitors ethylene diamine tetraacetic acid (EDTA), ebselen and clavulanic acid, with a fifty percent inhibitory concentration (IC50) of 1.2, 84.7 and 13.3 μM, and the VIM-2 expressed in E. coli cells was inhibited by EDTA, D-captopril and rhodanine with an IC50 of 1.3, 510.8 and 9.3 μM, respectively. This study provides an approach that could be applied to screen and evaluate small molecule inhibitors of β-lactamases in whole cells or to identify drug resistant bacteria.

The overuse of traditional antibiotics has resulted in a large number of bacteria that are resistant to antibiotics; therefore, the development of new antibacterial agents is urgently required to combat the drug-resistant bacteria. In this work, a series of cationic polyacrylate copolymers N1–N4 was synthesized by free-radical copolymerization. Amphiphilic N3 self-assembled into positively charged nanostructures (N3-NSs) with a diameter of 455.9 nm and a zeta potential of 33.4 mV in aqueous solution, which was confirmed by SEM and DLS. N3-NSs exhibited a broad-spectrum and high sterilization efficacy against both Gram-positive and Gram-negative bacteria, with a MIC value in the range of 2–8 μg mL−1 and a more than 94% sterilization rate against the drug-resistant E. coli, P. aeruginosa, E. faecalis, VRE, and B. subtilis tested. Most importantly, the nanostructures did not result in resistance of the VRE and P. aeruginosa even after their 16th passages. N3-NSs caused serious damage to E. coli and E. faecalis membranes, with significant loss of membrane potential from −43.9 ± 0.6 and −39.1 ± 1.4 to −6.3 ± 0.3 and −4.6 ± 0.2 mV, respectively. Fluorescence staining, SEM and membrane potential characterization reveal the action mechanism of the nanostructures, which is that N3-NSs initially bind to the surface of bacteria, then pierce into the bacterial membranes with their hydrophobic groups, and finally disrupt the membranes, therefore exhibiting broad-spectrum antibacterial efficacy. N3-NSs exhibit low toxicity on mouse fibroblasts (L929) at a dose of 40 μg mL−1. This work provides a promising way for the development of efficient antibacterial materials that do not cause drug-resistance.

We report a promising NDM-1 inhibitor, disulfiram, which can covalently bind to NDM-1 by forming an S–S bond with the Cys208 residue. Its copper-containing metabolite in vivo, Cu(DTC)2, also inactivated NDM-1 through oxidizing the Zn(II) thiolate site of the enzyme, therefore exhibiting dual functional inhibitory potential against B1 and B2 subclass MβLs.

The d , d -dipeptidase enzyme VanX is the main cause of vancomycin resistance in gram-positive bacteria because of hydrolysis of the D-Ala-D-Ala dipeptide used in cell-wall biosynthesis. Continuous assay of VanX has proven challenging due to lack of a chromophoric substrate. Here, we report a direct approach for continuous assay of VanX in vitro and in vivo from hydrolysis of D-Ala-D-Ala, based on the heat-rate changes measured with isothermal titration calorimetry (ITC). With the ITC approach, determination of kinetic parameters of VanX hydrolyzing D-Ala-D-Ala and the inhibition constant of d -cysteine inhibitor yielded KM of 0.10 mM, kcat of 11.5 s−1, and Ki of 18.8 μM, which are consistent with the data from ninhydrin/Cd(II) assays. Cell-based ITC studies demonstrated that the VanX expressed in E. coli and in clinical strain VRE was inhibited by d -cysteine with IC50 values of 29.8 and 28.6 μM, respectively. Also, the total heat from D-Ala-D-Ala (4 mM) hydrolysis decreases strongly (in absolute value) from 1.26 mJ for VRE to 0.031 mJ for E. faecalis, which is consistent with the large MIC value of vancomycin of 512 μg/mL for VRE and the much smaller value of 4 μg/mL for E. faecalis. The ITC approach proposed here could be applied to screen and evaluate small molecule inhibitors of VanX or to identify drug resistant bacteria.

