In vitro evaluation of CENTA, a new beta-lactamase-susceptible chromogenic cephalosporin reagent

Jones, Ronald N.; Wilson, Harold W.; Novick, William J.; Barry, Arthur L.; Thornsberry, C

doi:10.1128/jcm.15.5.954-958.1982

Cited by 51 publications

(29 citation statements)

References 10 publications

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“…In effect of OM permeabilization, the enzyme can hydrolyze CENTA’s β-lactam ring. The resulting color change can be measured spectrophotometrically at 405 nm [ 71 ]. The IM permeabilization was monitored with ONPG, a chromogenic β-galactosidase substrate.…”

Section: Methodsmentioning

confidence: 99%

Effect of Disulfide Cyclization of Ultrashort Cationic Lipopeptides on Antimicrobial Activity and Cytotoxicity

Neubauer

Jaśkiewicz

Sikorska

et al. 2020

IJMS

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Ultrashort cationic lipopeptides (USCLs) are considered to be a promising class of antimicrobials with high activity against a broad-spectrum of microorganisms. However, the majority of these compounds are characterized by significant toxicity toward human cells, which hinders their potential application. To overcome those limitations, several approaches have been advanced. One of these is disulfide cyclization that has been shown to improve drug-like characteristics of peptides. In this article the effect of disulfide cyclization of the polar head of N-palmitoylated USCLs on in vitro biological activity has been studied. Lipopeptides used in this study consisted of three or four basic amino acids (lysine and arginine) and cystine in a cyclic peptide. In general, disulfide cyclization of the lipopeptides resulted in peptides with reduced cytotoxicity. Disulfide-cyclized USCLs exhibited improved selectivity between Candida sp., Gram-positive strains and normal cells in contrast to their linear counterparts. Interactions between selected USCLs and membranes were studied by molecular dynamics simulations using a coarse-grained force field. Moreover, membrane permeabilization properties and kinetics were examined. Fluorescence and transmission electron microscopy revealed damage to Candida cell membrane and organelles. Concluding, USCLs are strong membrane disruptors and disulfide cyclization of polar head can have a beneficial effect on its in vitro selectivity between Candida sp. and normal human cells.

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Section: Methodsmentioning

confidence: 99%

Effect of Disulfide Cyclization of Ultrashort Cationic Lipopeptides on Antimicrobial Activity and Cytotoxicity

Neubauer

Jaśkiewicz

Sikorska

et al. 2020

IJMS

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“…β-Lactamase activity, and therefore protease activity, was measured by the change in absorbance from β-lactamase substrate, 7-(2-thienylacetamido)-3-((3-carboxy-4-nitrophenyl)thiomethyl)-cephalosporonic acid (CENTA) 50 to hydrolysed product 51. 204 To prevent the BLIP from remaining Please do not adjust margins Please do not adjust margins bound to the β-lactamase after proteasecatalysed amide hydrolysis, which would reduce the potential positive output signal, a number of β-lactamase mutants were synthesised and tested within the assay. Through this optimisation, a β-lactamase mutant with a lower binding affinity to BLIP was able to deliver a 68-fold signal increase from the background reaction.…”

