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Biochemical and structural analysis of inhibitors targeting the ADC-7 cephalosporinase of Acinetobacter baumannii.

ABSTRACT: ?-Lactam resistance in Acinetobacter baumannii presents one of the greatest challenges to contemporary antimicrobial chemotherapy. Much of this resistance to cephalosporins derives from the expression of the class C ?-lactamase enzymes, known as Acinetobacter-derived cephalosporinases (ADCs). Currently, ?-lactamase inhibitors are structurally similar to ?-lactam substrates and are not effective inactivators of this class C cephalosporinase. Herein, two boronic acid transition state inhibitors (BATSIs S02030 and SM23) that are chemically distinct from ?-lactams were designed and tested for inhibition of ADC enzymes. BATSIs SM23 and S02030 bind with high affinity to ADC-7, a chromosomal cephalosporinase from Acinetobacter baumannii (Ki = 21.1 ± 1.9 nM and 44.5 ± 2.2 nM, respectively). The X-ray crystal structures of ADC-7 were determined in both the apo form (1.73 Å resolution) and in complex with S02030 (2.0 Å resolution). In the complex, S02030 makes several canonical interactions: the O1 oxygen of S02030 is bound in the oxyanion hole, and the R1 amide group makes key interactions with conserved residues Asn152 and Gln120. In addition, the carboxylate group of the inhibitor is meant to mimic the C3/C4 carboxylate found in ?-lactams. The C3/C4 carboxylate recognition site in class C enzymes is comprised of Asn346 and Arg349 (AmpC numbering), and these residues are conserved in ADC-7. Interestingly, in the ADC-7/S02030 complex, the inhibitor carboxylate group is observed to interact with Arg340, a residue that distinguishes ADC-7 from the related class C enzyme AmpC. A thermodynamic analysis suggests that ?H driven compounds may be optimized to generate new lead agents. The ADC-7/BATSI complex provides insight into recognition of non-?-lactam inhibitors by ADC enzymes and offers a starting point for the structure-based optimization of this class of novel ?-lactamase inhibitors against a key resistance target.

SUBMITTER: Powers RA

PROVIDER: S-EPMC4263437 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Biochemical and structural analysis of inhibitors targeting the ADC-7 cephalosporinase of Acinetobacter baumannii.

Powers Rachel A RA Swanson Hollister C HC Taracila Magdalena A MA Florek Nicholas W NW Romagnoli Chiara C Caselli Emilia E Prati Fabio F Bonomo Robert A RA Wallar Bradley J BJ

Biochemistry 20141125 48

β-Lactam resistance in Acinetobacter baumannii presents one of the greatest challenges to contemporary antimicrobial chemotherapy. Much of this resistance to cephalosporins derives from the expression of the class C β-lactamase enzymes, known as Acinetobacter-derived cephalosporinases (ADCs). Currently, β-lactamase inhibitors are structurally similar to β-lactam substrates and are not effective inactivators of this class C cephalosporinase. Herein, two boronic acid transition state inhibitors (B ...[more]

PMID: 25380506

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Project description:The ADC (AmpC) β-lactamase is universally present in the Acinetobacter baumannii chromosome, suggesting it may have a yet-to-be-identified cellular function. Using peptidoglycan composition analysis, we show that overexpressing the ADC-7 β-lactamase in A. baumannii drives changes consistent with altered l,d-transpeptidase activity. Based on this, we tested whether cells overexpressing ADC-7 would exhibit new vulnerabilities. As proof of principle, a screen of transposon insertions revealed that an insertion in the distal 3' end of canB, encoding carbonic anhydrase, resulted in a significant loss of viability when the adc-7 gene was overexpressed. A canB deletion mutant exhibited a more pronounced loss of viability than the transposon insertion, and this became amplified when cells overexpressed ADC-7. Interestingly, overexpression of the OXA-23 or TEM-1 β-lactamases also led to a pronounced loss of viability in cells with reduced carbonic anhydrase activity. In addition, we demonstrate that reduced CanB activity led to increased sensitivity to peptidoglycan synthesis inhibitors and to the carbonic anhydrase inhibitor ethoxzolamide. Furthermore, this strain exhibited a synergistic interaction with the peptidoglycan inhibitor fosfomycin and ethoxzolamide. Our results highlight the impact of ADC-7 overexpression on cell physiology and reveal that the essential carbonic anhydrase CanB may represent a novel target for antimicrobial agents that would exhibit increased potency against β-lactamase-overexpressing A. baumannii. IMPORTANCE Acinetobacter baumannii has become resistant to all classes of antibiotics, with β-lactam resistance responsible for the majority of treatment failures. New classes of antimicrobials are needed to treat this high-priority pathogen. This study had uncovered a new genetic vulnerability in β-lactamase-expressing A. baumannii, where reduced carbonic anhydrase activity becomes lethal. Inhibitors of carbonic anhydrase could represent a new method for treating A. baumannii infections.

Dataset Information

Biochemical and structural analysis of inhibitors targeting the ADC-7 cephalosporinase of Acinetobacter baumannii.

Publications

Biochemical and structural analysis of inhibitors targeting the ADC-7 cephalosporinase of Acinetobacter baumannii.

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