A series of multivalent zwitterionic poly(methacrylate) copolymers (PMGT) containing different reactive ligands (epoxy and –Si–OCH3) are designed and synthesized for surface modification of 3-aminopropyltriethoxysilane activated PDMS (PDMS-A) via a simple incubation and subsequent post-crosslinking procedure. The co-existence of protonated primary amine (NH3+), terminal primary amine (−NH2) and Si–OH/Si–OC2H5 surface receptors on PDMS-A created a platform to fabricate stable PMGT coatings through multivalent interactions with epoxy and –Si–OCH3 ligands in the polymer chains. The effect of molar ratio of epoxy and –Si–(OCH3)3 ligands (ne/nSi) in the polymer chains on the cooperativity of multivalent interactions is studied. The results show that ne/nSi in the PMGT polymer chain plays a crucial role in the regulation of the coating formation. At higher ne/nSi (1.78), a relatively rough and thin biomimetic coating is formed. By contrast, lower ne/nSi (1.04) in the polymer chain is helpful to produce a more uniform and dense polymer coating. It is attributed to the good balance between the anchoring of polymer chains and the growth of coating thickness in the incubation step through suppressing the competitive autocatalytic crosslinking reactions of epoxy-amine system, demonstrating positive cooperativity of multivalent interactions. The following post-crosslinking procedure under moisture atmosphere containing triethylamine (TEA) facilitates to realize the formation of durable PMGT polymer coating, in which zwitterionic PC moieties are orientated towards the outer surface, providing the PDMS with greatly improved controllable hydrophilicity, coating stability and antifouling properties.

The superbug infection caused by New Delhi metallo-β-lactamase (NDM-1) has grown into an emerging threat, labelling and inhibition of NDM-1 has proven challenging due to its shuttling between pathogenic bacteria. Here, we report a potent covalent scaffold, ebsulfur, for targeting the protein in vitro and in vivo. Enzymatic kinetic study indicated that eighteen ebsulfurs gained except 1a–b and 1f inhibited NDM-1, exhibiting an IC50 value ranging of 0.16–9 μM, and 1g was found to be the best, dose- and time-dependent inhibitor with an IC50 of 0.16 μM. Also, these ebsulfurs effectively restored the antibacterial activity of cefazolin against E. coli expressing NDM-1, and the best effect was observed to be from 1g, 1i and 1n, resulting in an 256-fold reduction in MIC of the antibiotic at a dose of 16 μg/mL. The equilibrium dialysis study implied that the ebsulfur disrupted the coordination of one Zn(II) ion at active site of NDM-1. Labelling of NDM-1 using a constructed fluorescent ebsulfur Ebs-R suggested that the inhibitor covalently bound to the target through SDS-PAGE analysis in vitro. Also, labelling NDM-1 in living E. coli cells with Ebs-R by confocal microscopic imaging showed the real-time distribution change process of intracellular recombinant protein NDM-1. Moreover, the cytotoxicity of these ebsulfurs against L929 mouse fibroblastic cells was tested, and their capability to restore antibacterial activity of antibiotic against clinical strains E. coli EC08 producing NDM-1 was determined. The ebsulfur scaffold proposed here is valuable for development of the covalent irreversible inhibitors of NDM-1, and also for labelling the target in vitro and in vivo.

The superbug infection mediated by metallo-β-lactamases (MβLs) has grown into anemergent health threat, and development of MβL inhibitors is an ideal strategy to combat the infection. In this work, twenty-five thiosemicarbazones 1a-e, 2a-e, 3a-e, 4a-d, 5a-d and 6a-b were synthesized and assayed against MβLs ImiS, NDM-1 and L1. The gained molecules specifically inhibited NDM-1 and ImiS, exhibiting an IC

The irregular use of antibiotics has created a natural selection pressure for bacteria to adapt resistance. Bacterial resistance caused by metallo-β-lactamases (MβLs) has been the most prevalent in terms of posing a threat to human health. The New Delhi metallo-β-lactamase-1 (NDM-1) has been shown to be capable of hydrolyzing almost all β-lactams. In this work, eight aromatic Schiff bases 1-8 were prepared and identified by enzyme kinetic assays to be the potent inhibitors of NDM-1 (except 4). These molecules exhibited a more than 95 % inhibition, and an IC

To combat the superbug infection caused by metallo-β-lactamases (MβLs), a dipyridyl-substituted thiosemicarbazone (DpC), was identified to be the broad-spectrum inhibitor of MβLs (NDM-1, VIM-2, IMP-1, ImiS, L1), with an IC50 value in the range of 0.021–1.08 µM. It reversibly and competitively inhibited NDM-1 with a Ki value of 10.2 nM. DpC showed broad-spectrum antibacterial effect on clinical isolate K. pneumonia, CRE, VRE, CRPA and MRSA, with MIC value ranged from 16 to 32 µg/mL, and exhibited synergistic antibacterial effect with meropenem on MβLs-producing bacteria, resulting in a 2–16-, 2–8-, and 8-fold reduction in MIC of meropenem against EC-MβLs, EC01-EC24, K. pneumonia, respectively. Moreover, mice experiments showed that DpC also had synergistic antibacterial action with meropenem. In this work, DpC was identified to be a potent scaffold for the development of broad-spectrum inhibitors of MβLs.

