scholarly journals Mechanistic Investigations of Metallo-β-Lactamase Inhibitors: Strong Zinc Binding Is Not Required for Potent Enzyme Inhibition

2020 ◽  
Author(s):  
Nicola Wade ◽  
Kamaleddin Tehrani ◽  
Nora Brüchle ◽  
Matthijs J. van Haren ◽  
Vida Mashayekhi ◽  
...  
Keyword(s):  
Metal Binding ◽  
Bacterial Resistance ◽  
Mechanistic Study ◽  
Titration Calorimetry ◽  
E Coli ◽  
Inhibitory Mechanisms ◽  
Patent Literature ◽  
Pharmaceutical Agents ◽  
Nanomolar Range ◽  
Carbapenem Antibiotic

Metallo-β-lactamases (MBLs) are zinc-dependent bacterial resistance enzymes that can inactivate essentially all classes of β-lactam antibiotics. Infections due to multi-drug-resistant pathogens that express MBLs are difficult to treat and carry high mortality rates. At present there are no clinically approved MBL inhibitors underscoring the urgent need for pharmaceutical agents capable of counteracting the action of these enzymes. In order to develop effective MBL inhibitors it is essential to understand their inhibitory mechanisms. We here describe a comprehensive mechanistic study on a panel of structurally distinct MBL inhibitors drawn from the diverse collection of compounds reported to date in both the scientific and patent literature. Specifically, we determined the half-maximal inhibitory concentration value (IC<sub>50</sub>) for each inhibitor against purified NDM-1 and IMP-1 revealing clear differences in inhibitory potency among the compounds tested. Additional mechanistic insights into metal binding were also obtained by means of isothermal titration calorimetry (ITC) which was used to assess the affinity of the MBL inhibitors for Zn<sup>2+</sup>, Ca<sup>2+</sup> and Mg<sup>2+</sup>. These investigations revealed clear differences in metal binding among the MBL inhibitors evaluated. In addition, we directly compared the ability of these compounds to resensitize an NDM-1-expressing <i>E. coli</i> strain to the last resort carbapenem antibiotic meropenem. Notably, indole carboxylate <b>12</b> proved to be the most potent inhibitor tested in its ability to synergize with meropenem and with IC<sub>50</sub> values in the low nanomolar range against both enzymes. Interestingly, while compound <b>12</b> was found the most active MBL inhibitor, it exhibited no appreciable binding to any of the metals tested. These findings provide valuable insights into differences in mechanism and potency for the various classes of MBL inhibitors reported to date.

scholarly journals Mechanistic Investigations of Metallo-β-Lactamase Inhibitors: Strong Zinc Binding Is Not Required for Potent Enzyme Inhibition

2020 ◽  
Author(s):  
Nicola Wade ◽  
Kamaleddin Tehrani ◽  
Nora Brüchle ◽  
Matthijs J. van Haren ◽  
Vida Mashayekhi ◽  
...  
Keyword(s):  
Metal Binding ◽  
Bacterial Resistance ◽  
Mechanistic Study ◽  
Titration Calorimetry ◽  
E Coli ◽  
Inhibitory Mechanisms ◽  
Patent Literature ◽  
Pharmaceutical Agents ◽  
Nanomolar Range ◽  
Carbapenem Antibiotic

Metallo-β-lactamases (MBLs) are zinc-dependent bacterial resistance enzymes that can inactivate essentially all classes of β-lactam antibiotics. Infections due to multi-drug-resistant pathogens that express MBLs are difficult to treat and carry high mortality rates. At present there are no clinically approved MBL inhibitors underscoring the urgent need for pharmaceutical agents capable of counteracting the action of these enzymes. In order to develop effective MBL inhibitors it is essential to understand their inhibitory mechanisms. We here describe a comprehensive mechanistic study on a panel of structurally distinct MBL inhibitors drawn from the diverse collection of compounds reported to date in both the scientific and patent literature. Specifically, we determined the half-maximal inhibitory concentration value (IC<sub>50</sub>) for each inhibitor against purified NDM-1 and IMP-1 revealing clear differences in inhibitory potency among the compounds tested. Additional mechanistic insights into metal binding were also obtained by means of isothermal titration calorimetry (ITC) which was used to assess the affinity of the MBL inhibitors for Zn<sup>2+</sup>, Ca<sup>2+</sup> and Mg<sup>2+</sup>. These investigations revealed clear differences in metal binding among the MBL inhibitors evaluated. In addition, we directly compared the ability of these compounds to resensitize an NDM-1-expressing <i>E. coli</i> strain to the last resort carbapenem antibiotic meropenem. Notably, indole carboxylate <b>12</b> proved to be the most potent inhibitor tested in its ability to synergize with meropenem and with IC<sub>50</sub> values in the low nanomolar range against both enzymes. Interestingly, while compound <b>12</b> was found the most active MBL inhibitor, it exhibited no appreciable binding to any of the metals tested. These findings provide valuable insights into differences in mechanism and potency for the various classes of MBL inhibitors reported to date.

