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Structural insight into potent broad-spectrum inhibition with reversible recyclization mechanism: avibactam in complex with CTX-M-15 and Pseudomonas aeruginosa AmpC ?-lactamases.

ABSTRACT: Although ?-lactams have been the most effective class of antibacterial agents used in clinical practice for the past half century, their effectiveness on Gram-negative bacteria has been eroded due to the emergence and spread of ?-lactamase enzymes that are not affected by currently marketed ?-lactam/?-lactamase inhibitor combinations. Avibactam is a novel, covalent, non-?-lactam ?-lactamase inhibitor presently in clinical development in combination with either ceftaroline or ceftazidime. In vitro studies show that avibactam may restore the broad-spectrum activity of cephalosporins against class A, class C, and some class D ?-lactamases. Here we describe the structures of two clinically important ?-lactamase enzymes bound to avibactam, the class A CTX-M-15 extended-spectrum ?-lactamase and the class C Pseudomonas aeruginosa AmpC ?-lactamase, which together provide insight into the binding modes for the respective enzyme classes. The structures reveal similar binding modes in both enzymes and thus provide a rationale for the broad-spectrum inhibitory activity of avibactam. Identification of the key residues surrounding the binding pocket allows for a better understanding of the potency of this scaffold. Finally, avibactam has recently been shown to be a reversible inhibitor, and the structures provide insights into the mechanism of avibactam recyclization. Analysis of the ultra-high-resolution CTX-M-15 structure suggests how the deacylation mechanism favors recyclization over hydrolysis.

SUBMITTER: Lahiri SD

PROVIDER: S-EPMC3716117 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Structural insight into potent broad-spectrum inhibition with reversible recyclization mechanism: avibactam in complex with CTX-M-15 and Pseudomonas aeruginosa AmpC β-lactamases.

Lahiri Sushmita D SD Mangani Stefano S Durand-Reville Thomas T Benvenuti Manuela M De Luca Filomena F Sanyal Gautam G Docquier Jean-Denis JD

Antimicrobial agents and chemotherapy 20130225 6

Although β-lactams have been the most effective class of antibacterial agents used in clinical practice for the past half century, their effectiveness on Gram-negative bacteria has been eroded due to the emergence and spread of β-lactamase enzymes that are not affected by currently marketed β-lactam/β-lactamase inhibitor combinations. Avibactam is a novel, covalent, non-β-lactam β-lactamase inhibitor presently in clinical development in combination with either ceftaroline or ceftazidime. In vitr ...[more]

PMID: 23439634

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Project description:?-Lactamase enzymes (EC 3.5.2.6) are a significant threat to the continued use of ?-lactam antibiotics to treat infections. A novel non-?-lactam ?-lactamase inhibitor with activity against many class A and C and some class D ?-lactamase variants, avibactam, is now available in the clinic in partnership with ceftazidime. Here, we explored the activity of avibactam against a variety of characterized isogenic laboratory constructs of ?-lactamase inhibitor-resistant variants of the class A enzyme SHV (M69I/L/V, S130G, K234R, R244S, and N276D). We discovered that the S130G variant of SHV-1 shows the most significant resistance to inhibition by avibactam, based on both microbiological and biochemical characterizations. Using a constant concentration of 4 mg/liter of avibactam as a ?-lactamase inhibitor in combination with ampicillin, the MIC increased from 1 mg/liter for blaSHV-1 to 256 mg/liter for blaSHV S130G expressed in Escherichia coli DH10B. At steady state, the k2/K value of the S130G variant when inactivated by avibactam was 1.3 M(-1) s(-1), versus 60,300 M(-1) s(-1) for the SHV-1 ?-lactamase. Under timed inactivation conditions, we found that an approximately 1,700-fold-higher avibactam concentration was required to inhibit SHV S130G than the concentration that inhibited SHV-1. Molecular modeling suggested that the positioning of amino acids in the active site of SHV may result in an alternative pathway of inactivation when complexed with avibactam, compared to the structure of CTX-M-15-avibactam, and that S130 plays a role in the acylation of avibactam as a general acid/base. In addition, S130 may play a role in recyclization. As a result, we advance that the lack of a hydroxyl group at position 130 in the S130G variant of SHV-1 substantially slows carbamylation of the ?-lactamase by avibactam by (i) removing an important proton acceptor and donator in catalysis and (ii) decreasing the number of H bonds. In addition, recyclization is most likely also slow due to the lack of a general base to initiate the process. Considering other inhibitor-resistant mechanisms among class A ?-lactamases, S130 may be the most important amino acid for the inhibition of class A ?-lactamases, perhaps even for the novel diazabicyclooctane class of ?-lactamase inhibitors.

Dataset Information

Structural insight into potent broad-spectrum inhibition with reversible recyclization mechanism: avibactam in complex with CTX-M-15 and Pseudomonas aeruginosa AmpC ?-lactamases.

Publications

Structural insight into potent broad-spectrum inhibition with reversible recyclization mechanism: avibactam in complex with CTX-M-15 and Pseudomonas aeruginosa AmpC β-lactamases.

Similar Datasets

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