The "superbug" infection caused by metallo-β-lactamases (MβLs) has grown into anemergent health threat, and development of effective MβL inhibitors to restore existing antibiotic efficacy is an ideal alternative. Although the serine-β-lactamase inhibitors have been used in clinical settings, MβL inhibitors are not available to date. In this work, thirty-one quinolinyl sulfonamides 1a-p and sulphonyl esters 2a-o were synthesized and assayed against MβL NDM-1. The obtained molecules specifically inhibited NDM-1, 1a-p and 2a-o exhibited an IC

A biomimetic total synthesis of scabellone B is described. Through sequential regioselective introduction of a geranyl group by means of silyl protection, oxidative dimerization, and biomimetic oxo-6π electrocyclization with good cyclization selectivity, a biomimetic ­approach to scabellone B was achieved in five steps and 32% overall yield.

In an effort to develop new inhibitors of metallo-β-lactamases (MβLs), twenty-eight azolylthioacetamides were synthesized and assayed against MβLs. The obtained benzimidazolyl and benzioxazolyl substituted 1-19 specifically inhibited the enzyme ImiS, and 10 was found to be the most potent inhibitor of ImiS with an IC50 value of 15 nM. The nitrobenzimidazolyl substituted 20-28 specifically inhibited NDM-1, with 27 being the most potent inhibitor with an IC50 value of 170 nM. Further studies with 10, 11, and 27 revealed a mixed inhibition mode with competitive and uncompetitive inhibition constants in a similar range as the IC50 values. These inhibitors resulted in a 2-4-fold decrease in imipenem MIC values using E. coli cells producing ImiS or NDM-1. While the source of uncompetitive (possibly allosteric) inhibition remains unclear, docking studies indicate that 10 and 11 may interact orthosterically with Zn2 in the active site of CphA, while 27 could bridge the two Zn(II) ions in the active site of NDM-1 via its nitro group.

Antibiotic-resistant bacteria has become one of the greatest challenges to global human health today. Innovative strategies are needed to identify new therapeutic leads to tackle infections of drug-resistant Gram-negative bacteria. We herein synthesize a series of EB analogues to investigate their antibacterial activities. Select polar functionality at N-terminus of EB exhibited higher activities against multi-drug-resistant Gram-negative pathogens, including E. coli, P. aeruginosa and K. pneumoniae. EB analogue 4g and 4i exhibited potent antibacterial activities against E. coli-ESBL (MIC = 1–4 µg/mL) and E. coli producing NDM-1 (MIC = 4–32 µg/mL), which is superior to the traditional antibiotics (cefazolin, imipenem). Furthermore, the time-kill kinetics studies and the inhibition zone tests indicated that analogue 4i effectively and rapidly cause death of E. coli-ESBL and E. coli-NDM-1. Additionally, accumulation assays and SEM images showed that 4i could permeate bacterial membranes, leading to an irregular cell morphology. Importantly, bacterial resistance for analogue 4i was difficult to induce against E. coli-ESBL. EB analogues here reported low cytotoxicity against L-929 cells and mice model in vivo. We believe that EB analogues with polar functionality could play a pivotal role in the development of novel antibacterial agents in eradicating multi-drug-resistant Gram-negative pathogens infections.

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.

We discovered a promising metallo-β-lactamase inhibitor, a DNA nanoribbon, by enzymatic kinetics and isothermal titration calorimetry evaluations. Atomic force microscopy, gel electrophoresis, competitive binding experiments, circular dichroic and thermal denaturation studies suggested that the DNA nanoribbon could bind to the enzyme through a minor groove.

