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See detailStudies of the Domains II and III of Bacillus subtilis PBP4a in relation with the protein localization
Vanden Broeck, Arnaud ULg; Van Der Heiden, Edwige ULg; Sauvage, Eric ULg et al

Poster (2014, April 23)

Bacillus subtilis PBP4a belongs to the class-C1 PBPs characterized by two internal additional domains of unknown function. Seven lysine residues (K) are protruding from domain II. Four of them have been ... [more ▼]

Bacillus subtilis PBP4a belongs to the class-C1 PBPs characterized by two internal additional domains of unknown function. Seven lysine residues (K) are protruding from domain II. Four of them have been mutated in glutamine residues (Q). Both proteins (WT and Mut4KQ PBP4a) have been produced without signal peptide in E. coli and their sub-cellular localizations determined by measuring the DD-carboxypeptidase activities in the different compartments (cytoplasmic vs membrane attached proteins). In order to detect a possible influence of the PBP4a domain III in the localization of the protein, its encoding sequence has been cloned into pET-28b-BlaP, a vector allowing the production of WT BlaP β-lactamase or BlaP/DIII chimeric protein (with domain III inserted in a permissive loop of BlaP). The nitrocefin hydrolysis activities of BlaP or BlaP/DIII have been measured in the different cellular compartments. [less ▲]

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See detailThe crystal structure of the cell division amidase AmiC reveals the fold of the AMIN domain, a new peptidoglycan binding domain.
Rocaboy, Mathieu; Herman, Raphael; Sauvage, Eric ULg et al

in Molecular microbiology (2013)

Binary fission is the ultimate step of the prokaryotic cell cycle. In Gram-negative bacteria like Escherichia coli, this step implies the invagination of three biological layers (cytoplasmic membrane ... [more ▼]

Binary fission is the ultimate step of the prokaryotic cell cycle. In Gram-negative bacteria like Escherichia coli, this step implies the invagination of three biological layers (cytoplasmic membrane, peptidoglycan and outer membrane), biosynthesis of the new poles and eventually, daughter cells separation. The latter requires the coordinated action of the N-acetylmuramyl-L-alanine amidases AmiA/B/C and their LytM activators EnvC and NlpD to cleave the septal peptidoglycan. We present here the 2.5 A crystal structure of AmiC which includes the first report of an AMIN domain structure, a beta-sandwich of two symmetrical four-stranded beta-sheets exposing highly conserved motifs on the two outer faces. We show that this N-terminal domain, involved in the localization of AmiC at the division site, is a new peptidoglycan-binding domain. The C-terminal catalytic domain shows an auto-inhibitory alpha helix obstructing the active site. AmiC lacking this helix exhibits by itself an activity comparable to that of the wild type AmiC activated by NlpD. We also demonstrate the interaction between AmiC and NlpD by microscale thermophoresis and confirm the importance of the active site blocking alpha helix in the regulation of the amidase activity. [less ▲]

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See detailInhibition of dd-Peptidases by a Specific Trifluoroketone: Crystal Structure of a Complex with the Actinomadura R39 dd-Peptidase.
Dzhekieva, Liudmila; Adediran, S. A.; Herman, Raphael et al

in Biochemistry (2013)

Inhibitors of bacterial dd-peptidases represent potential antibiotics. In the search for alternatives to beta-lactams, we have investigated a series of compounds designed to generate transition state ... [more ▼]

Inhibitors of bacterial dd-peptidases represent potential antibiotics. In the search for alternatives to beta-lactams, we have investigated a series of compounds designed to generate transition state analogue structures upon reaction with dd-peptidases. The compounds contain a combination of a peptidoglycan-mimetic specificity handle and a warhead capable of delivering a tetrahedral anion to the enzyme active site. The latter includes a boronic acid, two alcohols, an aldehyde, and a trifluoroketone. The compounds were tested against two low-molecular mass class C dd-peptidases. As expected from previous observations, the boronic acid was a potent inhibitor, but rather unexpectedly from precedent, the trifluoroketone [d-alpha-aminopimelyl(1,1,1-trifluoro-3-amino)butan-2-one] was also very effective. Taking into account competing hydration, we found the trifluoroketone was the strongest inhibitor of the Actinomadura R39 dd-peptidase, with a subnanomolar (free ketone) inhibition constant. A crystal structure of the complex between the trifluoroketone and the R39 enzyme showed that a tetrahedral adduct had indeed formed with the active site serine nucleophile. The trifluoroketone moiety, therefore, should be considered along with boronic acids and phosphonates as a warhead that can be incorporated into new and effective dd-peptidase inhibitors and therefore, perhaps, antibiotics. [less ▲]