scholarly journals Zinc- and iron-dependent cytosolic metallo-β-lactamase domain proteins exhibit similar zinc-binding affinities, independent of an atypical glutamate at the metal-binding site

2004 ◽  
Vol 385 (1) ◽  
pp. 145-153 ◽  
Author(s):  
Oliver SCHILLING ◽  
Andreas VOGEL ◽  
Brenda KOSTELECKY ◽  
Hugo NATAL da LUZ ◽  
Daniel SPEMANN ◽  
...  
Keyword(s):  
Binding Site ◽  
Metal Binding ◽  
Dissociation Constants ◽  
Zinc Binding ◽  
Titration Calorimetry ◽  
Good Correspondence ◽  
Metal Binding Site ◽  
E Coli ◽  
Metal Ligands ◽  
Metal Selectivity

ZiPD (zinc phosphodiesterase; synonyms are ElaC, ecoZ, RNaseZ and 3′ tRNase) and the iron-dependent redox enzyme FlRd (flavorubredoxin) from Escherichia coli represent prototypical cases of proteins sharing the metallo-β-lactamase fold that require strict metal selectivity for catalytic activity, yet their metal selectivity has only been partially understood. In contrast with hydrolytic metallo-β-lactamase proteins, iron-dependent FlRd-like enzymes have an atypical glutamate ligand, which replaces one otherwise conserved histidine ligand. X-ray absorption spectroscopy revealed that the FlRd metallo-β-lactamase domain is capable of incorporating two zinc ions into the binuclear metal-binding site. Zinc dissociation constants, determined by isothermal titration calorimetry are similar for zinc binding to E. coli ZiPD (Kd1=2.2±0.2 μM and Kd2=23.0±0.6 μM) and to the E. coli FlRd metallo-β-lactamase domain (Kd1=0.7±0.1 μM and Kd2=26.0±0.1 μM). In good correspondence, apo-ZiPD requires incubation with 10 μM zinc for full reconstitution of the phosphodiesterase activity. Accordingly, metal selectivity of ZiPD and FlRd only partially relies on first shell metal ligands. Back mutation of the atypical glutamate in FlRd to a histidine unexpectedly resulted in an increased first zinc dissociation constant (Kd1=30±4 μM and Kd2=23±2 μM). In combination with a recent mutational study on ZiPD [Vogel, Schilling and Meyer-Klaucke (2004) Biochemistry 43, 10379–10386], we conclude that the atypical glutamate does not guide metal selectivity of the FlRd metallo-β-lactamase domain but suppresses possible hydrolytic cross-activity.

scholarly journals Investigation of metal binding and activation of Escherichia coli glyoxalase I: kinetic, thermodynamic and mutagenesis studies

2004 ◽  
Vol 377 (2) ◽  
pp. 309-316 ◽  
Author(s):  
Susan L. CLUGSTON ◽  
Rieko YAJIMA ◽  
John F. HONEK
Keyword(s):  
Escherichia Coli ◽  
Isothermal Titration Calorimetry ◽  
Metal Binding ◽  
Inductively Coupled Plasma ◽  
Metal Ion ◽  
Titration Calorimetry ◽  
Glyoxalase I ◽  
E Coli ◽  
Catalytic Metal ◽  
Bivalent Metals