The ‘superbug’ infection caused by metallo-β-lactamases (MβLs) including L1 has grown into an emerging threat. To probe whether mercaptoacetate thioesters inhibiting L1 is a contribution of the thioester itself or its hydrolysate, ten mercaptoacetate thioesters 1–10 were synthesized, which specifically inhibited L1, exhibiting IC(50) values ranging from 0.17 to 1.2 μM, and 8 was found to be the best inhibitor (IC(50) = 0.17 μM). These thioesters restored the antimicrobial activity of cefazolin against E. coli expressing L1 by 2–4-fold. UV-vis monitoring showed that 1, 8 and 9 were unhydrolyzed in Tris buffer (pH 6.0–8.5), but hydrolyzed by L1; further HPLC monitoring indicated that 1/3 of the thioester 9 was converted to mercaptoacetic acid. STD-NMR monitoring suggested that both the thioester and its hydrolysate mercaptoacetic acid jointly inhibited L1.

The emergence of drug-resistant pathogens is a global public health problem. With the increasing antibiotic resistance of bacteria, there is an obvious need for the development for alternative therapeutics and materials that can effectively control this situation. In this work, a new conjugate, 5,10,15,20-tetrakis (para-aminophenyl) porphyrin-vancomycin was successfully explored for the identification and photoinactivation of antibiotic resistant Gram-positive pathogens. The minimum inhibitory concentration assay and photodynamic inactivation evaluation results revealed that the conjugate can effective inhibit the growth of 6 tested pathogenic strains under white light. The interaction intensity of the conjugate with Gram-positive and Gram-negative bacterial cells was evaluated by surface plasmon resonance, and the results showed that its activity toward Staphylococcus aureus was 14-fold larger than its activity toward Escherichia coli at 20 mM, indicating the conjugate selectively gathers in Gram-positive cells.

We report an UV-Vis method for monitoring the hydrolysis of the β-lactam antibiotics inside living bacterial cells. Cell-based studies demonstrated that the hydrolysis of cefazolin was inhibited by three known NDM-1 inhibitors. This approach can be applied to the monitoring of reactions in a complex biological system, for instance in medical testing.

In an effort to understand the recombination of a B2 metallo-β-lactamase (MβL), the binding of metals to apo-ImiS was studied by isothermal titration calorimetry and fluorescence spectra. The binding of Zn(II), Co(II) to apo-ImiS resulted in activation free energies Δ G ≠ θ values of 93.719 and 92.948 kJ mol−1, respectively, and increasing of fluorescence intensity at maxima emission of 340 nm.

The ‘superbug’ infection caused by metallo-β-lactamases (MβLs) has grown into an emergent health threat. Given the clinical importance of MβLs, a novel scaffold, dithiocarbamate, was constructed. The obtained molecules, DC1, DC8 and DC10, inhibited MβLs NDM-1, VIM-2, IMP-1, ImiS and L1 from all three subclasses, exhibiting an IC50 < 26 μM. DC1 was found to be the best inhibitor of ImiS (IC50 < 0.22 μM). DC1-2, DC4, DC8 and DC10 restored antimicrobial effects of cefazolin and imipenem against E. coli-BL21, producing NDM-1, ImiS or L1, and DC1 showed the best inhibition of E. coli cells, expressing the three MβLs, resulting in a 2-16-fold reduction in the minimum inhibitory concentrations (MICs) of both antibiotics. Kinetics and isothermal titration calorimetry (ITC) assays showed that DC1 exhibited a reversible, and partially mixed inhibition, of NDM-1, ImiS and L1, with Ki values of 0.29, 0.14 and 5.06 µM, respectively. Docking studies suggest that the hydroxyl and carbonyl groups of DC1 form coordinate bonds with the Zn (II) ions, in the active center of NDM-1, ImiS and L1, thereby inhibiting the activity of the enzymes. Cytotoxicity assays showed that DC1, DC3, DC7 and DC9 have low toxicity in L929 mouse fibroblastic cells, at a dose of up to 250 μM. These studies revealed that the dithiocarbamate is a valuable scaffold for the development of MβLs inhibitors.

Given the enormous biomedical importance of screening inhibitor of the metallo-β-lactamases (MβLs), we determined the thermokinetic parameters of cefalexin hydrolysis with MβL L1 from Stenotrophomonas maltophilia in the absence and presence of 2,5-pyrrolidinedicarboxylic acid (PDA) as enzyme inhibitor. The presence of PDA caused a value decrease of the rate constant k, and a value increase of the apparent activation energy E (from 33.88 to 35.46 kJ mol−1), activation enthalpy Δ H ≠ θ (from 31.35 to 32.92 kJ mol−1), activation free energy Δ G ≠ θ and activation entropy Δ S ≠ θ (from −201.31 to −196.39 J mol−1 K−1) in comparison with the absence of PDA in the temperature range of 298.15–313.15 K, proposing that the changes of k, E, Δ H ≠ θ and Δ S ≠ θ values can be employed to identify the inhibitors of MβLs.