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See detail2-nitrobenzyl esters of penam and cephem derivatives as inhibitors of penicillin-binding proteins
Brulé, Cédric; Grugier, Jérôme; Brans, Alain ULg et al

in Asian Journal of Organic Chemistry (2013), 2

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See detailMacrocyle-embedded b-lactams as novel inhibitors of the Penicillin Binding Protein PBP2a from MRSA
Dive, Georges ULg; Bouillon, Camille; Sliwa, Aline et al

in European Journal of Medicinal Chemistry (2013), 64

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See detailPeptidoglycan glycosyltransferase substrate mimics as templates for the design of new antibacterial drugs.
Derouaux, Adeline ULg; Sauvage, Eric ULg; Terrak, Mohammed ULg

in Frontiers in immunology (2013), 4

Peptidoglycan (PG) is an essential net-like macromolecule that surrounds bacteria, gives them their shape, and protects them against their own high osmotic pressure. PG synthesis inhibition leads to ... [more ▼]

Peptidoglycan (PG) is an essential net-like macromolecule that surrounds bacteria, gives them their shape, and protects them against their own high osmotic pressure. PG synthesis inhibition leads to bacterial cell lysis, making it an important target for many antibiotics. The final two reactions in PG synthesis are performed by penicillin-binding proteins (PBPs). Their glycosyltransferase (GT) activity uses the lipid II precursor to synthesize glycan chains and their transpeptidase (TP) activity catalyzes the cross-linking of two glycan chains via the peptide side chains. Inhibition of either of these two reactions leads to bacterial cell death. beta-lactam antibiotics target the transpeptidation reaction while antibiotic therapy based on inhibition of the GTs remains to be developed. Ongoing research is trying to fill this gap by studying the interactions of GTs with inhibitors and substrate mimics and utilizing the latter as templates for the design of new antibiotics. In this review we present an updated overview on the GTs and describe the structure-activity relationship of recently developed synthetic ligands. [less ▲]

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See detailSynthesis and evaluation of boronic acids as inhibitors of Penicillin Binding Proteins of classes A, B and C
Zervosen, Astrid ULg; Sauvage, Eric ULg; Bouillez, André ULg et al

Poster (2012, April 18)

The widespread use of beta-lactam antibiotics has lead to the worldwide appearance of drug-resistant strains. Bacteria have developed resistance to beta-lactams by three main mechanisms: the production of ... [more ▼]

The widespread use of beta-lactam antibiotics has lead to the worldwide appearance of drug-resistant strains. Bacteria have developed resistance to beta-lactams by three main mechanisms: the production of beta-lactamases that catalyze hydrolysis of beta-lactams, the production of low-affinity, drug resistant Penicillin Binding Proteins (PBPs) and the over expression of resistant PBPs. PBPs are interesting targets because they catalyse the last steps of the biosynthesis of peptidoglycan, which is unique in bacteria and has no mammalian analogs, outside the cytoplasmic membrane. Various non-ß-lactam inhibitors of PBPs have been developed with the objective of attempting to stall the development of ß-lactam resistance. Boronic acids are potent beta-lactamase inhibitors and have been shown to display some specificity for soluble transpeptidases and PBPs, but their potential as inhibitors of the latter enzymes is yet to be widely explored. Recently, a (2, 6-dimethoxybenzamido)methylboronic acid was identified as being a potent inhibitor of Actinomadura sp. R39 transpeptidase (IC50: 1.3 µM). Here, we will discuss the synthesis of a number of acylaminomethylboronic acids, analogs of (2, 6-dimethoxybenzamido)methylboronic acid, and their potential as inhibitors of PBPs. Several boronic acids of this library were able to inhibit PBPs of classes A, B and C from penicillin sensitive strains. Thus (2-nitrobenzamido)methylboronic acid was identified as a good inhibitor of class A PBP (PBP1b from S. pneumoniae, IC50 = 26 µM), class B PBP (PBP2xR6 from S. pneumoniae, IC50 = 138 µM) and class C PBP (R39 from Actinomadura sp., IC50 = 0.6 µM). Crystal structures of complexes of R39 and PBP1b with boronic acid analogs of our library have already been solved and allowed an interpretation of results. We believe that this work opens new avenues towards the development of molecules that will inhibit PBPs, and eventually display bactericidal effect, on distinct bacterial species. [less ▲]

