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.Download high-res image (51KB)Download full-size image

The emergence of antibiotic resistance caused by β-lactamases, including serine β-lactamases (SβLs) and metallo-β-lactamases (MβLs), is a global public health threat. L1, a B3 subclass MβL, hydrolyzes almost all of known β-lactam antibiotics. We report a simple and straightforward UV-Vis approach for real-time activity assays of β-lactamases inside living bacterial cells, and this method has been exemplified by choosing antibiotics, L1 enzyme, Escherichia coli expressing L1 (L1 E. coli), Escherichia coli expressing extended-spectrum β-lactamases (ESBL-E. coli), clinical bacterial strains, and reported MβL and SβL inhibitors. The cell-based studies demonstrated that cefazolin was hydrolyzed by L1 E. coli and clinical strains, and confirmed the hydrolysis to be inhibited by two known L1 inhibitors EDTA and azolylthioacetamide (ATAA), with an IC50 value of 1.6 and 18.9 μM, respectively. Also, it has been confirmed that the breakdown of cefazolin caused by ESBL-E. coli was inhibited by clavulanic acid, the first SβL inhibitor approved by FDA. The data gained through this approach are closely related to the biological function of the target enzyme in its physiological environment. The UV-Vis method proposed here can be applied to target-based whole-cell screening to search for potent β-lactamase inhibitors, and to assays of reactions in complex biological systems, for instance in medical assays.

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.

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.

The metallo-β-lactamases (MβLs) cleave the β-lactam ring of β-lactam antibiotics, conferring resistance against these drugs to bacteria. Twenty-four triazolylthioacetamides were prepared and evaluated as inhibitors of representatives of the three subclasses of MβLs. All these compounds exhibited specific inhibitory activity against NDM-1 with an IC50 value range of 0.15–1.90 μM, but no activity against CcrA, ImiS, and L1 at inhibitor concentrations of up to 10 μM. Compounds 4d and 6c are partially mixed inhibitors with Ki values of 0.49 and 0.63 μM using cefazolin as the substrate. Structure–activity relationship studies reveal that replacement of hydrogen on the aromatic ring by chlorine, heteroatoms, or alkyl groups can affect bioactivity, while leaving the aromatic ring of the triazolylthiols unmodified maintains the inhibitory potency. Docking studies reveal that the typical potent inhibitors of NDM-1, 4d and 6c, form stable interactions in the active site of NDM-1, with the triazole bridging Zn1 and Zn2, and the amide interacting with Lys 211 (Lys224).Keywords: inhibitor; Metallo-β-lactamase; NDM-1; triazolylthioacetamide

A new scaffold, azolylthioacetamide, was constructed and assayed against metallo-β-lactamases (MβLs). The obtained molecules specifically inhibited MβL ImiS, and 1c was found to be the most potent inhibitor, with a Ki = 1.2 μM using imipenem as substrate. Structure–activity relationships reveal that the aromatic carboxyl improves inhibitory activity of the inhibitors, but the aliphatic carboxyl does not. Compounds 1c–d and 1h–i showed the best antibacterial activities against E. coli BL21(DE3) cells producing CcrA or ImiS, resulting in 32- and 8-fold reduction in MIC values, respectively; 1c and 1f–j resulted in a reduction in MIC against P. aeruginosa. Docking studies revealed that 1a, 1c, and 1d fit tightly into the substrate binding site of CphA as a proxy for ImiS with the aromatic carboxylate forming interactions with Lys224, the Zn(II) ion, the backbone of Asn233, and hydrophobic portions of the inhibitors aligning with hydrophobic patches of the protein surface.

In light of the biomedical significance of metallo-β-lactamases (MβLs), ten new mercaptoacetic acid thioester amino acid derivatives were synthesized and characterized. Biological activity assays indicated that all these synthesized compounds are very potent inhibitors of L1, exhibiting an IC50 value range of 0.018–2.9 μM and a Ki value range of 0.11–0.95 μM using cefazolin as substrate. Partial thioesters also showed effective inhibitory activities against NDM-1 and ImiS with an IC50 value range of 12–96 and 3.6–65 μM, respectively. Also, all these thioesters increased susceptibility of E. coli cells expressing L1 to cefazolin, indicated by a 2–4-fold reduction in MIC of the antibiotic. Docking studies revealed potential binding modes of the two most potent L1 inhibitors to the active site in which the carboxylate group interacts with both Zn(II) ions and Ser221. This work introduces a highly promising scaffold for the development of metallo-β-lactamase L1 inhibitors.Keywords: Antibiotic resistance; inhibitor; L1; mercaptoacetic acid thioester; metallo-β-lactamase; subclass B3

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 ΔG≠θ values of 93.719 and 92.948 kJ mol−1, respectively, and increasing of fluorescence intensity at maxima emission of 340 nm.Binding of Zn(II), Co(II) to apo-ImiS results in an activation free energy ΔG≠θ value of 92.948 and 93.908 kJ mol−1, respectively, and increasing of fluorescence intensity at maxima emission of 340 nm.

