Evolving Carbapenemases: Can Medicinal Chemists Advance One Step Ahead of the Coming Storm?

Oelschlaeger, Peter; Ni, Ai; DuPrez, Kevin; Welsh, William J.; Toney, Jeffrey H.

doi:10.1021/jm9012938

Cited by 57 publications

(68 citation statements)

References 130 publications

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“…IMP-type enzymes are clinically important due to their broad substrate spectra, insensitivity to currently used b-lactamase inhibitors (clavulanate, sulbactam, and tazobactam), occurrence throughout the world, and the ability of their encoding genes to spread rapidly in bacteria. [5][6][7][8][9][10] In addition, IMP…”

Section: Introductionmentioning

confidence: 99%

Understanding the determinants of substrate specificity in IMP family metallo‐β‐lactamases: The importance of residue 262

et al. 2014

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In Gram-negative bacteria, resistance to b-lactam antibacterials is largely due to b-lactamases and is a growing public health threat. One of the most concerning b-lactamases to evolve in bacteria are the Class B enzymes, the metallo-b-lactamases (MBLs). To date, penams and cephems resistant to hydrolysis by MBLs have not yet been found. As a result of this broad substrate specificity, a better understanding of the role of catalytically important amino acids in MBLs is necessary to design novel b-lactams and inhibitors. Two MBLs, the wild type IMP-1 with serine at position 262, and an engineered variant with valine at the same position (IMP-1-S262V), were previously found to exhibit very different substrate spectra. These findings compelled us to investigate the impact of a threonine at position 262 (IMP-1-S262T) on the substrate spectrum. Here, we explore MBL sequence-structureactivity relationships by predicting and experimentally validating the effect of the S262T substitution in IMP-1. Using site-directed mutagenesis, threonine was introduced at position 262, and the IMP-1-S262T enzyme, as well as the other two enzymes IMP-1 and IMP-1-S262V, were purified and kinetic constants were determined against a range of b-lactam antibacterials. Catalytic efficiencies (k cat /K M ) obtained with IMP-1-S262T and minimum inhibitory concentrations (MICs) observed with bacterial cells expressing the protein were intermediate or comparable to the corresponding values with IMP-1 and IMP-1-S262V, validating the role of this residue in catalysis. Our results reveal the important role of IMP residue 262 in b-lactam turnover and support this approach to predict activities of certain novel MBL variants.Keywords: metallo-b-lactamase; point mutation; antibiotic resistance; enzyme evolution; IMP-1 antibody Abbreviations: BSA, bovine serum albumin; ESI-MS, electrospray ionization mass spectrometry; IPTG, isopropyl b-D-1-thiogalactopyranoside; MBL, metallo-b-lactamase; MIC, minimum inhibitory concentration; MOPS, 3-(N-morpholino)propanesulfonic acid; PAR, 4-(2-pyridylazo)resorcinol; PCR, polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

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Section: Introductionmentioning

confidence: 99%

Understanding the determinants of substrate specificity in IMP family metallo‐β‐lactamases: The importance of residue 262

et al. 2014

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“…[3][4][5][6] One of these blactamases, KPC-2, is becoming a major healthcare threat in the United States and throughout the world. 5,7,8 KPC-2 manifests a very broad substrate profile including penicillins, extended-spectrum cephalosporins, cephamycins, and carbapenems. 9,10 Moreover, Papp-Wallace et al showed that this enzyme is resistant to b-lactamase inhibitors (clavulanic acid, sulbactam, and tazobactam), which are typically given in combination with a b-lactam for the treatment of infections caused by b-lactamase producing pathogens.…”

Section: Introductionmentioning

confidence: 99%

Elucidating the role of Trp105 in the KPC‐2 β‐lactamase

et al. 2010

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The molecular basis of resistance to b-lactams and b-lactam-b-lactamase inhibitor combinations in the KPC family of class A enzymes is of extreme importance to the future design of effective b-lactam therapy. Recent crystal structures of KPC-2 and other class A b-lactamases suggest that Ambler position Trp105 may be of importance in binding b-lactam compounds. Based on this notion, we explored the role of residue Trp105 in KPC-2 by conducting site-saturation mutagenesis at this position. Escherichia coli DH10B cells expressing the Trp105Phe, -Tyr, -Asn, and -His KPC-2 variants possessed minimal inhibitory concentrations (MICs) similar to E. coli cells expressing wild type (WT) KPC-2. Interestingly, most of the variants showed increased MICs to ampicillin-clavulanic acid but not to ampicillin-sulbactam or piperacillin-tazobactam. To explain the biochemical basis of this behavior, four variants (Trp105Phe, -Asn, -Leu, and -Val) were studied in detail. Consistent with the MIC data, the Trp105Phe b-lactamase displayed improved catalytic efficiencies, k cat /K m , toward piperacillin, cephalothin, and nitrocefin, but slightly decreased k cat /K m toward cefotaxime and imipenem when compared to WT b-lactamase. The Trp105Asn variant exhibited increased K m s for all substrates. In contrast, the Trp105Leu and -Val substituted enzymes demonstrated notably decreased catalytic efficiencies (k cat /K m ) for all substrates. With respect to clavulanic acid, the K i s and partition ratios were increased for the Trp105Phe, -Asn, and -Val variants. We conclude that interactions between Trp105 of KPC-2 and the b-lactam are essential for hydrolysis of substrates. Taken together, kinetic and molecular modeling studies define the role of Trp105 in b-lactam and b-lactamase inhibitor discrimination.