In an effort to prepare a fluorogenic substrate to be used in activity assays with metallo-β-lactamases, (6R,7R)-8-oxo-7-(2-oxo-2H-chromene-3-carboxamido)-3-((4-(2-oxo-2H-chromene-3-carboxamido)-phenylthio)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (CA) was synthesized and characterized. CA exhibited a fluorescence quantum yield (φ) of 0.0059, two fluorescence lifetimes of 3.63×10(-10) and 5.38×10(-9)s, and fluorescence intensity that is concentration-dependent. Steady-state kinetic assays revealed that CA is a substrate for metallo-β-lactamases (MβLs) L1 and CcrA, exhibiting Km and kcat values of 18μM and 5s(-1) and 11μM and 17s(-1), respectively.

One of the most common way that bacteria become resistant to antibiotics is by the production of metallo-β-lactamases (MβLs) to hydrolyze the β-lactam-containing antibiotics. In this paper, the thermodynamic parameters of benzylpenicillin hydrolysis with B1 subclasses MβL CcrA (carbapenem and cephamycin resistance) from Bacteroides fragilis were determined by microcalorimetry. The activation free energy Δ G ≠ θ is 87.90 ± 0.03, 88.99 ± 0.01, 89.93 ± 0.04 and 90.93 ± 0.05 kJ mol−1 at 293.15, 298.15, 303.15 and 308.15 K, activation enthalpy Δ H ≠ θ is 29.21 ± 0.03 kJ mol−1, activation entropy Δ S ≠ θ is −200.34 ± 0.08 J mol−1 K−1, the reaction order is 1.4, and the apparent activation energy E is 31.71 kJ mol−1. The thermodynamic characterization indicated that CcrA prefer to hydrolyze penicillins among three kinds of β-lactam-containing antibiotics.

In an effort to identify novel, broad-spectrum inhibitors against the metallo-β-lactamases (MβLs), several N-heterocyclic derivatives were tested as inhibitors of MβLs CcrA, ImiS, and L1, which are representative enzymes from the distinct MβL subclasses. Three N-heterocyclic dicarboxylic acid derivatives were competitive inhibitors of CcrA and L1, exhibiting K(i) values ≤2 μM, while only 2,4-thiazolidinedicarboxylic acid (1b) was a competitive inhibitor of ImiS. Two 2-mercapto-1,3,4-thiadiazole derivatives were noncompetitive inhibitors of CcrA and ImiS, exhibiting K(i) values

In an effort to study on the antibiotic resistance in bacteria, we first report the thermokinetic parameters of the imipenem hydrolysis with B1 subclasses metallo-β-lactamase CcrA from Bacteroides fragilis . The values of activation free energy Δ G ≠ θ is 87.54 ± 0.03, 88.78 ± 0.03, 89.90 ± 0.04 and 91.06 ± 0.02 kJ mol −1 at 293.15, 298.15, 303.15 and 308.15 K, respectively, the activation enthalpy Δ H ≠ θ is 19.10 ± 0.01 kJ mol −1 , the activation entropy Δ S ≠ θ is −233.56 ± 0.09 J mol −1 K −1 and the apparent activation energy E is 21.598 kJ mol −1 . The results reveal that the imipenem hydrolysis catalyzed by CcrA is an exothermic and spontaneous reaction with reaction order of 1.4, and has a lower Δ H ≠ θ and Δ S ≠ θ than the cefazolin hydrolysis with same enzyme.

