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International audience; Metallo-β-lactamases (MBLs) represent an increasingly serious threat to public health because of their increased prevalence worldwide in relevant opportunistic Gram-negative pathogens. MBLs efficiently inactivate widely used and most valuable β-lactam antibiotics, such as oxyiminocephalosporins (ceftriaxone, ceftazidime) and the last-resort carbapenems. To date, no MBL inhibitor has been approved for therapeutic applications. We are developing inhibitors characterized by a 1,2,4-triazole-3-thione scaffold as an original zinc ligand and few promising series were already reported. Here, we present the synthesis and evaluation of a new series of compounds characterized by the presence of an arylalkyl substituent at position 4 of the triazole ring. The alkyl link was mainly an ethylene, but a few compounds without alkyl or with an alkyl group of various lengths up to a butyl chain were also synthesized. Some compounds in both sub-series were micromolar to submicromolar inhibitors of tested VIM-type MBLs. A few of them were broad-spectrum inhibitors, as they showed significant inhibitory activity on NDM-1 and, to a lesser extent, IMP-1. Among these, several inhibitors were able to significantly reduce the meropenem MIC on VIM-1- and VIM-4- producing clinical isolates by up to 16-fold. In addition, ACE inhibition was absent or moderate and one promising compound did not show toxicity toward HeLa cells at concentrations up to 250 μM. This series represents a promising basis for further exploration. Finally, molecular modelling of representative compounds in complex with VIM-2 was performed to study their binding mode.

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 Ki 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.

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-&beta, lactamases (MBLs), which represent major resistance determinants to &beta, 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 Ki 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 &beta, 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.

Der p 5 is one of the important house dust mite allergens in Algeria; this allergen is frequently recognized by patients with allergic asthma. However, there is no information on its IgG–binding epitopes. In the present study, rabbits were immunized with recombinant Der p 5 allergen, and serum samples were obtained. Recognition of linear IgG epitopes of Der p 5 was determined using synthesized peptides derived from the allergen sequence. The results showed that serum from immunized rabbits recognized three linear epitopes from Der p 5 (28EDKKHDYQNEFDFLLMERIHEQIK43), (37IHEQIKKGELALFYLQEQ55) and (92LMQRKDLDIFEQYNLEMAKKS112). More interestingly, we observed that the 92L-S112 amino acid sequence is well recognized by both IgE and IgG antibodies. Der p 5 stimulates the synthesis of specific IgG antibodies which recognize common but also novel epitopes compared to IgE antibody binding. Indeed, the potential to induce IgG antibodies can be used to inhibit human IgE binding to allergens which may be part of the mechanism of action of specific immunotherapy. Keywords: Allergen, blocking antibodies, Der p 5, house dust mite, IgG-binding epitope

Three-dimensional modeling of the HMA4 protein in Arabidopsis reveals the zinc permeation pathway across the plasma membrane, and mutations in the pathway alter zinc and cadmium transport differently in plants., The P1B ATPase heavy metal ATPase 4 (HMA4) is responsible for zinc and cadmium translocation from roots to shoots in Arabidopsis thaliana. It couples ATP hydrolysis to cytosolic domain movements, enabling metal transport across the membrane. The detailed mechanism of metal permeation by HMA4 through the membrane remains elusive. Here, homology modeling of the HMA4 transmembrane region was conducted based on the crystal structure of a ZntA bacterial homolog. The analysis highlighted amino acids forming a metal permeation pathway, whose importance was subsequently investigated functionally through mutagenesis and complementation experiments in plants. Although the zinc pathway displayed overall conservation among the two proteins, significant differences were observed, especially in the entrance area with altered electronegativity and the presence of a ionic interaction/hydrogen bond network. The analysis also newly identified amino acids whose mutation results in total or partial loss of the protein function. In addition, comparison of zinc and cadmium accumulation in shoots of A. thaliana complemented lines revealed a number of HMA4 mutants exhibiting different abilities in zinc and cadmium translocation. These observations could be instrumental to design low cadmium-accumulating crops, hence decreasing human cadmium exposure.

