Publications by authors named "Jean-Denis Docquier"

85 Publications

4-Alkyl-1,2,4-triazole-3-thione analogues as metallo-β-lactamase inhibitors.

Bioorg Chem 2021 08 26;113:105024. Epub 2021 May 26.

Institut des Biomolécules Max Mousseron, UMR5247 CNRS, Université de Montpellier, ENSCM, Faculté de Pharmacie, 34093 Montpellier Cedex 5, France. Electronic address:

In Gram-negative bacteria, the major mechanism of resistance to β-lactam antibiotics is the production of one or several β-lactamases (BLs), including the highly worrying carbapenemases. Whereas inhibitors of these enzymes were recently marketed, they only target serine-carbapenemases (e.g. KPC-type), and no clinically useful inhibitor is available yet to neutralize the class of metallo-β-lactamases (MBLs). We are developing compounds based on the 1,2,4-triazole-3-thione scaffold, which binds to the di-zinc catalytic site of MBLs in an original fashion, and we previously reported its promising potential to yield broad-spectrum inhibitors. However, up to now only moderate antibiotic potentiation could be observed in microbiological assays and further exploration was needed to improve outer membrane penetration. Here, we synthesized and characterized a series of compounds possessing a diversely functionalized alkyl chain at the 4-position of the heterocycle. We found that the presence of a carboxylic group at the extremity of an alkyl chain yielded potent inhibitors of VIM-type enzymes with K values in the μM to sub-μM range, and that this alkyl chain had to be longer or equal to a propyl chain. This result confirmed the importance of a carboxylic function on the 4-substituent of 1,2,4-triazole-3-thione heterocycle. As observed in previous series, active compounds also preferentially contained phenyl, 2-hydroxy-5-methoxyphenyl, naphth-2-yl or m-biphenyl at position 5. However, none efficiently inhibited NDM-1 or IMP-1. Microbiological study on VIM-2-producing E. coli strains and on VIM-1/VIM-4-producing multidrug-resistant K. pneumoniae clinical isolates gave promising results, suggesting that the 1,2,4-triazole-3-thione scaffold worth continuing exploration to further improve penetration. Finally, docking experiments were performed to study the binding mode of alkanoic analogues in the active site of VIM-2.
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http://dx.doi.org/10.1016/j.bioorg.2021.105024DOI Listing
August 2021

Discovery of , a Novel Broad-Spectrum Serine β-Lactamase Inhibitor of the Diazabicyclooctane Class, Which Strongly Potentiates Meropenem Activity against Carbapenem-Resistant Enterobacterales and .

J Med Chem 2020 12 11;63(24):15802-15820. Epub 2020 Dec 11.

Antabio SAS, 436 rue Pierre et Marie Curie, 31670 Labège, France.

The diazabicyclooctanes (DBOs) are a class of serine β-lactamase (SBL) inhibitors that use a strained urea moiety as the warhead to react with the active serine residue in the active site of SBLs. The first in-class drug, avibactam, as well as several other recently approved DBOs (e.g., relebactam) or those in clinical development (e.g., nacubactam and zidebactam) potentiate activity of β-lactam antibiotics, to various extents, against carbapenem-resistant Enterobacterales (CRE) carrying class A, C, and D SBLs; however, none of these are able to rescue the activity of β-lactam antibiotics against carbapenem-resistant (CRAB), a WHO "critical priority pathogen" producing class D OXA-type SBLs. Herein, we describe the chemical optimization and resulting structure-activity relationship, leading to the discovery of a novel DBO, , which uniquely has a fluorine atom replacing the carboxamide and stands apart from the current DBOs in restoring carbapenem activity against OXA-CRAB as well as SBL-carrying CRE pathogens.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01535DOI Listing
December 2020

Intermolecular interactions of the extended recognition site of VIM-2 metallo-β-lactamase with 1,2,4-triazole-3-thione inhibitors. Validations of a polarizable molecular mechanics potential by ab initio QC.

J Comput Chem 2021 01 10;42(2):86-106. Epub 2020 Nov 10.

Laboratoire de Chimie Théorique, Paris, France.

Molecular dynamics on the complexes of inhibitors with Zn-metalloproteins are a privileged area of applications of polarizable molecular mechanics potentials. With which accuracy could these reproduce the QC intermolecular interaction energies in the two mono-zinc cores and in the dizinc core, toward full-fledged MD simulations on the entire protein complexes? We considered the complexes of the extended recognition site of a Zn-dependent metallo-β-lactamase, VIM-2, produced by bacteria responsible for nosocomial infections, with five newly synthesized inhibitors sharing an original dizinc binding group, 1,2,4-triazole-3-thione (TZT). We considered the energy-minimized structures of each of the five VIM-2 complexes obtained with the SIBFA potential. Energy decomposition analyses (EDA) at the HF level enabled to compare the QC and the SIBFA ΔE values and their contributions in the zinc cores, with and without TZT, totaling 30 complexes. With one exception, the ΔE(QC) values were reproduced with relative errors <1.5%. We next considered the complex of the entire inhibitors with an extended model of VIM-2 recognition site, totaling up to 280 atoms. ΔE(SIBFA) could closely reproduce ΔE(QC). EDA analyses were resumed on the complexes of each inhibitor arm with its interacting VIM-2 residues. As a last step, EDA results at correlated levels were analyzed for the mono- and dizinc sites enabling comparisons with dispersion-augmented ΔE(SIBFA) and correlated multipoles and polarizabilities. Closely reproducing ΔE(QC) and the contrasting trends of its individual contributions should enable for dependable free energy perturbation studies and comparisons to recent experimental ΔG values, limiting as much as possible the reliance on error compensations.
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http://dx.doi.org/10.1002/jcc.26437DOI Listing
January 2021

4-Amino-1,2,4-triazole-3-thione-derived Schiff bases as metallo-β-lactamase inhibitors.

