Crystal Structures of KPC-2 β-Lactamase in Complex with 3-Nitrophenyl Boronic Acid and the Penam Sulfone PSR-3-226

Wei, Ke; Bethel, Christopher R.; Papp-Wallace, Krisztina M.; Pagadala, Sundar Ram Reddy; Nottingham, Micheal; Fernández, Daniel; Buynak, John D.; Bonomo, Robert A.; Akker, F. van den

doi:10.1128/aac.06099-11

Cited by 45 publications

(73 citation statements)

References 35 publications

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“…They all have the same overall structure with similarities in the structural features surrounding the active site, such as two subdomains generating a cleft ( Fig. 7 and 8) (12,23,149,(185)(186)(187). One of the subdomains (the alpha subdomain) is largely ␣-helical.…”

Section: Structure-function Relationships Of Class a Enzymesmentioning

confidence: 99%

“…The hydrolysis substrate spectrum of KPCs includes aminothiazole-oxime cephalosporins such as cefotaxime and inhibitors such as clavulanate (148). Boronic acid was also recently identified as an inhibitor of KPCs (149). NMC-A-and IMI-type enzymes, which have amino acid sequences displaying ϳ97% identity, are related to SME-type enzymes, to which they are ϳ70% identical at the amino acid level.…”

Section: Toca P Vulgaris R Aquatilis and S Fonticola)mentioning

confidence: 99%

“…In the structure of TEM-1, the region of the extended Asn-Gly-Asp loop is occupied by K ϩ ions. Note that in (gray), OXY-1 (brown), and KPC-2 (green) (149,(185)(186)(187).…”

Section: Structure-function Relationships Of Class a Enzymesmentioning

confidence: 99%

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A Structure-Based Classification of Class A β-Lactamases, a Broadly Diverse Family of Enzymes

Slama²,

et al. 2016

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SUMMARYFor medical biologists, sequencing has become a commonplace technique to support diagnosis. Rapid changes in this field have led to the generation of large amounts of data, which are not always correctly listed in databases. This is particularly true for data concerning class A β-lactamases, a group of key antibiotic resistance enzymes produced by bacteria. Many genomes have been reported to contain putative β-lactamase genes, which can be compared with representative types. We analyzed several hundred amino acid sequences of class A β-lactamase enzymes for phylogenic relationships, the presence of specific residues, and cluster patterns. A clear distinction was first made between dd-peptidases and class A enzymes based on a small number of residues (S70, K73, P107, 130SDN132, G144, E166, 234K/R, 235T/S, and 236G [Ambler numbering]). Other residues clearly separated two main branches, which we named subclasses A1 and A2. Various clusters were identified on the major branch (subclass A1) on the basis of signature residues associated with catalytic properties (e.g., limited-spectrum β-lactamases, extended-spectrum β-lactamases, and carbapenemases). For subclass A2 enzymes (e.g., CfxA, CIA-1, CME-1, PER-1, and VEB-1), 43 conserved residues were characterized, and several significant insertions were detected. This diversity in the amino acid sequences of β-lactamases must be taken into account to ensure that new enzymes are accurately identified. However, with the exception of PER types, this diversity is poorly represented in existing X-ray crystallographic data.

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Section: Structure-function Relationships Of Class a Enzymesmentioning

confidence: 99%

Section: Toca P Vulgaris R Aquatilis and S Fonticola)mentioning

confidence: 99%

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A Structure-Based Classification of Class A β-Lactamases, a Broadly Diverse Family of Enzymes

Slama²,

et al. 2016

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“…As stated above, an emerging notion in the discovery of novel BLIs is to utilize boronic acid transition state analogs (BATSIs) to inhibit serine ␤-lactamases such as KPC-2 and SHV-1 (12)(13)(14). Such an approach has led to several potent inhibitor compounds (15,16).…”

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confidence: 99%

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

Nguyen

Krishnan

Rojas

et al. 2016

Antimicrob Agents Chemother

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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 COH. . .O 2.9-Å hydrogen bond with S130 in KPC-2.

