Dataset Information

Crystal Structures of KPC-2 and SHV-1 ?-Lactamases in Complex with the Boronic Acid Transition State Analog S02030.

ABSTRACT: Resistance to expanded-spectrum cephalosporins and carbapenems has rendered certain strains of Klebsiella pneumoniae the most problematic pathogens infecting patients in the hospital and community. This broad-spectrum resistance to ?-lactamases emerges in part via the expression of KPC-2 and SHV-1 ?-lactamases and variants thereof. KPC-2 carbapenemase is particularly worrisome, as the genetic determinant encoding this ?-lactamase is rapidly spread via plasmids. Moreover, KPC-2, a class A enzyme, is difficult to inhibit with mechanism-based inactivators (e.g., clavulanate). In order to develop new ?-lactamase inhibitors (BLIs) to add to the limited available armamentarium that can inhibit KPC-2, we have structurally probed the boronic acid transition state analog S02030 for its inhibition of KPC-2 and SHV-1. S02030 contains a boronic acid, a thiophene, and a carboxyl triazole moiety. We present here the 1.54- and 1.87-Å resolution crystal structures of S02030 bound to SHV-1 and KPC-2 ?-lactamases, respectively, as well as a comparative analysis of the S02030 binding modes, including a previously determined S02030 class C ADC-7 ?-lactamase complex. S02030 is able to inhibit vastly different serine ?-lactamases by interacting with the conserved features of these active sites, which includes (i) forming the bond with catalytic serine via the boron atom, (ii) positioning one of the boronic acid oxygens in the oxyanion hole, and (iii) utilizing its amide moiety to make conserved interactions across the width of the active site. In addition, S02030 is able to overcome more distantly located structural differences between the ?-lactamases. This unique feature is achieved by repositioning the more polar carboxyl-triazole moiety, generated by click chemistry, to create polar interactions as well as reorient the more hydrophobic thiophene moiety. The former is aided by the unusual polar nature of the triazole ring, allowing it to potentially form a unique C-H…O 2.9-Å hydrogen bond with S130 in KPC-2.

SUBMITTER: Nguyen NQ

PROVIDER: S-EPMC4775972 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Publications

Crystal Structures of KPC-2 and SHV-1 β-Lactamases in Complex with the Boronic Acid Transition State Analog S02030.

Nguyen Nhu Q NQ Krishnan Nikhil P NP Rojas Laura J LJ Prati Fabio F Caselli Emilia E Romagnoli Chiara C Bonomo Robert A RA van den Akker Focco F

Antimicrobial agents and chemotherapy 20160104 3

Resistance to expanded-spectrum cephalosporins and carbapenems has rendered certain strains of Klebsiella pneumoniae the most problematic pathogens infecting patients in the hospital and community. This broad-spectrum resistance to β-lactamases emerges in part via the expression of KPC-2 and SHV-1 β-lactamases and variants thereof. KPC-2 carbapenemase is particularly worrisome, as the genetic determinant encoding this β-lactamase is rapidly spread via plasmids. Moreover, KPC-2, a class A enzyme, ...[more]

PMID: 26729491

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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

Crystal Structures of KPC-2 and SHV-1 ?-Lactamases in Complex with the Boronic Acid Transition State Analog S02030.

Publications

Crystal Structures of KPC-2 and SHV-1 β-Lactamases in Complex with the Boronic Acid Transition State Analog S02030.

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