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The divergent sequences, protein structures, and catalytic mechanisms of serine-and metallo-βlactamases hamper the development of wide-spectrum β-lactamase inhibitors that can block both types of enzymes. The O-aryloxycarbonyl hydroxamate inactivators of Enterobacter cloacae P99 class C serine-β-lactamase are unusual covalent inhibitors in that they target both active-site Ser and Lys residues, resulting in a crosslink consisting of only two atoms. Many clinically-relevant metallo-β-lactamases have an analogous active-site Lys residue used to bind β-lactam substrates, suggesting a common site to target with covalent inhibitors. Here, we demonstrate that an Oaryloxycarbonyl hydroxamate inactivator of serine β-lactamases can also serve as a classical affinity label for New Delhi metallo-β-lactamase-1 (NDM-1). Rapid dilution assays, site-directed mutagenesis, and global kinetic fitting are used to map covalent modification at Lys211 and determine K I (140 μM) and k inact (0.045 min −1 ) values. Mass spectrometry of the intact protein and the use of ultraviolet photodissociation (UVPD) for extensive fragmentation confirm stoichiometric covalent labeling that occurs specifically at Lys211. A 2.0 Å resolution X-ray crystal structure of inactivated NDM-1 reveals that the covalent adduct is bound at the substratebinding site, but is not directly coordinated to the active-site zinc cluster. These results indicate that Lys-targeted affinity labels might be a successful strategy for developing compounds that can inactivate both serine and metallo-β-lactamases.
The divergent sequences, protein structures, and catalytic mechanisms of serine-and metallo-βlactamases hamper the development of wide-spectrum β-lactamase inhibitors that can block both types of enzymes. The O-aryloxycarbonyl hydroxamate inactivators of Enterobacter cloacae P99 class C serine-β-lactamase are unusual covalent inhibitors in that they target both active-site Ser and Lys residues, resulting in a crosslink consisting of only two atoms. Many clinically-relevant metallo-β-lactamases have an analogous active-site Lys residue used to bind β-lactam substrates, suggesting a common site to target with covalent inhibitors. Here, we demonstrate that an Oaryloxycarbonyl hydroxamate inactivator of serine β-lactamases can also serve as a classical affinity label for New Delhi metallo-β-lactamase-1 (NDM-1). Rapid dilution assays, site-directed mutagenesis, and global kinetic fitting are used to map covalent modification at Lys211 and determine K I (140 μM) and k inact (0.045 min −1 ) values. Mass spectrometry of the intact protein and the use of ultraviolet photodissociation (UVPD) for extensive fragmentation confirm stoichiometric covalent labeling that occurs specifically at Lys211. A 2.0 Å resolution X-ray crystal structure of inactivated NDM-1 reveals that the covalent adduct is bound at the substratebinding site, but is not directly coordinated to the active-site zinc cluster. These results indicate that Lys-targeted affinity labels might be a successful strategy for developing compounds that can inactivate both serine and metallo-β-lactamases.
An organocatalytic MBH cascade reaction was developed to construct new 3-(α-acrylic acid) benzoxaboroles, designed to mimic ‘anchoring’ pharmacophore features of carbapenems, with the aim of helping overcome carbapenemase resistance.
β-lactamases (BLs) represent the most frequent cause of antimicrobial resistance in Gram-negative bacteria. Despite the continuous efforts in the development of BL inhibitors (BLIs), new BLs able to hydrolyze the last developed antibiotics rapidly emerge. Moreover, the insurgence rate of effective mutations is far higher than the release of BLIs able to counteract them. This results in a shortage of antibiotics that is menacing the effective treating of infectious diseases. The situation is made even worse by the co-expression in bacteria of BLs with different mechanisms and hydrolysis spectra, and by the lack of inhibitors able to hit them all. Differently from other targets, BL flexibility has not been deeply exploited for drug design, possibly because of the small protein size, for their apparent rigidity and their high fold conservation. In this mini-review, we discuss the evidence for BL binding site dynamics being crucial for catalytic efficiency, mutation effect, and for the design of new inhibitors. Then, we report on identified allosteric sites in BLs and on possible allosteric inhibitors, as a strategy to overcome the frequent occurrence of mutations in BLs and the difficulty of competing efficaciously with substrates. Nevertheless, allosteric inhibitors could work synergistically with traditional inhibitors, increasing the chances of restoring bacterial susceptibility towards available antibiotics.
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