Eur J Med Chem 2020 Dec 20;208:112720. Epub 2020 Aug 20.

Institut des Biomolécules Max Mousseron, UMR5247 CNRS, Université de Montpellier, ENSCM, Faculté de Pharmacie, 34093, Montpellier Cedex 5, France. Electronic address:

Resistance to β-lactam antibiotics in Gram-negatives producing metallo-β-lactamases (MBLs) represents a major medical threat and there is an extremely urgent need to develop clinically useful inhibitors. We previously reported the original binding mode of 5-substituted-4-amino/H-1,2,4-triazole-3-thione compounds in the catalytic site of an MBL. Moreover, we showed that, although moderately potent, they represented a promising basis for the development of broad-spectrum MBL inhibitors. Here, we synthesized and characterized a large number of 4-amino-1,2,4-triazole-3-thione-derived Schiff bases. Compared to the previous series, the presence of an aryl moiety at position 4 afforded an average 10-fold increase in potency. Among 90 synthetic compounds, more than half inhibited at least one of the six tested MBLs (L1, VIM-4, VIM-2, NDM-1, IMP-1, CphA) with K values in the μM to sub-μM range. Several were broad-spectrum inhibitors, also inhibiting the most clinically relevant VIM-2 and NDM-1. Active compounds generally contained halogenated, bicyclic aryl or phenolic moieties at position 5, and one substituent among o-benzoic, 2,4-dihydroxyphenyl, p-benzyloxyphenyl or 3-(m-benzoyl)-phenyl at position 4. The crystallographic structure of VIM-2 in complex with an inhibitor showed the expected binding between the triazole-thione moiety and the dinuclear centre and also revealed a network of interactions involving Phe61, Tyr67, Trp87 and the conserved Asn233. Microbiological analysis suggested that the potentiation activity of the compounds was limited by poor outer membrane penetration or efflux. This was supported by the ability of one compound to restore the susceptibility of an NDM-1-producing E. coli clinical strain toward several β-lactams in the presence only of a sub-inhibitory concentration of colistin, a permeabilizing agent. Finally, some compounds were tested against the structurally similar di-zinc human glyoxalase II and found weaker inhibitors of the latter enzyme, thus showing a promising selectivity towards MBLs.
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http://dx.doi.org/10.1016/j.ejmech.2020.112720DOI Listing
December 2020

ANT2681: SAR Studies Leading to the Identification of a Metallo-β-lactamase Inhibitor with Potential for Clinical Use in Combination with Meropenem for the Treatment of Infections Caused by NDM-Producing .

ACS Infect Dis 2020 09 20;6(9):2419-2430. Epub 2020 Aug 20.

Antabio SAS, 436 rue Pierre et Marie Curied, 31670 Labège, France.

The clinical effectiveness of the important β-lactam class of antibiotics is under threat by the emergence of resistance, mostly due to the production of acquired serine- (SBL) and metallo-β-lactamase (MBL) enzymes. To address this resistance issue, multiple β-lactam/β-lactamase inhibitor combinations have been successfully introduced into the clinic over the past several decades. However, all of those combinations contain SBL inhibitors and, as yet, there are no MBL inhibitors in clinical use. Consequently, there exists an unaddressed yet growing healthcare problem due to the rise in recent years of highly resistant strains which produce New Delhi metallo (NDM)-type metallo-carbapenemases. Previously, we reported the characterization of an advanced MBL inhibitor lead compound, ANT431. Herein, we discuss the completion of a lead optimization campaign culminating in the discovery of the preclinical candidate ANT2681, a potent NDM inhibitor with strong potential for clinical development.
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http://dx.doi.org/10.1021/acsinfecdis.0c00207DOI Listing
September 2020

Virtual screening identifies broad-spectrum β-lactamase inhibitors with activity on clinically relevant serine- and metallo-carbapenemases.

Sci Rep 2020 07 29;10(1):12763. Epub 2020 Jul 29.

Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 103, 41125, Modena, Italy.

Bacteria are known to evade β-lactam antibiotic action by producing β-lactamases (BLs), including carbapenemases, which are able to hydrolyze nearly all available β-lactams. The production of BLs represents one of the best known and most targeted mechanisms of resistance in bacteria. We have performed the parallel screening of commercially available compounds against a panel of clinically relevant BLs: class A CTX-M-15 and KPC-2, subclass B1 NDM-1 and VIM-2 MBLs, and the class C P. aeruginosa AmpC. The results show that all BLs prefer scaffolds having electron pair donors: KPC-2 is preferentially inhibited by sulfonamide and tetrazole-based derivatives, NDM-1 by compounds bearing a thiol, a thiosemicarbazide or thiosemicarbazone moiety, while VIM-2 by triazole-containing molecules. Few broad-spectrum BLs inhibitors were identified; among these, compound 40 potentiates imipenem activity against an NDM-1-producing E. coli clinical strain. The binary complexes of the two most promising compounds binding NDM-1 and VIM-2 were obtained at high resolution, providing strong insights to improve molecular docking simulations, especially regarding the interaction of MBLs with inhibitors.
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http://dx.doi.org/10.1038/s41598-020-69431-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7391774PMC
July 2020

4-(-Alkyl- and -Acyl-amino)-1,2,4-triazole-3-thione Analogs as Metallo-β-Lactamase Inhibitors: Impact of 4-Linker on Potency and Spectrum of Inhibition.

Biomolecules 2020 07 23;10(8). Epub 2020 Jul 23.

Institut des Biomolécules Max Mousseron, UMR5247 CNRS, Université de Montpellier, ENSCM, Faculté de Pharmacie, 34093 Montpellier, France.