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See detailNovel fragments of clavulanate observed in the structure of the class A b-lactamase from Bacillus licheniformis BS3
Power, Pablo; Mercuri, Paola ULg; Herman, Raphaël ULg et al

in Journal of Antimicrobial Chemotherapy (2012), 67(10), 2379-2387

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See detailDevelopment of new drugs for an old target — the penicillin binding proteins.
Zervosen, Astrid ULg; Sauvage, Eric ULg; Frère, Jean-Marie ULg et al

in Molecules (2012), 17(11), 12478-505

The widespread use of β-lactam antibiotics has led to the worldwide appearance of drug-resistant strains. Bacteria have developed resistance to β-lactams by two main mechanisms: the production of β ... [more ▼]

The widespread use of β-lactam antibiotics has led to the worldwide appearance of drug-resistant strains. Bacteria have developed resistance to β-lactams by two main mechanisms: the production of β-lactamases, sometimes accompanied by a decrease of outer membrane permeability, and the production of low-affinity, drug resistant Penicillin Binding Proteins (PBPs). PBPs remain attractive targets for developing new antibiotic agents because they catalyse the last steps of the biosynthesis of peptidoglycan, which is unique to bacteria, and lies outside the cytoplasmic membrane. Here we summarize the “current state of the art” of non-β-lactam inhibitors of PBPs, which have being developed in an attempt to counter the emergence of β-lactam resistance. These molecules are not susceptible to hydrolysis by β-lactamases and thus present a real alternative to β-lactams. We present transition state analogs such as boronic acids, which can covalently bind to the active serine residue in the catalytic site. Molecules containing ring structures different from the β-lactam-ring like lactivicin are able to acylate the active serine residue. High throughput screening methods, in combination with virtual screening methods and structure based design, have allowed the development of new molecules. Some of these novel inhibitors are active against major pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and thus open avenues new for the discovery of novel antibiotics. [less ▲]

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See detailSynthesis and evaluation of boronic acids as inhibitors of Penicillin Binding Proteins of classes A, B and C.
Zervosen, Astrid ULg; Bouillez, André ULg; Herman, Alexandre et al

in Bioorganic & Medicinal Chemistry (2012), 20(12), 3915-24

In response to the widespread use of beta-lactam antibiotics bacteria have evolved drug resistance mechanisms that include the production of resistant Penicillin Binding Proteins (PBPs). Boronic acids are ... [more ▼]

In response to the widespread use of beta-lactam antibiotics bacteria have evolved drug resistance mechanisms that include the production of resistant Penicillin Binding Proteins (PBPs). Boronic acids are potent beta-lactamase inhibitors and have been shown to display some specificity for soluble transpeptidases and PBPs, but their potential as inhibitors of the latter enzymes is yet to be widely explored. Recently, a (2,6-dimethoxybenzamido)methylboronic acid was identified as being a potent inhibitor of Actinomadura sp. R39 transpeptidase (IC(50): 1.3muM). In this work, we synthesized and studied the potential of a number of acylaminomethylboronic acids as inhibitors of PBPs from different classes. Several derivatives inhibited PBPs of classes A, B and C from penicillin sensitive strains. The (2-nitrobenzamido)methylboronic acid was identified as a good inhibitor of a class A PBP (PBP1b from Streptococcus pneumoniae, IC(50)=26muM), a class B PBP (PBP2xR6 from Streptococcus pneumoniae, IC(50)=138muM) and a class C PBP (R39 from Actinomadura sp., IC(50)=0.6muM). This work opens new avenues towards the development of molecules that inhibit PBPs, and eventually display bactericidal effects, on distinct bacterial species. [less ▲]