International audience; The controlled delivery of proteins within calcium carbonate (CaCO 3) particles is currently widely investigated. The success of these carriers is driven by ionic interactions between the encapsulated proteins and the particles. This poses a great limitation on the successful loading of proteins that have no ionic affinity to CaCO 3. In this study, we explored the use of polysaccharide–protein interactions to strongly enhance the encapsulation of proteins in CaCO 3 microparticles. Previously, Vandevenne and colleagues inserted a human chitin binding domain (ChBD) that has intrinsic affinity for hyaluronic acid (HA) into a b-lactamase (BlaP). This generated chimeric protein, named BlaPChBD, was shown to be fully bifunctional. In this study we showed that this hybrid protein can associate with HA and be successfully loaded into vaterite CaCO 3 microparticles using supercritical CO 2 (ScCO 2) technology aided by the templating effect of HA on CaCO 3. The presence of ChBD inserted into BlaP increased the encapsulation of the protein by 6-fold when complexed with HA. Furthermore, thrombin cleavage sites were engineered on both sides of the inserted ChBD in the chimeric BlaP to achieve release of the protein from the micro-particles by protease cleavage. Our results showed that thrombin cleavage increased the release of the protein from the microparticles within 36 hours from o20% to 87%. In conclusion, the presence of ChBD successfully improved the encapsulation yield of the protein while retaining up to 82% of its activity and efficient release of the protein from the microparticles was achieved by protease cleavage.

52nd International Conference on Medicinal Chemistry (RICT) of the French-Medicinal-Chemistry-Society (SCT) - Interfacing Chemical Biology and Drug Discovery -- JUL 06-08, 2016 -- Caen, FRANCE, WOS: 000403905300014, PubMed ID: 28505394, Metallo-beta-lactamases (MBLs) cause resistance of Gram-negative bacteria to beta-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 3D structures suggested that the triazolethione 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 IC50 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, VIM2, 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 IC50 values toward plasmodial glyoxalase II were in the 10 mm range., French Med Chem Soc, Univ Caen, Agence Nationale de la Recherche ("ANTIMBL") [ANR-14-CE16-0028-01]; Deutsche Forschungsgemeinschaft [BE1540/15-2 within SPP 1710]; Agence Nationale de la Recherche [ANR-06-BLAN-0086], We thank Mr. Pierre Sanchez for mass spectrometry analyses and Wolfram Meyer-Klaucke for advice about tRNase Z. Part of this work was supported by the Agence Nationale de la Recherche ("ANTIMBL", ANR-14-CE16-0028-01, including a fellowship to L.S.), the Deutsche Forschungsgemeinschaft (BE1540/15-2 within SPP 1710 to K.B.), and Agence Nationale de la Recherche ("subtilRNA", ANR-06-BLAN-0086) to C.C.

International audience; Although commercialized inhibitors of active site serine β-lactamases are currently used in coadministration with antibiotic therapy, no clinically useful inhibitors of metallo-β-lactamases (MBLs) have yet been discovered. 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 1a, behaved as competitive inhibitors for the three subclasses. Apart from 13 and 21, all the mercapto-phosphonates 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. 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 Zn2+ ion, respectively. Molecular modeling studies of the interactions between compounds 10a and 18 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.

International audience; Mycobacterium fallax (M. fallax) is naturally sensitive to many beta-lactam antibiotics (MIC < 2 microg/ml) and devoid of beta-lactamase activity. In this paper, we show that the production of the beta-lactamase of Mycobacterium fortuitum by M. fallax significantly increased the MIC values for good substrates of the enzyme, whereas the potency of poor substrates or transient inactivators was not modified. The rates of diffusion of beta-lactams through the mycolic acid layer were low, but for all studied compounds the half-equilibration times were such that they would only marginally affect the MIC values in the absence of beta-lactamase production. These results emphasize the importance of enzymatic degradation as a major factor in the resistance of mycobacteria to penicillins.