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“…etallo-␤-lactamases (MBLs) efficiently inactivate most ␤-lactam antibacterials and have recently raised concerns due to this broad substrate spectrum, their global spread in various Gram-negative bacteria, and the absence of inhibitors for clinical use (1)(2)(3). Zn(II)-bound anionic intermediates of chromogenic ␤-lactams, such as nitrocefin, have been observed during their hydrolysis catalyzed by the MBLs CcrA (4), L1 (5), NDM-1 (6), and VIM-2 (7).…”

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Meropenem and Chromacef Intermediates Observed in IMP-25 Metallo-β-Lactamase-Catalyzed Hydrolysis

Oelschlaeger

Aitha

Yang

et al. 2015

Antimicrob Agents Chemother

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Metallo-␤-lactamases (MBLs) efficiently inactivate most ␤-lactam antibacterials and have recently raised concerns due to this broad substrate spectrum, their global spread in various Gram-negative bacteria, and the absence of inhibitors for clinical use (1-3). Zn(II)-bound anionic intermediates of chromogenic ␤-lactams, such as nitrocefin, have been observed during their hydrolysis catalyzed by the MBLs CcrA (4), L1 (5), NDM-1 (6), and VIM-2 (7). For imipenemase (IMP-1), there might be a nitrocefin intermediate (8), although other studies negate this (9, 10). Tioni and coworkers also observed imipenem and meropenem intermediates in BcII (11). Recently, we found that the variant IMP-25 hydrolyzes meropenem more efficiently than IMP-1 and IMP-6 (12). IMP-6 was previously reported to confer resistance to carbapenems, especially meropenem (13). The increasing k cat for meropenem hydrolysis in the order IMP-1 ¡ IMP-6 ¡ IMP-25 (22 Ϯ 1 s Ϫ1 ¡ 60 Ϯ 10 s Ϫ1 ¡ 100 Ϯ 10 s Ϫ1 , respectively) also translates into increasing MICs of meropenem (16 g/ml ¡ 64 g/ml ¡ 128 g/ml, respectively) (12).To study in more detail the basis of the increased k cat in IMP-25 versus IMP-1, we carried out pre-steady-state kinetic experiments (6) for the hydrolysis of meropenem [6°C, 25 M enzyme (E) and substrate (S) ([E]-to-[S] ratio of 1:1) in 50 mM MOPS (morpholinepropanesulfonic acid) (pH 7.0) supplemented with 100 M Zn(II) ions]. The meropenem substrate (S) was tracked at 310 nm (due to noise at 300 nm), product (P) was tracked at 342 nm, and intermediate (I) was tracked at 390 nm. Details on the determination of extinction coefficients and conversion of the spectral data into concentrations are provided in the supplemental material. With IMP-1, a very small amount of intermediate was observed (Fig. 1E, black line), and the data could not be fitted to a two-phase association/decay function. Substrate disappeared at approximately the same exponential rate (25 Ϯ 3 s Ϫ1 ) at which product appeared (38 Ϯ 12 s Ϫ1 ) (Table 1). In contrast, with IMP-25 (Fig. 1A and B and Table 1), an initial appearance of I at 300 Ϯ 100 s Ϫ1 was followed by its disappearance at 24 Ϯ 8 s Ϫ1 . As with IMP-1, the exponential disappearance rate of S (27 Ϯ 1 s Ϫ1 ) was similar to the appearance rate of P (30 Ϯ 20 s Ϫ1 ). Thus, the observed rates of S disappearance and P appearance are indistinguishable between IMP-1 and IMP-25 under these conditions, and the uncertainty of the product formation rates was rather high. However, a clear formation and decay of intermediate was observed in IMP-25. Intermediate accumulated to no more than 6% of the initial [S], which equals [E], meaning that only up to 6% of the E molecules were bound to I.In order to explore if a clearer picture could be obtained at conditions that are more similar to steady-state conditions, we increased [S] to 100 M, 200 M, and 500 M. Several complications became apparent for the accurate quantification of S and P and, consequently, their disappearance and appearance rates, respectively, at higher concentr...

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Evolving Carbapenemases: Can Medicinal Chemists Advance One Step Ahead of the Coming Storm?

Cited by 57 publications

References 130 publications

Understanding the determinants of substrate specificity in IMP family metallo‐β‐lactamases: The importance of residue 262

Understanding the determinants of substrate specificity in IMP family metallo‐β‐lactamases: The importance of residue 262

Elucidating the role of Trp105 in the KPC‐2 β‐lactamase

Meropenem and Chromacef Intermediates Observed in IMP-25 Metallo-β-Lactamase-Catalyzed Hydrolysis

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