Penicillin binding proteins (PBPs) catalyze steps in the biosynthesis of bacterial cell walls and are the targets for the beta-lactam antibiotics. Non-beta-lactam based antibiotics that target PBPs are of interest because bacteria have evolved resistance to the beta-lactam antibiotics. Boronic acids have been developed as inhibitors of the mechanistically related serine beta-lactamases and serine proteases; however, they have not been explored extensively as PBP inhibitors. Here we report aromatic boronic acid inhibitors of the D,D-carboxypeptidase R39 from Actinomadura sp. strain. Analogues of an initially identified inhibitor [3-(dihydroxyboryl)benzoic acid 1, IC(50) 400 microM] were prepared via routes involving pinacol boronate esters, which were deprotected via a two-stage procedure involving intermediate trifluorborate salts that were hydrolyzed to provide the free boronic acids. 3-(Dihydroxyboryl)benzoic acid analogues containing an amide substituent in the meta, but not ortho position were up to 17-fold more potent inhibitors of the R39 PBP and displayed some activity against other PBPs. These compounds may be useful for the development of even more potent boronic acid based PBP inhibitors with a broad spectrum of antibacterial activity.
To resist to β-lactam antibiotics Eubacteria either constitutively synthesize a β-lactamase or a low affinity penicillin-binding protein target, or induce its synthesis in response to the presence of antibiotic outside the cell. In Bacillus licheniformis and Staphylococcus aureus , a membrane-bound penicillin receptor (BlaR/MecR) detects the presence of β-lactam and launches a cytoplasmic signal leading to the inactivation of BlaI/MecI repressor, and the synthesis of a β-lactamase or a low affinity target. We identified a dipeptide, resulting from the peptidoglycan turnover and present in bacterial cytoplasm, which is able to directly bind to the BlaI/MecI repressor and to destabilize the BlaI/MecI-DNA complex. We propose a general model, in which the acylation of BlaR/MecR receptor and the cellular stress induced by the antibiotic, are both necessary to generate a cell wall-derived coactivator responsible for the expression of an inducible β-lactam-resistance factor. The new model proposed confirms and emphasizes the role of peptidoglycan degradation fragments in bacterial cell regulation.
The trypsin-like protease Der p 3, a major allergen of the house dust mite Dermatophagoides pteronyssinus, is synthesized as a zymogen, termed proDer p 3. No recombinant source of Der p 3 has been described yet, and the zymogen maturation mechanism remains to be elucidated. The Der p 3 zymogen was produced in Pichia pastoris. We demonstrated that the recombinant zymogen is glycosylated at the level of its propeptide. We showed that the activation mechanism of proDer p 3 is intermolecular and is mediated by the house dust mite cysteine protease Der p 1. The primary structure of the proDer p 3 propeptide is associated with a unique zymogen activation mechanism, which is different from those described for the trypsin-like family and relies on the house dust mite papain-like protease Der p 1. This is the first report of a recombinant source of Der p 3, with the same enzymatic activity as the natural enzyme and trypsin. Glycosylation of the propeptide was found to decrease the rate of maturation. Finally, we showed that recombinant Der p 3 is inhibited by the free modified prosequence T P1 R. House dust mite (HDM)6 allergens have been shown to be causative factors of allergic manifestations such as atopic dermatitis, perennial rhinitis, or bronchial asthma. More than 80% of patients suffering from allergic asthma are positive for mite extracts and have large amounts of IgE specific for mite allergens. In Europe, the most prevalent species of house dust mites are Dermatophagoides pteronyssinus and Dermatophagoides farinae, depending on relative humidity and temperature. No less than 23 groups of allergens were identified from extracts of these species (1, 2).Although the group 1 and 2 allergens were extensively studied, those from group 3 were poorly characterized. In D. pteronyssinus, the allergen of this group has been first identified by Stewart et al. (3) as a trypsin-like protease termed Der p 3. The binding of IgE from sera of allergic patients to Der f 3, a D. farinae protease homologous to Der p 3, appears to depend on the purity of the allergen, the tested patient populations, and the sensitivity of the technique (4). Although the frequency of IgE reactivity measured toward Der p 3 was ϳ70 -80%, suggesting that Der p 3 is a major allergen (5), a recent study determined a lower allergenic potency similar to mite allergen groups 8 and 10 (6). On the basis of both sequence comparisons and enzymatic studies, Der p 3 has been classified among the trypsin-like proteases from the S1A serine proteases family. Two other serine proteases with chymotryptic and collagenolytic activities and showing 36 and 76% of identity with Der p 3, were also identified in D. pteronyssinus extracts and were termed Der p 6 and Der p 9, respectively (7-9).The trypsin-like protease Der p 3 displays 47% of identity with salmon trypsin and 45% with bovine trypsin (3, 10, 11). All
The alkyne-azide Cu(I)-catalyzed Huisgen cycloaddition, a click-type reaction, was used to label a double-stranded oligonucleotide (siRNA) with fluorine-18. An alkyne solid support CPG for the preparation of monostranded oligonucleotides functionalized with alkyne has been developed. Two complementary azide labeling agents (1-(azidomethyl)-4-[(18)F]fluorobenzene) and 1-azido-4-(3-[(18)F]fluoropropoxy)benzene have been produced with 41% and 35% radiochemical yields (decay-corrected), respectively. After annealing with the complementary strand, the siRNA was directly labeled by click chemistry with [(18)F]fluoroazide to produce the [(18)F]-radiolabeled siRNA with excellent radiochemical yield and purity.
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