GlxI (glyoxalase I) isomerizes the hemithioacetal formed between glutathione and methylglyoxal. Unlike other GlxI enzymes, Escherichia coli GlxI exhibits no activity with Zn2+ but maximal activation with Ni2+. To elucidate further the metal site in E. coli GlxI, several approaches were undertaken. Kinetic studies indicate that the catalytic metal ion affects the kcat without significantly affecting the Km for the substrate. Inductively coupled plasma analysis and isothermal titration calorimetry confirmed one metal ion bound to the enzyme, including Zn2+, which produces an inactive enzyme. Isothermal titration calorimetry was utilized to determine the relative binding affinity of GlxI for various bivalent metals. Each metal ion examined bound very tightly to GlxI with an association constant (Ka)>107 M−1, with the exception of Mn2+ (Ka of the order of 106 M−1). One of the ligands to the catalytic metal, His5, was altered to glutamine, a side chain found in the Zn2+-active Homo sapiens GlxI. The affinity of the mutant protein for all bivalent metals was drastically decreased. However, low levels of activity were now observed for Zn2+-bound GlxI. Although this residue has a marked effect on metal binding and activation, it is not the sole factor determining the differential metal activation between the human and E. coli GlxI enzymes.

scholarly journals Independent Behavior of Commensal Flora for Carriage of Fluoroquinolone-Resistant Bacteria in Patients at Admission

2010 ◽  
Vol 54 (12) ◽  
pp. 5193-5200 ◽  
Author(s):  
Victoire de Lastours ◽  
Françoise Chau ◽  
Florence Tubach ◽  
Blandine Pasquet ◽  
Etienne Ruppé ◽  
...  
Keyword(s):  
Risk Factors ◽  
Bacterial Resistance ◽  
Care Facility ◽  
Health Care Facility ◽  
Epidemiological Data ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
E Coli ◽  
Commensal Flora ◽  
Resistant Strains

ABSTRACT The important role of commensal flora as a natural reservoir of bacterial resistance is now well established. However, whether the behavior of each commensal flora is similar to that of other floras in terms of rates of carriage and risk factors for bacterial resistance is unknown. During a 6-month period, we prospectively investigated colonization with fluoroquinolone-resistant bacteria in the three main commensal floras from hospitalized patients at admission, targeting Escherichia coli in the fecal flora, coagulase-negative Staphylococcus (CNS) in the nasal flora, and α-hemolytic streptococci in the pharyngeal flora. Resistant strains were detected on quinolone-containing selective agar. Clinical and epidemiological data were collected. A total of 555 patients were included. Carriage rates of resistance were 8.0% in E. coli, 30.3% in CNS for ciprofloxacin, and 27.2% in streptococci for levofloxacin; 56% of the patients carried resistance in at least one flora but only 0.9% simultaneously in all floras, which is no more than random. Risk factors associated with the carriage of fluoroquinolone-resistant strains differed between fecal E. coli (i.e., colonization by multidrug-resistant bacteria) and nasal CNS (i.e., age, coming from a health care facility, and previous antibiotic treatment with a fluoroquinolone) while no risk factors were identified for pharyngeal streptococci. Despite high rates of colonization with fluoroquinolone-resistant bacteria, each commensal flora behaved independently since simultaneous carriage of resistance in the three distinct floras was uncommon, and risk factors differed. Consequences of environmental selective pressures vary in each commensal flora according to its local specificities (clinical trial NCT00520715 [http://clinicaltrials.gov/ct2/show/NCT00520715 ]).

scholarly journals In VivoImpact of Met221 Substitution in GOB Metallo-β-Lactamase

2012 ◽  
Vol 56 (4) ◽  
pp. 1769-1773 ◽  
Author(s):  
Jorgelina Morán-Barrio ◽  
María-Natalia Lisa ◽  
Alejandro J. Vila
Keyword(s):  
Antibiotic Resistance ◽  
Protein Stability ◽  
Metal Binding ◽  
Active Sites ◽  
Bacterial Resistance ◽  
Structural Diversity ◽  
Hydrophobic Core

ABSTRACTMetallo-β-lactamases (MβLs) represent one of the main mechanisms of bacterial resistance against β-lactam antibiotics. The elucidation of their mechanism has been limited mostly by the structural diversity among their active sites. All MβLs structurally characterized so far present a Cys or a Ser residue at position 221, which is critical for catalysis. GOB lactamases stand as an exception within this picture, possessing a Met residue in this location. We studied different mutants in this position, and we show that Met221 is essential for protein stability, most likely due to its involvement in a hydrophobic core. In contrast to other known MβLs, residue 221 is not involved in metal binding or in catalysis in GOB enzymes, further highlighting the structural diversity of MβLs. We also demonstrate the usefulness of protein periplasmic profiles to assess the contribution of protein stability to antibiotic resistance.

