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See detailSélection et caractérisation de Nanobodies inhibiteurs de métallo-β-lactamases
Sohier, Jean ULg

Doctoral thesis (2013)

Les antibiotiques, et notamment les antibiotiques à noyau β-lactame, ont révolutionné la médecine du 20ième siècle, et sauvé un nombre incalculable de vies. Leur efficacité est cependant mise en péril par ... [more ▼]

Les antibiotiques, et notamment les antibiotiques à noyau β-lactame, ont révolutionné la médecine du 20ième siècle, et sauvé un nombre incalculable de vies. Leur efficacité est cependant mise en péril par la dissémination croissante des facteurs de résistance. La production de β-lactamases par des souches infectieuses est le principal mode de résistance aux antibiotiques à noyau β-lactame. Ces enzymes hydrolysent le noyau β-lactame de ces antibiotiques, les empêchant ainsi d’exercer leur action lytique. Parmi les β-lactamases, les métallo-β-lactamases (MβLs) attirent plus particulièrement l’attention en raison de leur capacité à hydrolyser les carbapénèmes, antibiotiques considérés comme les plus efficaces, et parce qu’il n’existe à l’heure actuelle aucun inhibiteur utile d’un point de vue clinique. Avec ce travail, nous avons voulu explorer le potentiel des Nanobodies (Nbs) de camélidé en tant qu’inhibiteurs de β-lactamases, et plus particulièrement de MβLs. Ces fragments d’anticorps correspondent aux domaines variables des anticorps à chaîne lourde produits par les camélidés (HCAbs). Aussi appelés VHHs, ou « single-domain antibodies » (sdAbs), ils sont considérés comme les plus petits fragments fonctionnels dérivés d’anticorps, et peuvent être facilement sélectionnés par phage-display. Des lamas et un dromadaire ont donc été immunisés avec la MβL VIM-4, ainsi qu’avec d’autres β-lactamases choisies comme modèles. Une sélection par phage-display nous a permis d’identifier un Nb inhibiteur de VIM-4, i.e. le Nanobody Nb_VIM38. Ce Nb a été fusionné à la protéine « cherry » pour augmenter sa production. Des concentrations de l’ordre du μM en cherry-NbVIM_38 inhibent l’hydrolyse de toutes les β-lactamines testées. Par ailleurs, un modèle d’inhibition mixte hyperbolique à tendance anticompétitive est proposé. L’épitope de NbVIM_38 a pu être déterminé par immunodétection en faisant usage de peptides chevauchants. Etant donné que le site de fixation de cherry-NbVIM_38 est distant du site actif, ce Nb peut être considéré comme un inhibiteur allostérique de la MβL VIM-4, et de probablement toutes les MβLs de type VIM. Notre hypothèse est que sa fixation interfère avec la dynamique de la boucle L7 du site actif. L’inhibition de la MβL BcII par le Nanobody cAbBcII10 a aussi été étudiée. Ce Nanobody a été obtenu par K. Conrath et al. en 2001. Il a une affinité de l’ordre du nM pour la MβL BcII (« tight-binding »), ce qui devrait être pris en compte si une caractérisation détaillée de l’inhibition devait être réalisée. De manière surprenante, l’inhibition observée est partielle et dépendante du substrat, ce qui suggère un modèle d’inhibition mixte. Ce Nb peut probablement aussi être considéré comme un inhibiteur allostérique. Ces Nanobodies illustrent la possibilité d’une inhibition allostérique des MβLs. Cette piste mériterait peut-être d’être explorée. [less ▲]

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See detailAllosteric inhibition of VIM metallo-beta-lactamase by a camelid nanobody
Sohier, Jean ULg; Laurent, Clémentine ULg; Chevigné, Andy et al

in Biochemical Journal (2013), 450(3), 477-486

Metallo-β-lactamase (MβL) enzymes are usually produced by multiresistant Gram-negative bacterial strains and have spread worldwide. An approach based on phage display was employed to select single-domain ... [more ▼]