Section: Double-catalyst Signal Amplificationmentioning

confidence: 99%

Approaches towards molecular amplification for sensing

Goggins

Frost

2016

Analyst

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Diagnostic assays that rely on molecular interactions have come a long way; from initial reversible detection systems towards irreversible reaction indicator-based methods. More recently, the emergence of innovative molecular amplification methodologies has revolutionised sensing, allowing diagnostic assays to achieve ultra-low limits of detection. There have been a significant number of molecular amplification approaches developed over recent years to accommodate the wide variety of analytes that require sensitive detection. To celebrate this achievement, this comprehensive critical review has been compiled to give a broad overview of the many different approaches used to attain amplification in sensing with an aim to inspire the next generation of diagnostic assays looking to achieve the ultimate detection limit. This review has been created with the focus on how each conceptually unique molecular amplification methodology achieves amplification, not just its sensitivity, while highlighting any key processes. Excluded are any references that were not found to contain an obvious molecular amplifier or amplification component, or that did not use an appropriate signal readout that could be incorporated into a sensing application. Additionally, methodologies where amplification is achieved through advances in instrumentation are also excluded. Depending upon the type of approach employed, amplification strategies are divided into four categories: target, label, signal or receptor amplification. More recent, more complex protocols combine a number of approaches and are therefore categorised by which amplification component described within was considered as the biggest advancement. The advantages and disadvantages of each methodology are discussed along with any limits of detection, if stated in the original article. Any subsequent use of the methodology within sensing or any other application is also mentioned to draw attention to its practicality. The importance of amplification within sensing is wholly emphasised while perspectives on the future direction of the field are also shared.

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“…Because the metallo-beta-lactamases, P2A and NDM, require zinc ions for activity [23], 0.1 mM ZnSO 4 was added to the bacterial growth media. Cell lysates from bacterial strains showing the highest protein expression levels by SDS-PAGE were evaluated for biological activity using CENTA as a chromogenic substrate for beta-lactamase activity [24,25]. Briefly, the assay was performed in a 50 mM NaH 2 PO 4 buffer, pH 7.0 with supplementation of 0.1 mM ZnSO 4 , with CENTA (Calbiochem) at 50 µg/mL.…”

Section: Methodsmentioning

confidence: 99%

Identification, Characterization, and Formulation of a Novel Carbapenemase Intended to Prevent Antibiotic-Mediated Gut Dysbiosis

Connelly

Parsley²,

Ge³

et al. 2019

Microorganisms

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Antibiotics can damage the gut microbiome leading to opportunistic infections and the emergence of antibiotic resistance. Microbiome protection via antibiotic inactivation in the gastrointestinal (GI) tract represents a strategy to limit antibiotic exposure of the colonic microbiota. Proof of concept for this approach was achieved with an orally-administered beta-lactamase enzyme, SYN-004 (ribaxamase), that was demonstrated to degrade ceftriaxone excreted into the GI tract and protect the gut microbiome from antibiotic-mediated dysbiosis. Ribaxamase efficiently degrades penicillin and cephalosporin beta-lactam antibiotics, but is not active against carbapenems. To expand this microbiome protection strategy to include all classes of beta-lactams, three distinct carbapenemases were evaluated for manufacturability, antibiotic degradation spectrum, and stability in human intestinal fluid. E. coli production strains were generated for P2A, a novel metallo-enzyme isolated from B. cereus, New Delhi metallo-beta-lactamase (NDM), and Klebsiella pneumoniae carbapenemase (KPC). While all three enzymes effectively inactivated a broad range of antibiotics, including penicillins, most cephalosporins, and carbapenems in vitro, only P2A retained biological activity when incubated with human chyme. As functional stability in the intestinal tract is a key requirement for an orally-delivered enzyme, P2A was chosen as a potential clinical candidate. An enteric formulation of P2A was developed, called SYN-006, that was inert under high acid conditions, with enzyme dissolution occurring at pH > 5.5. SYN-006 has the potential to expand microbiome protection via antibiotic inactivation to include all classes of beta-lactam antibiotics.

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In vitro evaluation of CENTA, a new beta-lactamase-susceptible chromogenic cephalosporin reagent

Cited by 51 publications

References 10 publications

Effect of Disulfide Cyclization of Ultrashort Cationic Lipopeptides on Antimicrobial Activity and Cytotoxicity

Effect of Disulfide Cyclization of Ultrashort Cationic Lipopeptides on Antimicrobial Activity and Cytotoxicity

Approaches towards molecular amplification for sensing

Identification, Characterization, and Formulation of a Novel Carbapenemase Intended to Prevent Antibiotic-Mediated Gut Dysbiosis

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