To fight the increasingly worrying bacterial resistance to antibiotics, the discovery and development of new therapeutics is urgently needed. Here, we report on a new series of 1,2,4-triazole-3-thione compounds as inhibitors of metallo-β-lactamases (MBLs), which represent major resistance determinants to β-lactams, and especially carbapenems, in Gram-negative bacteria. These molecules are stable analogs of 4-amino-1,2,4-triazole-derived Schiff bases, where the hydrazone-like bond has been reduced (hydrazine series) or the 4-amino group has been acylated (hydrazide series); the synthesis and physicochemical properties thereof are described. The inhibitory potency was determined on the most clinically relevant acquired MBLs (IMP-, VIM-, and NDM-types subclass B1 MBLs). When compared with the previously reported hydrazone series, hydrazine but not hydrazide analogs showed similarly potent inhibitory activity on VIM-type enzymes, especially VIM-2 and VIM-4, with values in the micromolar to submicromolar range. One of these showed broad-spectrum inhibition as it also significantly inhibited VIM-1 and NDM-1. Restoration of β-lactam activity in microbiological assays was observed for one selected compound. Finally, the binding to the VIM-2 active site was evaluated by isothermal titration calorimetry and a modeling study explored the effect of the linker structure on the mode of binding with this MBL.
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http://dx.doi.org/10.3390/biom10081094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7465886PMC
July 2020

Screen of Unfocused Libraries Identified Compounds with Direct or Synergistic Antibacterial Activity.

ACS Med Chem Lett 2020 May 6;11(5):899-905. Epub 2020 Mar 6.

Dipartimento di Biotecnologie Mediche, Università degli Studi di Siena, Viale Bracci 16, 53100 Siena, Italy.

Antibiotic resistance is an increasingly important global public health issue, as major opportunistic pathogens are evolving toward multidrug- and pan-drug resistance phenotypes. New antibiotics are thus needed to maintain our ability to treat bacterial infections. According to the WHO, carbapenem-resistant , , and are the most critical targets for the development of new antibacterial drugs. An automated phenotypic screen was implemented to screen 634 synthetic compounds obtained in-house for both their direct-acting and synergistic activity. Fourteen percent and 10% of the compounds showed growth inhibition against tested Gram-positive and Gram-negative bacteria, respectively. The most active direct-acting compounds showed a broad-spectrum antibacterial activity, including on some multidrug-resistant clinical isolates. In addition, 47 compounds were identified for their ability to potentiate the activity of other antibiotics. Compounds of three different scaffolds (2-quinolones, phenols, and pyrazoles) showed a strong potentiation of colistin, some being able to revert colistin resistance in .
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http://dx.doi.org/10.1021/acsmedchemlett.9b00674DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7236257PMC
May 2020

Isonitrile-Based Multicomponent Synthesis of β-Amino Boronic Acids as β-Lactamase Inhibitors.

Antibiotics (Basel) 2020 May 12;9(5). Epub 2020 May 12.

Dipartimento di Chimica, Università degli Studi di Milano, Via Golgi 19, 20133 Milan, Italy.

The application of various isonitrile-based multicomponent reactions to protected (2-oxoethyl)boronic acid (as the carbonyl component) is described. The Ugi reaction, both in the four components and in the four centers-three components versions, and the van Leusen reaction, proved effective at providing small libraries of MIDA-protected β-aminoboronic acids. The corresponding free β-aminoboronic acids, quantitatively recovered through basic mild deprotection, were found to be quite stable and were fully characterized, including by B-NMR spectroscopy. Single-crystal X-ray diffraction analysis, applied both to a MIDA-protected and a free β-aminoboronic acid derivative, provided evidence for different conformations in the solid-state. Finally, the antimicrobial activities of selected compounds were evaluated by measuring their minimal inhibitory concentration (MIC) values, and the binding mode of the most promising derivative on OXA-23 class D β-lactamase was predicted by a molecular modeling study.
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http://dx.doi.org/10.3390/antibiotics9050249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7277116PMC
May 2020

VNRX-5133 (Taniborbactam), a Broad-Spectrum Inhibitor of Serine- and Metallo-β-Lactamases, Restores Activity of Cefepime in and Pseudomonas aeruginosa.

Antimicrob Agents Chemother 2020 02 21;64(3). Epub 2020 Feb 21.

Venatorx Pharmaceuticals Incorporated, Malvern, Pennsylvania, USA

As shifts in the epidemiology of β-lactamase-mediated resistance continue, carbapenem-resistant (CRE) and carbapenem-resistant (CRPA) are the most urgent threats. Although approved β-lactam (BL)-β-lactamase inhibitor (BLI) combinations address widespread serine β-lactamases (SBLs), such as CTX-M-15, none provide broad coverage against either clinically important serine-β-lactamases (KPC, OXA-48) or clinically important metallo-β-lactamases (MBLs; e.g., NDM-1). VNRX-5133 (taniborbactam) is a new cyclic boronate BLI that is in clinical development combined with cefepime for the treatment of infections caused by β-lactamase-producing CRE and CRPA. Taniborbactam is the first BLI with direct inhibitory activity against Ambler class A, B, C, and D enzymes. From biochemical and structural analyses, taniborbactam exploits substrate mimicry while employing distinct mechanisms to inhibit both SBLs and MBLs. It is a reversible covalent inhibitor of SBLs with slow dissociation and a prolonged active-site residence time (half-life, 30 to 105 min), while in MBLs, it behaves as a competitive inhibitor, with inhibitor constant ( ) values ranging from 0.019 to 0.081 μM. Inhibition is achieved by mimicking the transition state structure and exploiting interactions with highly conserved active-site residues. In microbiological testing, taniborbactam restored cefepime activity in 33/34 engineered strains overproducing individual enzymes covering Ambler classes A, B, C, and D, providing up to a 1,024-fold shift in the MIC. Addition of taniborbactam restored the antibacterial activity of cefepime against all 102 clinical isolates tested and 38/41 clinical isolates tested with MICs of 1 and 4 μg/ml, respectively, representing ≥256- and ≥32-fold improvements, respectively, in antibacterial activity over that of cefepime alone. The data demonstrate the potent, broad-spectrum rescue of cefepime activity by taniborbactam against clinical isolates of CRE and CRPA.
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http://dx.doi.org/10.1128/AAC.01963-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7038240PMC
February 2020

Discovery of Taniborbactam (VNRX-5133): A Broad-Spectrum Serine- and Metallo-β-lactamase Inhibitor for Carbapenem-Resistant Bacterial Infections.