Metallo-β-lactamase (MβL) enzymes are usually produced by multiresistant Gram-negative bacterial strains and have spread worldwide. An approach based on phage display was employed to select single-domain antibody fragments (VHHs also called Nanobodies) that would inhibit the clinically relevant VIM-4 MβL. Out of more than 50 selected nanobodies, only the NbVIM_38 nanobody inhibited VIM-4. The paratope, inhibition mechanism and epitope of NbVIM_38 nanobody were then characterised. An alanine scan of the NbVIM_38 paratope showed that its binding was driven by hydrophobic amino acids. The inhibitory concentration was in the µM range for all tested β-lactams. In addition, the inhibition was found to follow a mixed hyperbolic profile with a predominantly uncompetitive component. Moreover, substrate inhibition was recorded only after nanobody binding. These kinetic data are indicative of a binding site that is distant from the active site. This finding was confirmed by epitope mapping analysis that was performed using peptides, and which identified two stretches of amino acids in the L6 loop and at the end of the alpha2 helix. Because this binding site is distant from the active site and alters both the substrate binding and catalytic properties of VIM-4, this nanobody can be considered as an allosteric inhibitor. [less ▲]

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See detailLe retour de la phagothérapie. Et pourquoi pas…
Sohier, Jean ULg

Conference (2012, September 28)

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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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See detailCA1838, A NANOBODY INHIBITING THE METALLO-β-LACTAMASE VIM-4.
Sohier, Jean ULg; Laurent, Clémentine ULg; Pardon, Els et al

Poster (2010)

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See detailcAbVIM4, a nanobody inhibiting the metallo-β-lactamase VIM-4
Sohier, Jean ULg; Laurent, Clémentine ULg; Pardon, Els et al

Poster (2010)

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See detailCharacterisation of B1 metallo-beta-lactamase inhibition by VHHs
Sohier, Jean ULg; Laurent, Clémentine ULg; Chevigné, Andy et al

Poster (2010)

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See detailKinetics and energetics of ligand binding determined by microcalorimetry: Insights into active site mobility in a psychrophilic alpha-amylase
D'Amico, Salvino ULg; Sohier, Jean ULg; Feller, Georges ULg

in Journal of Molecular Biology (2006), 358(5), 1296-1304

A new microcalorimetric method for recording the kinetic parameters k(cat)/K-m and K-i of alpha-amylases using polysaccharides and oligosaccharides as substrates is described. This method is based on the ... [more ▼]

A new microcalorimetric method for recording the kinetic parameters k(cat)/K-m and K-i of alpha-amylases using polysaccharides and oligosaccharides as substrates is described. This method is based on the heat released by glycosidic bond hydrolysis. The method has been developed to study the active site properties of the cold-active alpha-amylase produced by an Antarctic psychrophilic bacterium in comparison with its closest structural homolog from pig pancreas. It is shown that the psychrophilic a-amylase is more active on large macromolecular substrates and that the higher rate constants k(cat) are gained at the expense of a lower affinity for the substrate. The active site is able to accommodate larger inhibitory complexes, resulting in a mixed-type inhibition of starch hydrolysis by maltose. A method for recording the binding enthalpies by isothermal titration calorimetry in a low-affinity system has been developed, allowing analysis of the energetics of weak ligand binding using the allosteric activator chloride. It is shown that the low affinity of the psychrophilic a-amylase for chloride is entropically driven. The high enthalpic and entropic contributions of activator binding suggest large structural fluctuations between the free and the bound states of the cold-active enzyme. The kinetic and thermodynamic data for the psychrophilic a-amylase indicate that the strictly conserved side-chains involved in substrate binding and catalysis possess an improved mobility, responsible for activity in the cold, and resulting from the disappearance of stabilizing interactions far from the active site. (c) 2006 Elsevier Ltd. All rights reserved. [less ▲]

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