References of "Lassaux, Patricia"
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See detailRedefining the PF06864 Pfam family based on Burkholderia pseudomallei PilO2(Bp) S-SAD crystal structure.
Lassaux, Patricia ULg; Conchillo-Sole, Oscar; Manjasetty, Babu A. et al

in PloS one (2014), 9(4), 94981

Type IV pili are surface-exposed filaments and bacterial virulence factors, represented by the Tfpa and Tfpb types, which assemble via specific machineries. The Tfpb group is further divided into seven ... [more ▼]

Type IV pili are surface-exposed filaments and bacterial virulence factors, represented by the Tfpa and Tfpb types, which assemble via specific machineries. The Tfpb group is further divided into seven variants, linked to heterogeneity in the assembly machineries. Here we focus on PilO2(Bp), a protein component of the Tfpb R64 thin pilus variant assembly machinery from the pathogen Burkholderia pseudomallei. PilO2(Bp) belongs to the PF06864 Pfam family, for which an improved definition is presented based on newly derived Hidden Markov Model (HMM) profiles. The 3D structure of the N-terminal domain of PilO2(Bp) (N-PilO2(Bp)), here reported, is the first structural representative of the PF06864 family. N-PilO2(Bp) presents an actin-like ATPase fold that is shown to be present in BfpC, a different variant assembly protein; the new HMM profiles classify BfpC as a PF06864 member. Our results provide structural insight into the PF06864 family and on the Type IV pili assembly machinery. [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 detailA structure-based strategy for epitope discovery in Burkholderia pseudomallei OppA antigen.
Lassaux, Patricia ULg; Peri, Claudio; Ferrer-Navarro, Mario et al

in Structure (London, England : 1993) (2013), 21(1), 167-75

We present an approach integrating structural and computational biology with immunological tests to identify epitopes in the OppA antigen from the Gram-negative pathogen Burkholderia pseudomallei, the ... [more ▼]

We present an approach integrating structural and computational biology with immunological tests to identify epitopes in the OppA antigen from the Gram-negative pathogen Burkholderia pseudomallei, the etiological agent of melioidosis. The crystal structure of OppA(Bp), reported here at 2.1 A resolution, was the basis for a computational analysis that identified three potential epitopes. In parallel, antigen proteolysis and immunocapturing allowed us to identify three additional peptides. All six potential epitopes were synthesized as free peptides and tested for their immunoreactivity against sera from healthy seronegative, healthy seropositive, and recovered melioidosis patients. Three synthetic peptides allowed the different patient groups to be distinguished, underlining the potential of this approach. Extension of the computational analysis, including energy-based decomposition methods, allowed rationalizing results of the predictive analyses and the immunocapture epitope mapping. Our results illustrate a structure-based epitope discovery process, whose application may expand our perspectives in the diagnostic and vaccine design fields. [less ▲]

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See detailExploiting the Burkholderia pseudomallei acute phase antigen BPSL2765 for structure-based epitope discovery/design in structural vaccinology.
Gourlay, Louise J.; Peri, Claudio; Ferrer-Navarro, Mario et al

in Chemistry & biology (2013), 20(9), 1147-56

We solved the crystal structure of Burkholderia pseudomallei acute phase antigen BPSL2765 in the context of a structural vaccinology study, in the area of melioidosis vaccine development. Based on the ... [more ▼]

We solved the crystal structure of Burkholderia pseudomallei acute phase antigen BPSL2765 in the context of a structural vaccinology study, in the area of melioidosis vaccine development. Based on the structure, we applied a recently developed method for epitope design that combines computational epitope predictions with in vitro mapping experiments and successfully identified a consensus sequence within the antigen that, when engineered as a synthetic peptide, was selectively immunorecognized to the same extent as the recombinant protein in sera from melioidosis-affected subjects. Antibodies raised against the consensus peptide were successfully tested in opsonization bacterial killing experiments and antibody-dependent agglutination tests of B. pseudomallei. Our strategy represents a step in the development of immunodiagnostics, in the production of specific antibodies and in the optimization of antigens for vaccine development, starting from structural and physicochemical principles. [less ▲]

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See detailBroad antibiotic resistance profile of the subclass B3 metallo-β-lactamase GOB-1, a di-zinc enzyme.
Horsfall, Louise; Izougarhane, Youssef; Lassaux, Patricia ULg et al

in FEBS Journal (2011), 278(8)

The metallo-β-lactamase (MBL) GOB-1 was expressed via a T7 expression system in Escherichia coli BL21(DE3). The MBL was purified to homogeneity and shown to exhibit a broad substrate profile, hydrolyzing ... [more ▼]

The metallo-β-lactamase (MBL) GOB-1 was expressed via a T7 expression system in Escherichia coli BL21(DE3). The MBL was purified to homogeneity and shown to exhibit a broad substrate profile, hydrolyzing all the tested β-lactam compounds efficiently. The GOB enzymes are unique among MBLs due to the presence of a glutamine residue at position 116, a zinc-binding residue in all known class B1 and B3 MBL structures. Here we produced and studied the Q116A, Q116N and Q116H mutants. The substrate profiles were similar for each mutant, but with significantly reduced activity compared with that of the wild-type. In contrast to the Q116H enzyme, which bound two zinc ions just like the wild-type, only one zinc ion is present in Q116A and Q116N. These results suggest that the Q116 residue plays a role in the binding of the zinc ion in the QHH site. [less ▲]