scholarly journals PSVIII-8 Effect of Lactobacillus Casei Zhang on E. coli induced injury of blood and milk barrier in dairy cow

2020 ◽  
Vol 98 (Supplement_4) ◽  
pp. 259-259
Author(s):  
Yuhui Zheng ◽  
Shengli Li
Keyword(s):  
Bacterial Infection ◽  
Lactobacillus Casei ◽  
Bacterial Resistance ◽  
Bovine Mastitis ◽  
Production Performance ◽  
Economic Losses ◽  
Control Group ◽  
Bovine Mammary Epithelial Cells ◽  
E Coli ◽  
Lactobacillus Casei Zhang

Abstract Bovine mastitis is one of the major diseases which directly affects the milk production performance and it causes huge economic losses in the dairy industry. Bacterial infection is the main risk factor of bovine mastitis and the antibiotic therapy is the primary choice to control the disease. However, persistence use of antibiotic increases the incidence of bacterial resistance and traces of antibiotic residues in animal products. Lactobacillus casei Zhang is one of the probiotics with multiple biological functions, which has certain bacteriostatic effect on pathogenic microorganism. The purpose of this study was to explore the effect of Lactobacillus casei Zhang (L. casei Zhang) on the prevention of E. coli-induced milk-blood barrier damage. Bovine mammary epithelial cells (BMECs) were used to establish a milk-blood model and Control group (PBS), E. coli group, and L. casei Zhang pretreatment plus E. coli group were set up respectively. The results showed that: L. casei Zhang could significantly reduce the increase of LDH release caused by E. coli treatment (P&lt; 0.05). And it can also significantly reduce the decrease of transmembrane resistance of monolayer cells caused by E. coli treatment (P&lt; 0.05). In addition, L. casei Zhang could significantly reduce the expression of tight junction proteins ZO-1, Claudin-1, Claudin-4 and Occludin (P &lt; 0.05). In conclusion, L. casei Zhang could effectively improve the damage of the blood-milk barrier caused by E. coli and could protect BMECs during bacterial infection.

scholarly journals Alkaloid-Rich Crude Extracts, Fractions and Piperamide Alkaloids of Piper guineense Possess Promising Antibacterial Effects

Antibiotics ◽  
2018 ◽  
Vol 7 (4) ◽  
pp. 98 ◽  
Author(s):  
Eunice Mgbeahuruike ◽  
Pia Fyhrquist ◽  
Heikki Vuorela ◽  
Riitta Julkunen-Tiitto ◽  
Yvonne Holm
Keyword(s):  
Antibacterial Activity ◽  
Pathogenic Bacteria ◽  
Bacterial Resistance ◽  
Hot Water ◽  
Bacterial Strains ◽  
Inhibitory Effects ◽  
Piper Guineense ◽  
E Coli ◽  
Growth Inhibitory ◽  
Derivatives Of

Piper guineense is a food and medicinal plant commonly used to treat infectious diseases in West-African traditional medicine. In a bid to identify new antibacterial compounds due to bacterial resistance to antibiotics, twelve extracts of P. guineense fruits and leaves, obtained by sequential extraction, as well as the piperine and piperlongumine commercial compounds were evaluated for antibacterial activity against human pathogenic bacteria. HPLC-DAD and UHPLC/Q-TOF MS analysis were conducted to characterize and identify the compounds present in the extracts with promising antibacterial activity. The extracts, with the exception of the hot water decoctions and macerations, contained piperamide alkaloids as their main constituents. Piperine, dihydropiperine, piperylin, dihydropiperylin or piperlonguminine, dihydropiperlonguminine, wisanine, dihydrowisanine and derivatives of piperine and piperidine were identified in a hexane extract of the leaf. In addition, some new piperamide alkaloids were identified, such as a piperine and a piperidine alkaloid derivative and two unknown piperamide alkaloids. To the best of our knowledge, there are no piperamides reported in the literature with similar UVλ absorption maxima and masses. A piperamide alkaloid-rich hexane leaf extract recorded the lowest MIC of 19 µg/mL against Sarcina sp. and gave promising growth inhibitory effects against S. aureus and E. aerogenes as well, inhibiting the growth of both bacteria with a MIC of 78 µg/mL. Moreover, this is the first report of the antibacterial activity of P. guineense extracts against Sarcina sp. and E. aerogenes. Marked growth inhibition was also obtained for chloroform extracts of the leaves and fruits against P. aeruginosa with a MIC value of 78 µg/mL. Piperine and piperlongumine were active against E. aerogenes, S. aureus, E. coli, S. enterica, P. mirabilis and B. cereus with MIC values ranging from 39–1250 µg/mL. Notably, the water extracts, which were almost devoid of piperamide alkaloids, were not active against the bacterial strains. Our results demonstrate that P. guineense contains antibacterial alkaloids that could be relevant for the discovery of new natural antibiotics.