J Med Chem 2020 03 16;63(6):2789-2801. Epub 2019 Dec 16.

Venatorx Pharmaceuticals, Inc., 30 Spring Mill Drive, Malvern, Pennsylvania 19355, United States.

A major resistance mechanism in Gram-negative bacteria is the production of β-lactamase enzymes. Originally recognized for their ability to hydrolyze penicillins, emergent β-lactamases can now confer resistance to other β-lactam drugs, including both cephalosporins and carbapenems. The emergence and global spread of β-lactamase-producing multi-drug-resistant "superbugs" has caused increased alarm within the medical community due to the high mortality rate associated with these difficult-to-treat bacterial infections. To address this unmet medical need, we initiated an iterative program combining medicinal chemistry, structural biology, biochemical testing, and microbiological profiling to identify broad-spectrum inhibitors of both serine- and metallo-β-lactamase enzymes. Lead optimization, beginning with narrower-spectrum, weakly active compounds, provided (VNRX-5133, taniborbactam), a boronic-acid-containing pan-spectrum β-lactamase inhibitor. In vitro and in vivo studies demonstrated that restored the activity of β-lactam antibiotics against carbapenem-resistant and carbapenem-resistant Enterobacteriaceae. Taniborbactam is the first pan-spectrum β-lactamase inhibitor to enter clinical development.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7104248PMC
March 2020

A Standard Numbering Scheme for Class C β-Lactamases.

Antimicrob Agents Chemother 2020 02 21;64(3). Epub 2020 Feb 21.

Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio, USA

Unlike for classes A and B, a standardized amino acid numbering scheme has not been proposed for the class C (AmpC) β-lactamases, which complicates communication in the field. Here, we propose a scheme developed through a collaborative approach that considers both sequence and structure, preserves traditional numbering of catalytically important residues (Ser, Lys, Tyr, and Lys), is adaptable to new variants or enzymes yet to be discovered and includes a variation for genetic and epidemiological applications.
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http://dx.doi.org/10.1128/AAC.01841-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7038296PMC
February 2020

SAR Studies Leading to the Identification of a Novel Series of Metallo-β-lactamase Inhibitors for the Treatment of Carbapenem-Resistant Enterobacteriaceae Infections That Display Efficacy in an Animal Infection Model.

ACS Infect Dis 2019 01 30;5(1):131-140. Epub 2018 Nov 30.

Antabio SAS , 436 rue Pierre et Marie Curie , 31670 Labège , France.

The clinical effectiveness of carbapenem antibiotics such as meropenem is becoming increasingly compromised by the spread of both metallo-β-lactamase (MBL) and serine-β-lactamase (SBL) enzymes on mobile genetic elements, stimulating research to find new β-lactamase inhibitors to be used in conjunction with carbapenems and other β-lactam antibiotics. Herein, we describe our initial exploration of a novel chemical series of metallo-β-lactamase inhibitors, from concept to efficacy, in a survival model using an advanced tool compound (ANT431) in conjunction with meropenem.
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http://dx.doi.org/10.1021/acsinfecdis.8b00246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6332448PMC
January 2019

Alkyl-guanidine Compounds as Potent Broad-Spectrum Antibacterial Agents: Chemical Library Extension and Biological Characterization.

J Med Chem 2018 10 11;61(20):9162-9176. Epub 2018 Oct 11.

Department of Biotechnology, Chemistry, and Pharmacy , University of Siena , I-53100 Siena , Italy.

Nowadays, the increasing of multidrug-resistant pathogenic bacteria represents a serious threat to public health, and the lack of new antibiotics is becoming a global emergency. Therefore, research in antibacterial fields is urgently needed to expand the currently available arsenal of drugs. We have recently reported an alkyl-guanidine derivative (2), characterized by a symmetrical dimeric structure, as a good candidate for further developments, with a high antibacterial activity against both Gram-positive and Gram-negative strains. In this study, starting from its chemical scaffold, we synthesized a small library of analogues. Moreover, biological and in vitro pharmacokinetic characterizations were conducted on some selected derivatives, revealing notable properties: broad-spectrum profile, activity against resistant clinical isolates, and appreciable aqueous solubility. Interestingly, 2 seems neither to select for resistant strains nor to macroscopically alter the membranes, but further studies are required to determine the mode of action.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00619DOI Listing
October 2018

Type M Resistance to Macrolides Is Due to a Two-Gene Efflux Transport System of the ATP-Binding Cassette (ABC) Superfamily.

Front Microbiol 2018 31;9:1670. Epub 2018 Jul 31.

Department of Medical Biotechnologies, University of Siena, Siena, Italy.

The (A) gene was originally identified as the resistance determinant responsible for type M resistance to macrolides, a phenotype frequently found in clinical isolates of and . MefA was defined as a secondary transporter of the major facilitator superfamily driven by proton-motive force. However, when characterizing the (A)-carrying elements Tn and Φ1207.3, another macrolide resistance gene, (D), was found adjacent to (A). To define the respective contribution of (A) and (D) to macrolide resistance, three isogenic deletion mutants were constructed by transformation of a strain carrying Φ1207.3: (i) Δ(A)-Δ(D); (ii) Δ(A)-(D); and (iii) (A)-Δ(D). Susceptibility testing of mutants clearly showed that (D) is required for macrolide resistance, while deletion of (A) produced only a twofold reduction in the minimal inhibitory concentration (MIC) for erythromycin. The contribution of (D) to macrolide resistance was also studied in , which is the original host of Φ1207.3. Two isogenic strains of were constructed: (i) FR156, carrying Φ1207.3, and (ii) FR155, carrying Φ1207.3/Δ(D). FR155 was susceptible to erythromycin, whereas FR156 was resistant, with an MIC value of 8 μg/ml. Complementation experiments showed that reintroduction of the (D) gene could restore macrolide resistance in Δ(D) mutants. Radiolabeled erythromycin was retained by strains lacking (D), while (D)-carrying strains showed erythromycin efflux. Deletion of (A) did not affect erythromycin efflux. This data suggest that type M resistance to macrolides in streptococci is due to an efflux transport system of the ATP-binding cassette (ABC) superfamily, in which (A) encodes the transmembrane channel, and (D) the two ATP-binding domains.
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http://dx.doi.org/10.3389/fmicb.2018.01670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6079230PMC
July 2018

Atomic-Resolution Structure of a Class C β-Lactamase and Its Complex with Avibactam.