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See detailBiochemical and structural characterization of the subclass B1 metallo-β-lactamase VIM-4.
Lassaux, Patricia ULg; Traoré, Daouda; Loisel, Elodie et al

in Antimicrobial Agents and Chemotherapy (2011)

The metallo-β-lactamase VIM-4, mainly found in Pseudomonas aeruginosa or Acinetobacter baumannii, was produced in Escherichia coli and characterized by biochemical and X-ray techniques. A detailed kinetic ... [more ▼]

The metallo-β-lactamase VIM-4, mainly found in Pseudomonas aeruginosa or Acinetobacter baumannii, was produced in Escherichia coli and characterized by biochemical and X-ray techniques. A detailed kinetic study performed in the presence of Zn²+ at concentrations ranging from 0.4 to 100 μM showed that VIM-4 exhibits a kinetic profile similar to the profiles of VIM-2 and VIM-1. However, VIM-4 is more active than VIM-1 against benzylpenicillin, cephalothin, nitrocefin, and imipenem and is less active than VIM-2 against ampicillin and meropenem. The crystal structure of the dizinc form of VIM-4 was solved at 1.9 Å. The sole difference between VIM-4 and VIM-1 is found at residue 228, which is Ser in VIM-1 and Arg in VIM-4. This substitution has a major impact on the VIM-4 catalytic efficiency compared to that of VIM-1. In contrast, the differences between VIM-2 and VIM-4 seem to be due to a different position of the flapping loop and two substitutions in loop 2. Study of the thermal stability and the activity of the holo- and apo-VIM-4 enzymes revealed that Zn²+ ions have a pronounced stabilizing effect on the enzyme and are necessary for preserving the structure. [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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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 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 detailMercaptophosphonate Compounds as Broad-Spectrum Inhibitors of the Metallo-β-lactamases
Lassaux, Patricia ULg; Hamel, Matthieu; Gulea, Mihaela et al

in Journal of Medicinal Chemistry (2010), 53

In this paper, we investigated the inhibitory effect of mercaptophosphonate derivatives against the three subclasses of MBLs (B1, B2, and B3). All 14 tested mercaptophosphonates, with the exception of one ... [more ▼]

In this paper, we investigated the inhibitory effect of mercaptophosphonate derivatives against the three subclasses of MBLs (B1, B2, and B3). All 14 tested mercaptophosphonates, with the exception of one, behaved as competitive inhibitors for the three subclasses. <br />Apart from two compounds, all the mercaptophosphonates tested exhibit a good inhibitory effect on the subclass B2 MBL CphA with low inhibition constants (Ki<15 μM). Interestingly, compound 18 turned out to be a potent broad spectrum MBL inhibitor. <br />The crystallographic structures of the CphA-10a and CphA-18 complexes indicated that the sulfur atom of 10a and the phosphonato group of 18 interact with the Zn2þ ion, respectively. Molecular modeling studies of the interactions between two compounds and the VIM-4 (B1), CphA (B2), and FEZ-1 (B3) enzymes brought to light different binding modes depending on the enzyme and the inhibitor, consistent with the crystallographic structures. [less ▲]

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See detailInhibitors of VIM-2 by screening pharmacologically active and click-chemistry compound libraries
Minond, D.; Saldanha, S. A.; Spaargaren, M. et al

in Bioorganic & Medicinal Chemistry (2009), 17

VIM-2 is an Ambler class B metallo-beta-lactamase (MBL) capable of hydrolyzing a broad-spectrum of beta-lactam antibiotics. Although the discovery and development of MBL inhibitors continue to be an area ... [more ▼]

VIM-2 is an Ambler class B metallo-beta-lactamase (MBL) capable of hydrolyzing a broad-spectrum of beta-lactam antibiotics. Although the discovery and development of MBL inhibitors continue to be an area of active research, an array of potent, small molecule inhibitors is yet to be fully characterized for VIM-2. In the presented research, a compound library screening approach was used to identify and characterize VIM-2 inhibitors from a library of pharmacologically active compounds as well as a focused 'click' chemistry library. The four most potent VIM-2 inhibitors resulting from a VIM-2 screen were characterized by kinetic studies in order to determine K(i) and mechanism of enzyme inhibition. As a result, two previously described pharmacologic agents, mitoxantrone (1,4-dihydroxy-5,8-bis([2-([2-hydroxyethyl]amino)ethyl]amino)-9,10-anthracenedione) and 4-chloromercuribenzoic acid (pCMB) were found to be active, the former as a non-competitive inhibitor (K(i)=K(i)(')=1.5+/-0.2microM) and the latter as a slowly reversible or irreversible inhibitor. Additionally, two novel sulfonyl-triazole analogs from the click library were identified as potent, competitive VIM-2 inhibitors: N-((4-((but-3-ynyloxy)methyl)-1H-1,2,3-triazol-5-yl)methyl)-4-iodobenzenesulfonamide (1, K(i)=0.41+/-0.03microM) and 4-iodo-N-((4-(methoxymethyl)-1H-1,2,3-triazol-5-yl)methyl)benzenesulfonamide (2, K(i)=1.4+/-0.10microM). Mitoxantrone and pCMB were also found to potentiate imipenem efficacy in MIC and synergy assays employing Escherichia coli. Taken together, all four compounds represent useful chemical probes to further investigate mechanisms of VIM-2 inhibition in biochemical and microbiology-based assays. [less ▲]