scholarly journals Studies on genes expression pattern of antioxidant enzymes and enzymes involved into the genetic information implementation in E.coli cells due to the antibiotic resistance against apramycin and cefatoxime

2020 ◽  
Vol 17 ◽  
pp. 00103
Author(s):  
Oleg Fomenko ◽  
Evgeny Mikhailov ◽  
Nadezhda Pasko ◽  
Svetlana Grin ◽  
Andrey Koshchaev ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Antioxidant Enzymes ◽  
Genetic Information ◽  
Bacterial Resistance ◽  
Resistant Bacteria ◽  
Control Group ◽  
Bacterial Cells ◽  
E Coli ◽  
Antimicrobial Substances ◽  
Dna And Rna

The emergence of antibiotic-resistant bacteria is considered a serious problem. The resistance of bacteria against antimicrobial substances becomes important in the repair systems for damage to DNA and RNA molecules. The role of the antioxidant system in the development of bacterial resistance against antibiotics is not yet practically studied. The article studied the expression regulation of the genes of antioxidant enzymes and enzymes involved in the genetic information in E. coli cells with the antibiotic resistance against apramycin and cefatoxime. The study was conducted on bacterial cells resistant against these two antibiotics. The genes blaOXA-1, blaSHV, blaTEM, mdtK, aadA1, aadA2, sat, strA, blaCTX, blaPER-2, tnpA, tnpR, intC1 and intC1c were identified in bacterial cell case. This indicates the presence of plasmids in bacteria with these genes, which provide bacterial resistance to apramycin and cefatoxime. It was established that during the formation of cefotaxime resistance, there was a sharp increase in the expression of the Cu, Zn superoxide dismutase gene: in comparison with the control group, the representation of its transcripts increased 141.04 times for cefotoxime and 155.42 times for apramycin. It has been established that during the formation of resistance to the studied antibiotics in E. coli, an increase in the expression of the end4 and end3 genes is observed. There is tendency toward an increase in the number of transcripts of the pol3E gene observed in the formation of resistance against cefotaxime and apromycin.

scholarly journals Mycobacterial resistance to zinc poisoning requires assembly of P-ATPase-containing membrane metal efflux platforms

2021 ◽  
Author(s):  
Yves-Marie Boudehen ◽  
Marion Faucher ◽  
Xavier Marechal ◽  
Roger Miras ◽  
Jerome Rech ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Metal Binding ◽  
Bacterial Resistance ◽  
Binding Motif ◽  
Membrane Microdomain ◽  
High Zinc ◽  
Functional Membrane ◽  
Zinc Concentrations ◽  
Metal Efflux ◽  
High Concentrations

Transition metals are toxic at high concentrations. The P1B-ATPase metal exporter CtpC/Rv3270 is required for resistance to zinc poisoning in the human pathogen Mycobacterium tuberculosis. Here, we discovered that zinc resistance also depends on the chaperone-like protein PacL1/Rv3269. PacL1 bound Zn2+, but unlike PacL1 and CtpC, the PacL1 metal-binding motif (MBM) was required only at high zinc concentrations. PacL1 co-localized with CtpC in dynamic microdomains within the mycobacterial plasma membrane. Microdomain formation did not require flotillins nor the PacL1 MBM. Instead, loss of the PacL1 Glutamine/Alanine repeats led to loss of CtpC and sensitivity to zinc. PacL1 and CtpC are within the same operon, and homologous PacL1-P1B-ATPase pairs are widely distributed within and across prokaryotes. PacL1 colocalized and functioned redundantly with PacL orthologs in Mycobacterium tuberculosis. Overall, our study suggests that PacL proteins are scaffolds that assemble P-ATPase-containing metal efflux platforms, a novel type of functional membrane microdomain that underlies bacterial resistance to metal poisoning.

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