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.

In an effort to test whether a transition state analog is an inhibitor of the metallo-β-lactamases, a phospholactam analog of carbapenem has been synthesized and characterized. The phospholactam 1 proved to be a weak, time-dependent inhibitor of IMP-1 (70%), CcrA (70%), L1 (70%), NDM-1 (53%), and Bla2 (94%) at an inhibitor concentration of 100 μM. The phospholactam 1 activated ImiS and BcII at the same concentration. Docking studies were used to explain binding and to offer suggestions for modifications to the phospholactam scaffold to improve binding affinities.

•Determined thermokinetic parameters of cefalexin hydrolysis with metallo-β-lactamase (MβL) L1.•First evaluated thermokinetic effect of the antibiotic hydrolysis with MβL that enzyme inhibitor caused.•Proposed a thermokinetic approach to identify inhibitor of MβLs.•Summarized that antibiotic hydrolysis with B1 and B3 MβLs has a higher value of E   and ΔH≠θ than that with B2 MβLs.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 ΔH≠θ (from 31.35 to 32.92 kJ mol−1), activation free energy ΔG≠θ and activation entropy ΔS≠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  , ΔH≠θ and ΔS≠θ values can be employed to identify the inhibitors of MβLs.The enzyme inhibitor PDA caused a value decrease of rate constant k, and a value increase of thermokinetic parameters of E  , ΔH≠θ, ΔG≠θ and ΔS≠θ during cefalexin hydrolysis with metallo-β-lactamase (MβL) L1.

Novel fluorescently-labeled conjugates of risedronate were synthesized using an epoxide linker, enabling conjugation of risedronate via its pyridyl nitrogen with the aromatic succinimidyl esters. The compounds were characterized by using 1H NMR, 13C NMR, 31P NMR, UV–vis and fluorescence emission spectroscopies. Biological activity assays showed that the conjugates 14 and 15 exhibited photodynamic inactivation of Bacillus subtilis (ATCC 6633) with 91% and 47% bacterial lethality at 10 μM upon visible light irradiation, respectively. Both 14 and 15 could be also used for fluorescence imaging of Bacillus subtilis.

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 18 μM and 5 s−1 and 11 μM and 17 s−1, respectively.

In an effort to explore antibiotic resistance based on enzyme hydrolysis, the thermodynamic parameters of the D-Ala-D-Ala hydrolysis catalyzed by dipeptidase VanX and occurred in Gram-positive vancomycin-resistant pathogens were determined by microcalorimetry. The values of activation free energy \( \Updelta G_{ \ne }^{\theta } \) are 87.140 ± 0.055, 88.413 ± 0.067, 89.611 ± 0.051, and 90.823 ± 0.042 kJ mol−1 at 293.15, 298.15, 303.15, and 308.15 K, respectively, activation enthalpy \( \Updelta H_{ \ne }^{\theta } \) is 15.332 ± 0.006 kJ mol−1, activation entropy \( \Updelta S_{ \ne }^{\theta } \) is −245.02 ± 0.20 J mol−1 K−1, apparent activation energy E is 17.830 kJ mol−1, and the reaction order is 1.5. These thermodynamic data reveal that D-Ala-D-Ala hydrolysis with VanX is an exothermic and spontaneous reaction and has an approximative reaction rate with the imipenem hydrolysis with metallo-β-lactamase ImiS in vitro.