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See detailDiscovery of novel lipophilic inhibitors of OXA-10 enzyme (class D beta-lactamase) by screening amino analogs and homologs of citrate and isocitrate.
Beck, Joséphine; Vercheval, Lionel ULg; Bebrone, Carine ULg et al

in Bioorganic & Medicinal Chemistry Letters (2009), 19

Aminocitrate (and homolog) derivatives have been prepared by bis-alkylation of glycinate Schiff bases with bromoacetates (and ethyl acrylate), followed by N-acylation and esters (partial or complete ... [more ▼]

Aminocitrate (and homolog) derivatives have been prepared by bis-alkylation of glycinate Schiff bases with bromoacetates (and ethyl acrylate), followed by N-acylation and esters (partial or complete) deprotection. Aminoisocitrate was similarly obtained by mono-alkylation with diethyl fumarate. Evaluation against representative beta-lactamases revealed that the free acid derivatives are modest inhibitors of class A enzymes, whilst their benzyl esters showed a good inhibition of OXA-10 (class D enzyme). A docking experiment featured hydrophobic interactions in the active site. [less ▲]

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See detailAminophosphonic Acids and Aminobis(phosphonic acids) as Potential Inhibitors of Penicillin-Binding Proteins
Beck, Josephine; Gharbi, Sonia; Herteg-Fernea, Adriana et al

in European Journal of Organic Chemistry (2009), (1), 85-97

Abstract Aminophosphonic acids and aminobis(phosphonic acids) have been prepared by the alkylation of Schiff bases with methyl bromoacetate or ethyl acrylate. Other pathways, like the modified Pudovik ... [more ▼]

Abstract Aminophosphonic acids and aminobis(phosphonic acids) have been prepared by the alkylation of Schiff bases with methyl bromoacetate or ethyl acrylate. Other pathways, like the modified Pudovik reaction and Kabachnik-Fields reaction, have been considered for the synthesis of the -phosphonic bioisoster of aminocitrate. Partial or complete deprotection of the phosphonate ester have been realised by either acidic hydrolysis or by treatment with trimethylsilyl bromide. Evaluation against penicillin-binding proteins has shown that our compounds are modest inhibitors of class A -lactamases, but have an interesting activity against R39 (D,D-peptidase/carboxypeptidase). [less ▲]

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See detailDynamic combinatorial mass spectrometry leads to metallo-beta-lactamase inhibitors.
Lienard, Benoit M. R.; Huting, Rebekka; Lassaux, Patricia ULg et al

in Journal of medicinal chemistry (2008), 51(3), 684-8

The use of protein ESI mass spectrometry under non-denaturing conditions to analyze a dynamic combinatorial library of thiols/disulfides with the BcII metallo-beta-lactamase enabled the rapid ... [more ▼]

The use of protein ESI mass spectrometry under non-denaturing conditions to analyze a dynamic combinatorial library of thiols/disulfides with the BcII metallo-beta-lactamase enabled the rapid identification of an inhibitor with a K(i) of < 1 microM. The study exemplifies the utility of protein-MS for screening dynamic mixtures of potential enzyme-inhibitors. [less ▲]

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See detailStructural basis for the broad-spectrum inhibition of metallo-beta-lactamases by thiols
Lienard, Benoit M R; Garau, Gianpiero; Horsfall, Louise et al

in Organic & Biomolecular Chemistry (2008), 6(13), 2282-2294

The development of broad-spectrum metallo-beta-lactamase (MBL) inhibitors is challenging due to structural diversity and differences in metal utilisation by these enzymes. Analysis of structural data ... [more ▼]

The development of broad-spectrum metallo-beta-lactamase (MBL) inhibitors is challenging due to structural diversity and differences in metal utilisation by these enzymes. Analysis of structural data, followed by non-denturing mass spectrometric analyses, identified thiols proposed to inhibit representative MBLs from all three sub-classes: B1, B2 and B3. Solution analyses led to the identification of broad spectrum inhibitors, including potent inhibitors of the CphA MBL (Aeromonas hydrophila). Structural studies revealed that, as observed for other B1 and B3 MBLs, inhibition of the L1 MBL thiols involves metal chelation. Evidence is reported that this is not the case for inhibition of the CphA enzyme by some thiols; the crystal structure of the CphA-Zn-inhibitor complex reveals a binding mode in which the thiol does not interact with the zinc. The structural data enabled the design and the production of further more potent inhibitors. Overall the results suggest that the development of reasonably broad-spectrum MBL inhibitors should be possible. [less ▲]

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