X-ray absorption spectroscopy was used to investigate the metal-binding sites of ImiS from Aeromonas veronii bv. sobria in catalytically active (1-Zn), product-inhibited (1-Zn plus imipenem), and inactive (2-Zn) forms. The first equivalent of zinc(II) was found to bind to the consensus Zn{sub 2} site. The reaction of 1-Zn ImiS with imipenem leads to a product-bound species, coordinated to Zn via a carboxylate group. The inhibitory binding site of ImiS was examined by a comparison of wild-type ImiS with 1 and 2 equiv of bound zinc. 2-Zn ImiS extended X-ray absorption fine structure data support a binding site that is distant from the active site and contains both one sulfur donor and one histidine ligand. On the basis of the amino acid sequence of ImiS and the crystal structure of CphA [Garau et al. (2005) J. Mol. Biol. 345, 785-795], we propose that the inhibitory binding site is formed by M146, found on the B2-distinct {alpha}3 helix, and H118, a canonical Zn{sub 1} ligand, proposed to help activate the nucleophilic water. The mutation of M146 to isoleucine abolishes metal inhibition. This is the first characterization of ImiS with the native metal Zn and establishes, for the first time, the location ofmore » the inhibitory metal site.« less

Extended X-ray absorption fine structure studies of the metallo-beta-lactamase L1 from Stenotrophomonas maltophilia containing 1 and 2 equiv of Zn(II) and containing 2 equiv of Zn(II) plus hydrolyzed nitrocefin are presented. The data indicate that the first, catalytically dominant metal ion is bound by L1 at the consensus Zn1 site. The data further suggest that binding of the first metal helps preorganize the ligands for binding of the second metal ion. The di-Zn enzyme displays a well-defined metal-metal interaction at 3.42 A. Reaction with the beta-lactam antibiotic nitrocefin results in a product-bound species, in which the ring-opened lactam rotates in the active site to present the S1 sulfur atom of nitrocefin to one of the metal ions for coordination. The product bridges the two metal ions, with a concomitant lengthening of the Zn-Zn interaction to 3.62 A.

Glyoxalase 2 is a beta-lactamase fold-containing enzyme that appears to be involved with cellular chemical detoxification. Although the cytoplasmic isozyme has been characterized from several organisms, essentially nothing is known about the mitochondrial proteins. As a first step in understanding the structure and function of mitochondrial glyoxalase 2 enzymes, a mitochondrial isozyme (GLX2-5) from Arabidopsis thaliana was cloned, overexpressed, purified, and characterized using metal analyses, EPR and (1)H NMR spectroscopies, and x-ray crystallography. The recombinant enzyme was shown to bind 1.04 +/- 0.15 eq of iron and 1.31 +/- 0.05 eq of Zn(II) and to exhibit k(cat) and K(m) values of 129 +/- 10 s(-1) and 391 +/- 48 microm, respectively, when using S-d-lactoylglutathione as the substrate. EPR spectra revealed that recombinant GLX2-5 contains multiple metal centers, including a predominant Fe(III)Z-n(II) center and an anti-ferromagnetically coupled Fe(III)Fe(II) center. Unlike cytosolic glyoxalase 2 from A. thaliana, GLX2-5 does not appear to specifically bind manganese. (1)H NMR spectra revealed the presence of at least eight paramagnetically shifted resonances that arise from protons in close proximity to a Fe(III)Fe(II) center. Five of these resonances arose from solvent-exchangeable protons, and four of these have been assigned to NH protons on metal-bound histidines. A 1.74-A resolution crystal structure of the enzyme revealed that although GLX2-5 shares a number of structural features with human GLX2, several important differences exist. These data demonstrate that mitochondrial glyoxalase 2 can accommodate a number of different metal centers and that the predominant metal center is Fe(III)Zn(II).

In an effort to probe the structure of a group Bb metallo-beta-lactamase, Co(II)-substituted ImiS was prepared and characterized by electronic absorption, NMR, and EPR spectroscopies. ImiS containing 1 equiv of Co(II) (Co(II)(1)-ImiS) was shown to be catalytically active. Electronic absorption studies of Co(II)(1)-ImiS revealed the presence of two distinct features: (1) an intense sulfur to Co(II) ligand to metal charge transfer band and (2) less intense, Co(II) ligand field transitions that suggest 4-coordinate Co(II) in Co(II)(1)-ImiS. (1)H NMR studies of Co(II)(1)-ImiS suggest that one histidine, one aspartic acid, and one cysteine coordinate the metal ion in Co(II)(1)-ImiS. The addition of a second Co(II) to Co(II)(1)-ImiS did not result in any additional solvent-exchangeable NMR resonances, strongly suggesting that the second Co(II) does not bind to a site with histidine ligands. EPR studies reveal that the metal ion in Co(II)(1)-ImiS is 4-coordinate and that the second Co(II) is 5/6 coordinate. Taken together, these data indicate that the catalytic site in ImiS is the consensus Zn(2) site, in which Co(II) (and by extrapolation Zn(II)) is 4-coordinate and bound by Cys221, His263, Asp120, and probably one solvent water molecule. These studies also show that the second, inhibitory metal ion does not bind to the consensus Zn(1) site and that the metal ion binds at a site significantly removed from the active site. These results give the first structural information on metallo-beta-lactamase ImiS and suggest that the second metal binding site in ImiS may be targeted for inhibitors.