In an effort to understand the reactions of antibiotics hydrolysis with metallo-β-lactamases (MβLs), the thermokinetic parameters of cefazolin hydrolysis with B1 subclass MβL CcrA from Bacteroides fragilis were determined by microcalorimetric method. The values of activation free energy \( \Updelta G_{ \ne }^{\theta } \) are 88.032 ± 0.038, 89.075 ± 0.025, 90.095 ± 0.034, and 91.261 ± 0.044 kJ mol−1 at 293.15, 298.15, 303.15, and 308.15 K, respectively, the activation enthalpy \( \Updelta H_{ \ne }^{\theta } \) is 25.278 ± 0.005 kJ mol−1, the activation entropy \( \Updelta S_{ \ne }^{\theta } \) is −213.99 ± 0.14 J mol−1 K−1, the apparent activation energy E is 27.776 kJ mol−1, and the reaction order is 1.4. The results indicated that the cefazolin hydrolysis with CcrA is an exothermic and spontaneous reaction. An association between the thermokinetic and kinetic parameters was revealed, which is that the catalytic constant Kcat increase with increase in \( \Updelta H_{ \ne }^{\theta } \).

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 Ki 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 Ki values <7 μM; however, these same compounds did not inhibit L1. Two 2-mercapto-1,3,4-triazole derivatives were shown not to inhibit any of the tested MβLs. The N-heterocyclic derivatives were tested for antibacterial activity by examining the MIC values for existing antibiotics in the presence/absence of these derivatives. Consistent with the steady-state inhibition data, the inclusion of three N-heterocyclic dicarboxylic acid derivatives resulted in lower MIC values when using Escherichia coli BL21(DE3) cells containing the CcrA or L1 plasmids or Klebsiella pneumoniae (ATCC 700603), while 1b was the only dicarboxylic acid derivative to lower the MIC value of E. coli cells containing the ImiS plasmid. Inclusion of the 2-mercapto-1,3,4-thiadiazole derivatives resulted in lower MIC values for E. coli cells containing ImiS or L1 plasmids; however, these derivatives did not alter the MIC values for K. pneumoniae or E. coli cells containing the L1 plasmid. None of the N-heterocyclic derivatives affected the MIC of two methicillin resistant Staphylococcus aureus (MRSA) strains. Taken together, these studies demonstrate that N-heterocyclic dicarboxylic acids 1a–c and pyridylmercaptothiadiazoles 2a,b are good scaffolds for future broad-spectrum inhibitors of the MβLs.

VanX, a Zn(II)-dependent D-ala-D-ala dipeptidase, is essential for vancomycin resistance in Enterococcus faecium. The enzymatic activity of VanX was previously found to be inhibited competitively by 2-{[(1-aminoethyl) (hydroxy) phosphoryl]oxy} propanoic acid (1B). Here we report the synthesis and characterization of seven phosphonate dipeptide analogs of D-ala-D-ala with various substituent, the activity evaluation indicated that six of these phosphonate analogs inhibit VanX with IC50 of 0.48–8.21 mM. These data revealed a structure–activity relationship which is that the large substituent group on β-carbon resulted in low binding affinity of the phonphonate analog to VanX. This information will be helpful to guide the design and synthesis of the tightly-binding inhibitors for VanX.Six phosphonate dipeptide analogs of D-ala-D-ala with various substituent displayed inhibitory activity against VanX with IC50 values of 0.48–8.21 mM.

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 ΔG≠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 ΔH≠θ is 19.10 ± 0.01 kJ mol−1, the activation entropy ΔS≠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 ΔH≠θ and ΔS≠θ than the cefazolin hydrolysis with same enzyme.Highlights► First report the thermokinetic parameters of imipenem hydrolysis with CcrA. ► The hydrolysis is a spontaneous and exothermic reaction with order of 1.4. ► Imipenem hydrolysis with CcrA has a lower ΔH≠θ and ΔS≠θ than the cefazolin hydrolysis with same enzyme. ► Imipenem hydrolysis with CcrA had a 46-fold faster reaction rate than with ImiS.

A simple and unique conjugation of norvancomycin–fluorescein (VanF) has been achieved. It was characterized by UV–vis and fluorescence spectra and confirmed by MALDI-TOF mass spectrum. The photodynamic assay indicated that VanF effectively inactivated the Gram-positive Bacillus subtilis (ATCC 6633) from clinic with inactivation rate of 30–70% within 1–7.5 μM. In vitro, VanF showed low antimicrobial activity with value of >128 μg/mL, binding affinity with value of 180 nM per 108 cells/mL against the bacteria strains. The fluorescence imaging showed that VanF could label the B. subtilis strain, but not the Escherichia coli (ATCC 25922), Enterococcus faecalis (ATCC 51299, VanD), and VRE strains from clinic.

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.