BLIP-II Is a Highly Potent Inhibitor of Klebsiella pneumoniae Carbapenemase (KPC-2)

Brown, Nicholas G.; Chow, Dar‐Chone; Palzkill, Timothy

doi:10.1128/aac.00215-13

Cited by 15 publications

(12 citation statements)

References 29 publications

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“…The resulting plasmid was used to express and purify the KPC-2 enzyme and also used as a template for site-directed mutagenesis and subsequent expression of mutant enzymes in E . coli RB791 [ 47 ].…”

Section: Methodsmentioning

confidence: 99%

Natural Variants of the KPC-2 Carbapenemase have Evolved Increased Catalytic Efficiency for Ceftazidime Hydrolysis at the Cost of Enzyme Stability

2015

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The spread of β-lactamases that hydrolyze penicillins, cephalosporins and carbapenems among Gram-negative bacteria has limited options for treating bacterial infections. Initially, Klebsiella pneumoniae carbapenemase-2 (KPC-2) emerged as a widespread carbapenem hydrolyzing β-lactamase that also hydrolyzes penicillins and cephalosporins but not cephamycins and ceftazidime. In recent years, single and double amino acid substitution variants of KPC-2 have emerged among clinical isolates that show increased resistance to ceftazidime. Because it confers multi-drug resistance, KPC β-lactamase is a threat to public health. In this study, the evolution of KPC-2 function was determined in nine clinically isolated variants by examining the effects of the substitutions on enzyme kinetic parameters, protein stability and antibiotic resistance profile. The results indicate that the amino acid substitutions associated with KPC-2 natural variants lead to increased catalytic efficiency for ceftazidime hydrolysis and a consequent increase in ceftazidime resistance. Single substitutions lead to modest increases in catalytic activity while the double mutants exhibit significantly increased ceftazidime hydrolysis and resistance levels. The P104R, V240G and H274Y substitutions in single and double mutant combinations lead to the largest increases in ceftazidime hydrolysis and resistance. Molecular modeling suggests that the P104R and H274Y mutations could facilitate ceftazidime hydrolysis through increased hydrogen bonding interactions with the substrate while the V240G substitution may enhance backbone flexibility so that larger substrates might be accommodated in the active site. Additionally, we observed a strong correlation between gain of catalytic function for ceftazidime hydrolysis and loss of enzyme stability, which is in agreement with the ‘stability-function tradeoff’ phenomenon. The high Tm of KPC-2 (66.5°C) provides an evolutionary advantage as compared to other class A enzymes such as TEM (51.5°C) and CTX-M (51°C) in that it can acquire multiple destabilizing substitutions without losing the ability to fold into a functional enzyme.

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Section: Methodsmentioning

confidence: 99%

Natural Variants of the KPC-2 Carbapenemase have Evolved Increased Catalytic Efficiency for Ceftazidime Hydrolysis at the Cost of Enzyme Stability

2015

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“…Alternatively, “narrow spectrum” inhibitors should not be discounted for therapeutic purposes as they will likely cause less damage to the patients beneficial microbiome (Boucher et al, 2017 ). Another approach is to utilize naturally observed protein inhibitors of β-lactamases from Streptomyces , termed β-lactamase inhibitor proteins (BLIPs), which can be altered to modulate β-lactamase specificity (Brown et al, 2013 ; Chow et al, 2016 ; Adamski and Palzkill, 2017a , b ); peptides derived from BLIPs have been shown to have antimicrobial activity (Alaybeyoglu et al, 2015 ).…”

Section: Additional Inhibitor Design Approachesmentioning

confidence: 99%

Exploring Additional Dimensions of Complexity in Inhibitor Design for Serine β-Lactamases: Mechanistic and Intra- and Inter-molecular Chemistry Approaches

Akker

Bonomo

2018

Front. Microbiol.

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As a bacterial resistance strategy, serine β-lactamases have evolved from cell wall synthesizing enzymes known as penicillin-binding proteins (PBP), by not only covalently binding β-lactam antibiotics but, also acquiring mechanisms of deacylating these antibiotics. This critical deacylation step leads to release of hydrolyzed and inactivated β-lactams, thereby providing resistance for the bacteria against these antibiotics targeting the cell wall. To combat β-lactamase-mediated antibiotic resistance, numerous β-lactamase inhibitors were developed that utilize various strategies to inactivate the β-lactamase. Most of these compounds are “mechanism-based” inhibitors that in some manner mimic the β-lactam substrate, having a carbonyl moiety and a negatively charged carboxyl or sulfate group. These compounds form a covalent adduct with the catalytic serine via an initial acylation step. To increase the life-time of the inhibitory covalent adduct intermediates, a remarkable array of different strategies was employed to improve inhibition potency. Such approaches include post-acylation intra- and intermolecular chemical rearrangements as well as affecting the deacylation water. These approaches transform the inhibitor design process from a 3-dimensional problem (i.e., XYZ coordinates) to one with additional dimensions of complexity as the reaction coordinate and time spent at each chemical state need to be taken into consideration. This review highlights the mechanistic intricacies of the design efforts of the β-lactamase inhibitors which so far have resulted in the development of “two generations” and 5 clinically available inhibitors.

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“…11 The first member of the KPC family was identified in North Carolina in 1996 and has since spread to many other countries. 12 Although initially identified in K. pneumoniae , KPC has also been discovered in other Gram-negative pathogens, mainly belonging to the Enterobacteriaceae family (Carbapenem-resistant Enterobacteriaceae or CRE). 13 The bla KPC gene encodes a 293 amino acid enzyme, and there are 23 variants of KPC (KPC-2 through KPC-24) that differ from one another by one or two amino acid changes.…”

Section: Introductionmentioning

confidence: 99%

Molecular Basis of Substrate Recognition and Product Release by the Klebsiella pneumoniae Carbapenemase (KPC-2)

2017

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Carbapenem-resistant Enterobacteriaceae (CRE) are resistant to most β-lactam antibiotics due to production of the KPC-2 class A β-lactamase. Here we present the first product complex crystal structures of KPC-2 with β-lactam antibiotics, containing hydrolyzed cefotaxime and faropenem. They provide experimental insights into substrate recognition by KPC-2 and its unique cephalosporinase/carbapenemase activity. These structures also represent the first product complexes for a wild type serine β-lactamase, elucidating the product release mechanism of these enzymes in general.

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BLIP-II Is a Highly Potent Inhibitor of Klebsiella pneumoniae Carbapenemase (KPC-2)

Cited by 15 publications

References 29 publications

Natural Variants of the KPC-2 Carbapenemase have Evolved Increased Catalytic Efficiency for Ceftazidime Hydrolysis at the Cost of Enzyme Stability

Natural Variants of the KPC-2 Carbapenemase have Evolved Increased Catalytic Efficiency for Ceftazidime Hydrolysis at the Cost of Enzyme Stability

Exploring Additional Dimensions of Complexity in Inhibitor Design for Serine β-Lactamases: Mechanistic and Intra- and Inter-molecular Chemistry Approaches

Molecular Basis of Substrate Recognition and Product Release by the Klebsiella pneumoniae Carbapenemase (KPC-2)

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