ChemMedChem 2018 07 20;13(14):1437-1446. Epub 2018 Jun 20.

Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via Aldo Moro 2, 53100, Siena, Italy.

β-Lactamases (BLs) are important antibiotic-resistance determinants that significantly compromise the efficacy of valuable β-lactam antibacterial drugs. Thus, combinations with BL inhibitor were developed. Avibactam is the first non-β-lactam BL inhibitor introduced into clinical practice. Ceftazidime-avibactam represents one of the few last-resort antibiotics available for the treatment of infections caused by near-pandrug-resistant bacteria. TRU-1 is a chromosomally encoded AmpC-type BL of Aeromonas enteropelogenes, related to the FOX-type BLs and constitutes a good model for class C BLs. TRU-1 crystals provided ultrahigh-resolution diffraction data for the native enzyme and for its complex with avibactam. A comparison of the native and avibactam-bound structures revealed new details in the conformations of residues relevant for substrate and/or inhibitor binding. Furthermore, a comparison of the TRU-1 and Pseudomonas aeruginosa AmpC avibactam-bound structures revealed two inhibitor conformations that were likely to correspond to two different states occurring during inhibitor carbamylation/recyclization.
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http://dx.doi.org/10.1002/cmdc.201800213DOI Listing
July 2018

Discovery of a Novel Metallo-β-Lactamase Inhibitor That Potentiates Meropenem Activity against Carbapenem-Resistant Enterobacteriaceae.

Antimicrob Agents Chemother 2018 05 26;62(5). Epub 2018 Apr 26.

Antabio SAS, Labège, France.

Infections caused by carbapenem-resistant (CRE) are increasingly prevalent and have become a major worldwide threat to human health. Carbapenem resistance is driven primarily by the acquisition of β-lactamase enzymes, which are able to degrade carbapenem antibiotics (hence termed carbapenemases) and result in high levels of resistance and treatment failure. Clinically relevant carbapenemases include both serine β-lactamases (SBLs; e.g., KPC-2 and OXA-48) and metallo-β-lactamases (MBLs), such as NDM-1. MBL-producing strains are endemic within the community in many Asian countries, have successfully spread worldwide, and account for many significant CRE outbreaks. Recently approved combinations of β-lactam antibiotics with β-lactamase inhibitors are active only against SBL-producing pathogens. Therefore, new drugs that specifically target MBLs and which restore carbapenem efficacy against MBL-producing CRE pathogens are urgently needed. Here we report the discovery of a novel MBL inhibitor, ANT431, that can potentiate the activity of meropenem (MEM) against a broad range of MBL-producing CRE and restore its efficacy against an NDM-1-producing strain in a murine thigh infection model. This is a strong starting point for a chemistry lead optimization program that could deliver a first-in-class MBL inhibitor-carbapenem combination. This would complement the existing weaponry against CRE and address an important and growing unmet medical need.
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http://dx.doi.org/10.1128/AAC.00074-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5923143PMC
May 2018

An update on β-lactamase inhibitor discovery and development.

Drug Resist Updat 2018 01 7;36:13-29. Epub 2017 Nov 7.

Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via Aldo Moro 2, 53100 Siena, Italy. Electronic address:

Antibiotic resistance, and the emergence of pan-resistant clinical isolates, seriously threatens our capability to treat bacterial diseases, including potentially deadly hospital-acquired infections. This growing issue certainly requires multiple adequate responses, including the improvement of both diagnosis methods and use of antibacterial agents, and obviously the development of novel antibacterial drugs, especially active against Gram-negative pathogens, which represent an urgent medical need. Considering the clinical relevance of both β-lactam antibiotics and β-lactamase-mediated resistance, the discovery and development of combinations including a β-lactamase inhibitor seems to be particularly attractive, despite being extremely challenging due to the enormous diversity, both structurally and mechanistically, of the potential β-lactamase targets. This review will cover the evolution of currently available β-lactamase inhibitors along with the most recent research leading to new β-lactamase inhibitors of potential clinical interest or already in the stage of clinical development.
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http://dx.doi.org/10.1016/j.drup.2017.11.002DOI Listing
January 2018

Deciphering Multifactorial Resistance Phenotypes in by Genomics and Targeted Label-free Proteomics.

Mol Cell Proteomics 2018 03 19;17(3):442-456. Epub 2017 Dec 19.