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See detailUnexpected tricovalent binding mode of boronic acids within the active site of a penicillin binding protein.
Zervosen, Astrid ULg; Herman, Raphaël ULg; Kerff, Frédéric ULg et al

in Journal of the American Chemical Society (2011)

Boronic acids bearing appropriate side chains are good inhibitors of serine amidohydrolases. The boron usually adopts a tetrahedral conformation, bound to the nucleophilic serine of the active site and ... [more ▼]

Boronic acids bearing appropriate side chains are good inhibitors of serine amidohydrolases. The boron usually adopts a tetrahedral conformation, bound to the nucleophilic serine of the active site and mimicking the transition state of the enzymatic reaction. We have solved the structures of complexes of a penicillin-binding protein, the DD-peptidase from Actinomadura sp. R39, with four amidomethylboronic acids (2,6 dimethoxybenzamidomethylboronic acid, phenylacetamidomethylboronic acid, 2-chlorobenzamidomethylboronic acid, and 2-nitrobenzamidomethylboronic acid) and the pinacol ester derived from phenylacetamidomethylboronic acid. We found that, in each case, the boron forms a tricovalent adduct with Ogamma of Ser49, Ser298, and the terminal amine group of Lys410, three key residues involved in the catalytic mechanism of penicillin-binding proteins. This represents the first tricovalent enzyme-inhibitor adducts observed by crystallography. In two of the five R39-boronate structures, the boronic acid is found as a tricovalent adduct in two monomers of the asymmetric unit and as a monocovalent adduct with the active serine in the two remaining monomers of the asymmetric unit. Formation of the tricovalent complex from a classical monocovalent complex may involve rotation around the Ser49 Calpha-Cbeta bond to place the boron in a position to interact with Ser298 and Lys410, and a twisting of the side chain amide such that its carbonyl oxygen is able to hydrogen bond to the oxyanion hole NH of Thr413. Biphasic kinetics were observed in three of the five cases and details of the reaction between R39 and 2,6-dimethoxybenzamidomethylboronic acid were studied. Observation of biphasic kinetics was not, however, thought to be correlated to formation of tricovalent complexes, assuming that the latter do form in solution. Based on the crystallographic and kinetic results, a reaction scheme for this unexpected inhibition by boronic acids is proposed. [less ▲]

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See detailStructure Guided Development of Potent Reversibly Binding Penicillin Binding Protein Inhibitors
Woon, Esther C. Y.; Zervosen, Astrid ULg; Sauvage, Eric ULg et al

in ACS Medicinal Chemistry letters (2011), 2 (3)

Following from the evaluation of different types of electrophiles, combined modeling and crystallographic analyses are used to generate potent boronic acid based inhibitors of a penicillin binding protein ... [more ▼]

Following from the evaluation of different types of electrophiles, combined modeling and crystallographic analyses are used to generate potent boronic acid based inhibitors of a penicillin binding protein. The results suggest that a structurally informed approach to penicillin binding protein inhibition will be useful for the development of both improved reversibly binding inhibitors, including boronic acids, and acylating inhibitors, such as β-lactams. [less ▲]

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See detailStructures of class D β-lactamases
Kerff, Frédéric ULg; Sauvage, Eric ULg; Vercheval, Lionel ULg et al

in Frère, Jean-Marie (Ed.) Beta-lactamases (2011)

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See detailKinetics of Reactions of the Actinomadura R39 dd-Peptidase with Specific Substrates.
Adediran, S. A.; Kumar, Ish; Nagarajan et al

in Biochemistry (2011), 50(3), 376-387

The Actinomadura R39 dd-peptidase catalyzes the hydrolysis and aminolysis of a number of small peptides and depsipeptides. Details of its substrate specificity and the nature of its in vivo substrate are ... [more ▼]