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“…The first KPC channel contained 16 g/ml of MEM (Sigma-Aldrich), the second contained 32 g/ml of MEM, the third contained 16 g/ml of MEM plus 32 g/ml 3-nitrophenylboronic acid (NPBA; Sigma-Aldrich), and the fourth contained 32 g/ml of MEM plus 32 g/ml NPBA. NPBA was included because it is an inhibitor of the KPC enzyme (12). Interpretation algorithms in the image analyzer computed the difference between resistance scores in MEM alone and those in MEM plus NPBA to obtain an inhibition score, weighted by the number of growing clones in each condition.…”

mentioning

confidence: 99%

Rapid Ertapenem Susceptibility Testing and Klebsiella pneumoniae Carbapenemase Phenotype Detection in Klebsiella pneumoniae Isolates by Use of Automated Microscopy of Immobilized Live Bacterial Cells

et al. 2014

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We evaluated detection of ertapenem (ETP) resistance and Klebsiella pneumoniae carbapenemase (KPC) in 47 Klebsiella pneumoniae isolates using a novel automated microscopy system. Automated microscopy correctly classified 22/23 isolates as ETP resistant and 24/24 as ETP susceptible and correctly classified 21/21 isolates as KPC positive and 26/26 as KPC negative. C arbapenem-resistant Enterobacteriaceae (CRE) are emerging as a global threat; the plasmid-borne carbapenemase gene bla KPC is the predominant mechanism conferring carbapenem resistance in North America (1-5). This resistance gene has been reported in most species of Enterobacteriaceae, but it is most commonly found in Klebsiella pneumoniae. Timely detection of carbapenem resistance is critical for prompt optimization of antimicrobial therapy, but the sensitivity of antimicrobial susceptibility testing methods for CRE detection is variable and turnaround time can be slow (6-10). It has been demonstrated that in vitro detection of K. pneumoniae carbapenemase (KPC) expression can be difficult, varying by bacterial species and level of enzyme expression.(This study was presented in part at the 113th American Society for Microbiology General Meeting, Denver, CO, May 2013.)The objective of our study was to evaluate automated microscopy for detection of ertapenem (ETP) resistance in K. pneumoniae and the ability to attribute the mechanism of this resistance to the KPC enzyme. Forty-six K. pneumoniae isolates recovered from clinical specimens obtained at Barnes-Jewish Hospital (St. Louis, MO) and one KPC-negative, extended-spectrum ␤-lactamase (ESBL)-positive K. pneumoniae quality control strain, ATCC 700603, were tested (Table 1). The ertapenem and meropenem (MEM) susceptibility profiles of the isolates were determined by disk diffusion according to CLSI standards (11), and isolates were characterized for bla KPC using a laboratory-developed real-time PCR assay (6).For automated microscopy, bacterial suspensions were centrifuged (12,000 ϫ g for 4 min), washed in 1 mM L-histidine buffer at a pH of 7.2, and resuspended in a low-ionic-strength electrokinetic buffer containing 10 mM L-3,4-dihydroxyphenylalanine (L-DOPA) and 1 mM L-histidine at a pH of 7.0 (reagents from SigmaAldrich, St. Louis, MO). This created an inoculum of approximately 1 ϫ 10 6 CFU/ml for testing, and then automated microscopy was performed. Bacterial inocula were pipetted into independent flow cells of a multichannel disposable fluidic cassette (Accelerate Diagnostics Inc., Tucson, AZ). Bacteria were immobilized on the transparent lower surface of each flow cell using electrokinetic concentration (Fig. 1). Mixtures of antibiotics in Mueller-Hinton broth (Becton, Dickinson, Franklin Lakes, NJ) containing 0.85% noble agar (Affymetrix, Santa Clara, CA) were introduced into each flow cell channel. Dark-field images of each flow cell channel were taken at 10-min intervals during a fixed 3-h antibiotic exposure period. An offline image analyzer tracked each immobilized bacterial progenitor cell a...

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Crystal Structures of KPC-2 β-Lactamase in Complex with 3-Nitrophenyl Boronic Acid and the Penam Sulfone PSR-3-226

Cited by 45 publications

References 35 publications

A Structure-Based Classification of Class A β-Lactamases, a Broadly Diverse Family of Enzymes

A Structure-Based Classification of Class A β-Lactamases, a Broadly Diverse Family of Enzymes

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

Rapid Ertapenem Susceptibility Testing and Klebsiella pneumoniae Carbapenemase Phenotype Detection in Klebsiella pneumoniae Isolates by Use of Automated Microscopy of Immobilized Live Bacterial Cells

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