From the ‡Technology Research Department, Innovation Unit, bioMérieux SA, Marcy l'Etoile, France;

Resistance to β-lactams in involves various mechanisms. To decipher them, whole genome sequencing (WGS) and real-time quantitative polymerase chain reaction (RT-qPCR) were complemented by mass spectrometry (MS) in selected reaction monitoring mode (SRM) in 39 clinical isolates. The targeted label-free proteomic approach enabled, in one hour and using a single method, the quantitative detection of 16 proteins associated with antibiotic resistance: eight acquired β-lactamases ( GES, NDM-1, OXA-23, OXA-24, OXA-58, PER, TEM-1, and VEB), two resident β-lactamases ( ADC and OXA-51-like) and six components of the two major efflux systems ( AdeABC and AdeIJK). Results were normalized using "bacterial quantotypic peptides," peptide markers of the bacterial quantity, to obtain precise protein quantitation (on average 8.93% coefficient of variation for three biological replicates). This allowed to correlate the levels of resistance to β-lactam with those of the production of acquired as well as resident β-lactamases or of efflux systems. SRM detected enhanced ADC or OXA-51-like production and absence or increased efflux pump production. Precise protein quantitation was particularly valuable to detect resistance mechanisms mediated by regulated genes or by overexpression of chromosomal genes. Combination of WGS and MS, two orthogonal and complementary techniques, allows thereby interpretation of the resistance phenotypes at the molecular level.
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http://dx.doi.org/10.1074/mcp.RA117.000107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5836370PMC
March 2018

Computational and biological profile of boronic acids for the detection of bacterial serine- and metallo-β-lactamases.

Sci Rep 2017 12 18;7(1):17716. Epub 2017 Dec 18.

Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 103, 41125, Modena, Italy.

β-Lactamases (BLs) able to hydrolyze β-lactam antibiotics and more importantly the last resort carbapenems, represent a major mechanism of resistance in Gram-negative bacteria showing multi-drug or extensively drug resistant phenotypes. The early detection of BLs responsible of resistant infections is challenging: approaches aiming at the identification of new BLs inhibitors (BLI) can thus serve as the basis for the development of highly needed diagnostic tools. Starting from benzo-[b]-thiophene-2-boronic acid (BZB), a nanomolar inhibitor of AmpC β-lactamase (K  = 27 nM), we have identified and characterized a set of BZB analogues able to inhibit clinically-relevant β-lactamases, including AmpC, Extended-Spectrum BLs (ESBL), KPC- and OXA-type carbapenemases and metallo-β-lactamases (MBL). A multiligand set of boronic acid (BA) β-lactamase inhibitors was obtained using covalent molecular modeling, synthetic chemistry, enzyme kinetics and antibacterial susceptibility testing. Data confirmed the possibility to discriminate between clinically-relevant β-lactamases on the basis of their inhibition profile. Interestingly, this work also allowed the identification of potent KPC-2 and NDM-1 inhibitors able to potentiate the activity of cefotaxime (CTX) and ceftazidime (CAZ) against resistant clinical isolates (MIC reduction, 32-fold). Our results open the way to the potential use of our set of compounds as a diagnostic tool for the sensitive detection of clinically-relevant β-lactamases.
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http://dx.doi.org/10.1038/s41598-017-17399-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5735191PMC
December 2017

Design and synthesis of a novel inhibitor of T. Viride chitinase through an in silico target fishing protocol.

Bioorg Med Chem Lett 2017 08 10;27(15):3332-3336. Epub 2017 Jun 10.

Department of Biotechnology, Chemistry and Pharmacy, University of Siena, I-53100 Siena, Italy; Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, BioLife Science Building, Suite 333, 1900 N 12th Street, Philadelphia, PA 19122, USA; Lead Discovery Siena s.r.l, Via Vittorio Alfieri 31, I-53019 Castelnuovo Berardenga, Italy. Electronic address:

In the last ten years, we identified and developed a new therapeutic class of antifungal agents, the macrocyclic amidinoureas. These compounds are active against several Candida species, including clinical isolates resistant to currently available antifungal drugs. The mode of action of these molecules is still unknown. In this work, we developed an in-silico target fishing procedure to identify a possible target for this class of compounds based on shape similarity, inverse docking procedure and consensus score rank-by-rank. Chitinase enzyme emerged as possible target. To confirm this hypothesis a novel macrocyclic derivative has been produced, specifically designed to increase the inhibition of the chitinase. Biological evaluation highlights a stronger enzymatic inhibition for the new derivative, while its antifungal activity drops probably because of pharmacokinetic issues. Collectively, our data suggest that chitinase represent at least one of the main target of macrocyclic amidinoureas.
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http://dx.doi.org/10.1016/j.bmcl.2017.06.016DOI Listing
August 2017

Genetic and biochemical characterisation of CTX-M-37 extended-spectrum β-lactamase from an Enterobacter cloacae clinical isolate from Mongolia.

J Glob Antimicrob Resist 2017 09 3;10:3-7. Epub 2017 Jun 3.

Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 03722, South Korea. Electronic address:

Objectives: The aims of this study were to determine the resistance level of a bla-carrying Enterobacter cloacae isolate from Mongolia, to analyse kinetic parameters of the purified enzyme and to compare the genetic environment of the gene.

Methods: Minimum inhibitory concentrations (MICs) were determined using the Clinical and Laboratory Standards Institute (CLSI) agar dilution method. Purified CTX-M-37 enzyme was used to determined kinetic parameters. The genetic environment of the blaCTX-M-37 gene in E. cloacae was compared with a Kluyvera cryocrescens isolate.

Results: The E. cloacae isolate showed relatively low-level resistance to cefotaxime (MIC=16mg/L) compared with a CTX-M-3-producing strain (MIC=256mg/L), and CTX-M-37 had a lower k/K value for cefotaxime (2.0μMs) compared with CTX-M-3 (3.5μMs), possibly due to Asn114Asp substitution. The bla gene in the E. cloacae isolate was carried on a conjugative plasmid and was associated with an ISEcp1 element containing the -35 and -10 putative promoter sequences TTGAAA and TACAAT, respectively, unlike in the K. cryocrescens isolate.

Conclusions: The CTX-M-37-producing E. cloacae isolate showed relatively low-level resistance to cefotaxime and the purified enzyme had lower kinetic parameters as the result of Asn114Asp substitution. Presence of an ISEcp1 element and putative promoters upstream of the bla gene in E. cloacae, but not in the K. cryocrescens isolate, indicated their roles in mobilisation and expression of the gene.
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http://dx.doi.org/10.1016/j.jgar.2017.03.007DOI Listing
September 2017

1,2,4-Triazole-3-thione Compounds as Inhibitors of Dizinc Metallo-β-lactamases.