The Actinomadura R39 dd-peptidase catalyzes the hydrolysis and aminolysis of a number of small peptides and depsipeptides. Details of its substrate specificity and the nature of its in vivo substrate are not, however, well understood. This paper describes the interactions of the R39 enzyme with two peptidoglycan-mimetic substrates 3-(d-cysteinyl)propanoyl-d-alanyl-d-alanine and 3-(d-cysteinyl)propanoyl-d-alanyl-d-thiolactate. A detailed study of the reactions of the former substrate, catalyzed by the enzyme, showed dd-carboxypeptidase, dd-transpeptidase, and dd-endopeptidase activities. These results confirm the specificity of the enzyme for a free d-amino acid at the N-terminus of good substrates and indicated a preference for extended d-amino acid leaving groups. The latter was supported by determination of the structural specificity of amine nucleophiles for the acyl-enzyme generated by reaction of the enzyme with the thiolactate substrate. It was concluded that a specific substrate for this enzyme, and possibly the in vivo substrate, may consist of a partly cross-linked peptidoglycan polymer where a free side chain N-terminal un-cross-linked amino acid serves as the specific acyl group in an endopeptidase reaction. The enzyme is most likely a dd-endopeptidase in vivo. pH−rate profiles for reactions of the enzyme with peptides, the thiolactate named above, and β-lactams indicated the presence of complex proton dissociation pathways with sticky substrates and/or protons. The local structure of the active site may differ significantly for reactions of peptides and β-lactams. Solvent kinetic deuterium isotope effects indicate the presence of classical general acid/base catalysis in both acylation and deacylation; there is no evidence of the low fractionation factor active site hydrogen found previously in class A and C β-lactamases. [less ▲]

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See detailThree factors that modulate the activity of class D β-lactamases and interfere with the post-translational carboxylation of Lys 70
Vercheval, Lionel ULg; Di Paolo, Alexandre ULg; Borel, Franck et al

in Biochemical Journal (2010), 432(3), 495-504

Lys-70 carboxylation in the active site of class D β lactamases is essential for their activity. Structural, kinetic and affinity studies show that this post-translational modification can be affected by ... [more ▼]

Lys-70 carboxylation in the active site of class D β lactamases is essential for their activity. Structural, kinetic and affinity studies show that this post-translational modification can be affected by the presence of a poor substrate such as moxalactam but also by the V117T substitution. Val-117 is a strictly conserved hydrophobic residue located in the active site. In addition, inhibition of class D β lactamases by chloride ions is due to a competition between the side chain carboxylate of the modified Lys 70 and chloride ions. Determination of the individual kinetic constants shows that the deacylation of the acyl-enzyme is the rate limiting step for the wild type OXA 10 β lactamase. [less ▲]

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See detailCurrent challenges in antimicrobial chemotherapy: focus on beta-lactamase inhibition.
Bebrone, Carine ULg; Lassaux, Patricia ULg; Vercheval, Lionel ULg et al

in Drugs (2010), 70(6)

The use of the three classical beta-lactamase inhibitors (clavulanic acid, tazobactam, sulbactam) in combination with beta-lactam antibiotics is currently the most successful strategy to combat the beta ... [more ▼]

The use of the three classical beta-lactamase inhibitors (clavulanic acid, tazobactam, sulbactam) in combination with beta-lactam antibiotics is currently the most successful strategy to combat the beta-lactamase mediated resistance. However, these inhibitors are efficient in inactivating class A beta-lactamases only and the efficiency of the inhibitor/antibiotic combination can be compromised by several mechanisms among which the production of naturally resistant class B or class D enzymes, the hyperproduction of AmpC or even the production of evolved inhibitor-resistant class A enzymes. There is thus an urgent need in the development of novel inhibitors. For serine active enzymes (classes A, C and D), derivatives of the beta-lactam ring such as 6-beta-halogenopenicillanates, beta-lactam sulfones, penems and oxapenems, monobactams or trinems seem to be potential starting points to design efficient molecules (among which AM-112 and LK-157). Moreover, a promising non-beta-lactam molecule, NXL-104 is now under clinical trial. In contrast, an ideal inhibitor of metallo-beta-lactamases (class B) remains to be found, despite the huge number of potential molecules already described (biphenyl tetrazoles, cysteinyl peptides, mercaptocarboxylates, succinic acid derivatives, etc). The search for such an inhibitor is complicated by the absence of a covalent intermediate in their catalytic mechanisms and the fact that beta-lactam derivatives often behave as substrates rather than as inhibitors. Currently, the most promising broad spectrum inhibitors of class B enzymes are molecules presenting chelating groups (thiols, carboxylates, etc) combined with an aromatic group. This review describes all the types of molecules already tested as potential beta-lactamase inhibitors and thus constitutes an update of the current status in beta-lactamase inhibitor discovery. [less ▲]

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