In an effort to prepare Co(II)-substituted metallo-beta-lactamase L1 from Stenotrophomonas maltophilia for future spectroscopic and mechanistic studies, two methods for the preparation of Co(II)-L1 were tested. Method A involved adding CoCl2 directly to apo-L1 under anaerobic conditions. The resulting enzyme contained 1.9 moles of cobalt and exhibited very little activity, and UV-Vis, 1H NMR, and EPR studies indicated that most of the cobalt in this sample was Co(III). Method B involved reducing the single and unique disulfide bridge in L1 with tris(carboxyethyl)phosphine prior to adding CoCl2. The resulting enzyme was pink, contained 2.5 moles of cobalt per mole of enzyme, and exhibited kcat and Km values of 18+1 s(-1) and 10+/-1 microM, respectively, when using nitrocefin as the substrate. UV-Vis, 1H NMR, and EPR studies revealed that this enzyme sample contained high-spin Co(II). The UV-Vis spectra also provided evidence for Co(II) bound to one or both of the reduced cysteines. Efforts to block this non-specific Co(II) binding site using a chemical modification agent or site-directed mutagenesis were unsuccessful. The data presented here demonstrate the problem of solvent-exposed disulfides when preparing Co(II)-substituted enzymes and offers a convenient method to circumvent the problem.

In an effort to prepare novel inhibitors of bacterial aminopeptidase N (PepN), the phosphinate, propenylphosphinate, decylphosphinate, sulfonate, and sulfonamidate analogs of Ala-Ala were synthesized and tested as inhibitors. Phosphinate 1 was shown to inhibit PepN with a K(i) of 10microM, and propenylphosphinate 2 and decylphosphinate 3 inhibited PepN with a K(i) of ca. 1microM. Sulfonate and sulfonamidate analogs did not inhibit PepN.

In an effort to probe the inhibition of glyoxalase II (GLX2-2) from Arabidopsis thaliana , a series of N - and S -blocked glutathione compounds containing 9-fluorenylmethoxycarbonyl (FMOC) and Cbz protecting groups were synthesized and tested. The di-FMOC and di-Cbz compounds were the best inhibitors of GLX2-2 with K i values of 0.89 ± 0.05 and 2.3 ± 0.5 μM, respectively. The removal of protecting groups from either position resulted in comparable, diminished binding affinities. Analyses of site-directed mutants of GLX2-2 demonstrated that tight binding of these inhibitors is not due to interactions of the protecting groups with hydrophobic amino acids on the surface of the enzyme. Instead, MM2 calculations predict that the lowest energy structures of the unbound, doubly substituted inhibitors are similar to those of a bound inhibitor. These studies represent the first systematic attempt to understand the peculiar inhibition of GLX2 by N - and S -blocked glutathiones.

In order to accumulate photosensitizers on the cell walls of vancomycin-sensitive and vancomycin-resistant bacterial strains for labeling or/and photoinactivation of bacteria upon light irradiation, a simple and novel norvancomycin–rhodamine B (Van–Rh) conjugate was synthesized, characterized and confirmed using MALDI-TOF mass spectrometry. The properties of Van–Rh in photodynamic inactivation and fluorescent imaging of vancomycin-sensitive and vancomycin-resistant Enterococci (VRE) strains were investigated. The photodynamic assay indicated that Van–Rh effectively inactivated Bacillus subtilis (ATCC 6633), clinical isolates of VRE and Enterococcus faecalis (ATCC 51299, Van B genotype) with inactivation rates of 71, 54 and 47% at 9 μM upon 3 min of light exposure, respectively. Van–Rh enabled the fluorescent imaging of B. subtilis at 5 μM and two VRE strains at 20 μM, but not E. coli. The phototoxicity and binding affinity of rhodamine B were enhanced by conjugation with norvancomycin as an affinity ligand.