ChemMedChem 2017 06 12;12(12):972-985. Epub 2017 Jun 12.

Institut des Biomolécules Max Mousseron, UMR5247 CNRS, Université de Montpellier, ENSCM, Faculté de Pharmacie, 15 avenue Charles Flahault, 34093, Montpellier cedex 5, France.

Metallo-β-lactamases (MBLs) cause resistance of Gram-negative bacteria to β-lactam antibiotics and are of serious concern, because they can inactivate the last-resort carbapenems and because MBL inhibitors of clinical value are still lacking. We previously identified the original binding mode of 4-amino-2,4-dihydro-5-(2-methylphenyl)-3H-1,2,4-triazole-3-thione (compound IIIA) within the dizinc active site of the L1 MBL. Herein we present the crystallographic structure of a complex of L1 with the corresponding non-amino compound IIIB (1,2-dihydro-5-(2-methylphenyl)-3H-1,2,4-triazole-3-thione). Unexpectedly, the binding mode of IIIB was similar but reverse to that of IIIA. The 3 D structures suggested that the triazole-thione scaffold was suitable to bind to the catalytic site of dizinc metalloenzymes. On the basis of these results, we synthesized 54 analogues of IIIA or IIIB. Nineteen showed IC values in the micromolar range toward at least one of five representative MBLs (i.e., L1, VIM-4, VIM-2, NDM-1, and IMP-1). Five of these exhibited a significant inhibition of at least four enzymes, including NDM-1, VIM-2, and IMP-1. Active compounds mainly featured either halogen or bulky bicyclic aryl substituents. Finally, some compounds were also tested on several microbial dinuclear zinc-dependent hydrolases belonging to the MBL-fold superfamily (i.e., endonucleases and glyoxalase II) to explore their activity toward structurally similar but functionally distinct enzymes. Whereas the bacterial tRNases were not inhibited, the best IC values toward plasmodial glyoxalase II were in the 10 μm range.
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http://dx.doi.org/10.1002/cmdc.201700186DOI Listing
June 2017

Expanding the Repertoire of Carbapenem-Hydrolyzing Metallo-ß-Lactamases by Functional Metagenomic Analysis of Soil Microbiota.

Front Microbiol 2016 26;7:1985. Epub 2016 Dec 26.

Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of CopenhagenFrederiksberg, Denmark; Department of Biomedical Sciences, Ross University School of Veterinary MedicineSt. Kitts, West Indies.

Carbapenemases are bacterial enzymes that hydrolyze carbapenems, a group of last-resort β-lactam antibiotics used for treatment of severe bacterial infections. They belong to three β-lactamase classes based amino acid sequence (A, B, and D). The aim of this study was to elucidate occurrence, diversity and functionality of carbapenemase-encoding genes in soil microbiota by functional metagenomics. Ten plasmid libraries were generated by cloning metagenomic DNA from agricultural ( = 6) and grassland ( = 4) soil into . The libraries were cultured on amoxicillin-containing agar and up to 100 colonies per library were screened for carbapenemase production by CarbaNP test. Presumptive carbapenemases were characterized with regard to DNA sequence, minimum inhibitory concentration (MIC) of β-lactams, and imipenem hydrolysis. Nine distinct class B carbapenemases, also known as metallo-beta-lactamases (MBLs), were identified in six soil samples, including two subclass B1 (GRD23-1 and SPN79-1) and seven subclass B3 (CRD3-1, PEDO-1, GRD33-1, ESP-2, ALG6-1, ALG11-1, and DHT2-1). Except PEDO-1 and ESP-2, these enzymes were distantly related to any previously described MBLs (33 to 59% identity). RAIphy analysis indicated that six enzymes (CRD3-1, GRD23-1, DHT2-1, SPN79-1, ALG6-1, and ALG11-1) originated from , two (PEDO-1 and ESP-2) from and one (GRD33-1) from . All MBLs detected in soil microbiota were functional when expressed in , resulting in detectable imipenem-hydrolyzing activity and significantly increased MICs of clinically relevant ß-lactams. Interestingly, the MBLs yielded by functional metagenomics generally differed from those detected in the same soil samples by antibiotic selective culture, showing that the two approaches targeted different subpopulations in soil microbiota.
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http://dx.doi.org/10.3389/fmicb.2016.01985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5184135PMC
December 2016

Effect of High N-Acetylcysteine Concentrations on Antibiotic Activity against a Large Collection of Respiratory Pathogens.

Antimicrob Agents Chemother 2016 12 21;60(12):7513-7517. Epub 2016 Nov 21.

Department of Medical Biotechnologies, University of Siena, Siena, Italy

The effect of high N-acetylcysteine (NAC) concentrations (10 and 50 mM) on antibiotic activity against 40 strains of respiratory pathogens was investigated. NAC compromised the activity of carbapenems (of mostly imipenem and, to lesser extents, meropenem and ertapenem) in a dose-dependent fashion. We demonstrated chemical instability of carbapenems in the presence of NAC. With other antibiotics, 10 mM NAC had no major effects, while 50 mM NAC sporadically decreased (ceftriaxone and aminoglycosides) or increased (penicillins) antibiotic activity.
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http://dx.doi.org/10.1128/AAC.01334-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5119039PMC
December 2016

Structure-based approach for identification of novel phenylboronic acids as serine-β-lactamase inhibitors.

J Comput Aided Mol Des 2016 10 8;30(10):851-861. Epub 2016 Sep 8.

Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, Via L. Mangiagalli 25, 20133, Milan, Italy.

β-Lactamases are bacterial enzymes conferring resistance to β-lactam antibiotics in clinically-relevant pathogens, and represent relevant drug targets. Recently, the identification of new boronic acids (i.e. RPX7009) paved the way to the clinical application of these molecules as potential drugs. Here, we screened in silico a library of ~1400 boronic acids as potential AmpC β-lactamase inhibitors. Six of the most promising candidates were evaluated in biochemical assays leading to the identification of potent inhibitors of clinically-relevant β-lactamases like AmpC, KPC-2 and CTX-M-15. One of the selected compounds showed nanomolar K value with the clinically-relevant KPC-2 carbapenemase, while another one exhibited broad spectrum inhibition, being also active on Enterobacter AmpC and the OXA-48 class D carbapenemase.
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http://dx.doi.org/10.1007/s10822-016-9962-8DOI Listing
October 2016

Crystal Structure of the Pseudomonas aeruginosa BEL-1 Extended-Spectrum β-Lactamase and Its Complexes with Moxalactam and Imipenem.

Antimicrob Agents Chemother 2016 12 21;60(12):7189-7199. Epub 2016 Nov 21.

Dipartimento di Biotecnologie Mediche, Università di Siena, Siena, Italy

BEL-1 is an acquired class A extended-spectrum β-lactamase (ESBL) found in Pseudomonas aeruginosa clinical isolates from Belgium which is divergent from other ESBLs (maximum identity of 54% with GES-type enzymes). This enzyme is efficiently inhibited by clavulanate, imipenem, and moxalactam. Crystals of BEL-1 were obtained at pH 5.6, and the structure of native BEL-1 was determined from orthorhombic and monoclinic crystal forms at 1.60-Å and 1.48-Å resolution, respectively. By soaking native BEL-1 crystals, complexes with imipenem (monoclinic form, 1.79-Å resolution) and moxalactam (orthorhombic form, 1.85-Å resolution) were also obtained. In the acyl-enzyme complexes, imipenem and moxalactam differ by the position of the α-substituent and of the carbonyl oxygen (in or out of the oxyanion hole). More surprisingly, the Ω-loop, which includes the catalytically relevant residue Glu166, was found in different conformations in the various subunits, resulting in the Glu166 side chain being rotated out of the active site or even in displacement of its Cα atom up to approximately 10 Å. A BEL-1 variant showing the single Leu162Phe substitution (BEL-2) confers a higher level of resistance to CAZ, CTX, and FEP and shows significantly lower K values than BEL-1, especially with oxyiminocephalosporins. BEL-1 Leu162 is located at the beginning of the Ω-loop and is surrounded by Phe72, Leu139, and Leu148 (contact distances, 3.5 to 3.9 Å). This small hydrophobic cavity could not reasonably accommodate the bulkier Phe162 found in BEL-2 without altering neighboring residues or the Ω-loop itself, thus likely causing an important alteration of the enzyme kinetic properties.
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http://dx.doi.org/10.1128/AAC.00936-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5118998PMC
December 2016

Targeting clinically-relevant metallo-β-lactamases: from high-throughput docking to broad-spectrum inhibitors.

J Enzyme Inhib Med Chem 2016 28;31(sup1):98-109. Epub 2016 Apr 28.

a European Research Centre for Drug Discovery and Development (NatSynDrugs), University of Siena , Siena , Italy.

Metallo-β-lactamases (MBLs) represent one of the most important and widespread mechanisms of resistance to β-lactam antibiotics (including the life-saving carbapenems), against which no clinically useful inhibitors are currently available. We report herein a structure-based high-throughput docking (HTD) campaign on three clinically-relevant acquired MBLs (IMP-1, NDM-1 and VIM-2). The initial hit NF1810 (1) was optimized providing the broad-spectrum inhibitor 3i, which is able to potentiate the in vitro activity of cefoxitin on a VIM-2-producing E. coli strain.
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http://dx.doi.org/10.3109/14756366.2016.1172575DOI Listing
February 2017

Biological Characterization and in Vivo Assessment of the Activity of a New Synthetic Macrocyclic Antifungal Compound.

J Med Chem 2016 04 19;59(8):3854-66. Epub 2016 Apr 19.

Department of Biotechnology Chemistry and Pharmacy, University of Siena , I-53100 Siena, Italy.

We recently identified a novel family of macrocyclic amidinoureas showing potent antifungal activity against Candida spp. In this study, we demonstrate the fungicidal effect of these compounds as well as their killing activity in a dose-dependent manner. Transcriptional analysis data indicate that our molecules induce a significant change in the transcriptome involving ATP binding cassette (ABC) transporter genes. Notably, experiments against Candida albicans mutants lacking those genes showed resistance to the compound, suggesting the involvement of ABC transporters in the uptake or intracellular accumulation of the molecule. To probe the mode of action, we performed fluorescence microscopy experiments on fungal cells treated with an ad-hoc synthesized fluorescent derivative. Fluorescence microscopy images confirm the ability of the compound to cross the membrane and show a consistent accumulation within the cytoplasm. Finally, we provide data supporting the in vivo efficacy in a systemic infection murine model setup with a drug-resistant strain of C. albicans.
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http://dx.doi.org/10.1021/acs.jmedchem.6b00018DOI Listing
April 2016

Biochemical Characterization of CPS-1, a Subclass B3 Metallo-β-Lactamase from a Chryseobacterium piscium Soil Isolate.

Antimicrob Agents Chemother 2015 Dec 14;60(3):1869-73. Epub 2015 Dec 14.

Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg, Denmark Department of Biomedical Sciences, Ross University School of Veterinary Medicine, St. Kitts, West Indies

CPS-1 is a subclass B3 metallo-β-lactamase from a Chryseobacterium piscium isolate collected from soil, showing 68% amino acid identity to the GOB-1 enzyme. CPS-1 was overproduced in Escherichia coli Rosetta (DE3), purified by chromatography, and biochemically characterized. This enzyme exhibits a broad-spectrum substrate profile, including penicillins, cephalosporins, and carbapenems, which overall resembles those of L1, GOB-1, and acquired subclass B3 enzymes AIM-1 and SMB-1.
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http://dx.doi.org/10.1128/AAC.01924-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4775976PMC
December 2015
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