Facile, reagentless and in situ release of Escherichia coli intracellular enzymes by heat-inducible autolytic vector for high-throughput screening

ABSTRACTThe envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measureEscherichia colienvelope permeability to a β-galactosidase chromogenic substrate. The signal produced by cytoplasmic β-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds andE. coligene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinctE. colistrains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 μM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R> 0.5 μg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

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Integration of an In Situ MALDI-Based High-Throughput Screening Process: A Case Study with Receptor Tyrosine Kinase c-MET

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555217727701 ◽

2017 ◽

Vol 22 (10) ◽

pp. 1203-1210 ◽

Cited By ~ 5

Author(s):

Katrin Beeman ◽

Jens Baumgärtner ◽

Manuel Laubenheimer ◽

Karlheinz Hergesell ◽

Martin Hoffmann ◽

...

Keyword(s):

Drug Discovery ◽

High Throughput ◽

High Throughput Screening ◽

Maldi Ms ◽

Kinase Assay ◽

Label Free ◽

Screening Process ◽

Label Free Detection ◽

Ic50 Values

Mass spectrometry (MS) is known for its label-free detection of substrates and products from a variety of enzyme reactions. Recent hardware improvements have increased interest in the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS for high-throughput drug discovery. Despite interest in this technology, several challenges remain and must be overcome before MALDI-MS can be integrated as an automated “in-line reader” for high-throughput drug discovery. Two such hurdles include in situ sample processing and deposition, as well as integration of MALDI-MS for enzymatic screening assays that usually contain high levels of MS-incompatible components. Here we adapt our c-MET kinase assay to optimize for MALDI-MS compatibility and test its feasibility for compound screening. The pros and cons of the Echo (Labcyte) as a transfer system for in situ MALDI-MS sample preparation are discussed. We demonstrate that this method generates robust data in a 1536-grid format. We use the MALDI-MS to directly measure the ratio of c-MET substrate and phosphorylated product to acquire IC50 curves and demonstrate that the pharmacology is unaffected. The resulting IC50 values correlate well between the common label-based capillary electrophoresis and the label-free MALDI-MS detection method. We predict that label-free MALDI-MS-based high-throughput screening will become increasingly important and more widely used for drug discovery.

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Development of a Simple Assay Protocol for High-Throughput Screening of Mycobacterium tuberculosis Glutamine Synthetase for the Identification of Novel Inhibitors

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057105278013 ◽

2005 ◽

Vol 10 (7) ◽

pp. 725-729 ◽

Cited By ~ 4

Author(s):

Upasana Singh ◽

Vinita Panchanadikar ◽

Dhiman Sarkar

Keyword(s):

Escherichia Coli ◽

Mycobacterium Tuberculosis ◽

Glutamine Synthetase ◽

Small Molecules ◽

High Throughput ◽

High Throughput Screening ◽

Coli Strain ◽

Compound Library ◽

Essential Enzyme ◽

Assay Protocol

Mycobacterium tuberculosis glutamine synthetase (GS) is an essential enzyme involved in the pathogenicity of the organism. The screening of a compound library using a robust high-throughput screening (HTS) assay is currently thought to be the most efficient way of getting lead molecules, which are potent inhibitors for this enzyme. The authors have purified the enzyme to a >90% level from the recombinant Escherichia coli strain YMC21E, and it was used for partial characterization as well as standardization experiments. The results indicated that the Kmof the enzyme for L-glutamine and hydroxylamine were 60 mM and 8.3 mM, respectively. The Km for ADP, arsenate, and Mn2+ were 2 [.proportional]M, 5 [.proportional]M, and 25 [.proportional]M, respectively. When the components were adjusted according to their Km values, the activity remained constant for at least 3 h at both 25° C and 37° C. The Z′ factor determined in microplate format indicated robustness of the assay. When the signal/noise ratios were determined for different assay volumes, it was observed that the 200-[.proportional]l volume was found to be optimum. The DMSO tolerance of the enzyme was checked up to 10%, with minimal inhibition. The IC50 value determined for L-methionine S-sulfoximine on the enzyme activity was 3 mM. Approximately 18,000 small molecules could be screened per day using this protocol by a Beckman Coulter HTS setup.

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Method with high-throughput screening potential for antioxidative substances using Escherichia coli biosensor katG′::lux

Journal of Microbiological Methods ◽

10.1016/j.mimet.2015.08.018 ◽

2015 ◽

Vol 118 ◽

pp. 78-80 ◽

Cited By ~ 4

Author(s):

Jenni Tienaho ◽

Tytti Sarjala ◽

Robert Franzén ◽

Matti Karp

Keyword(s):

Escherichia Coli ◽

High Throughput ◽

High Throughput Screening

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Fluorescence Detection-Based Functional Assay for High-Throughput Screening for MraY

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.3.897-902.2004 ◽

2004 ◽

Vol 48 (3) ◽

pp. 897-902 ◽

Cited By ~ 51

Author(s):

Thérèse Stachyra ◽

Christophe Dini ◽

Paul Ferrari ◽

Ahmed Bouhss ◽

Jean van Heijenoort ◽

...

Keyword(s):

Escherichia Coli ◽

High Pressure ◽

Cell Wall ◽

High Throughput ◽

High Throughput Screening ◽

Reaction Medium ◽

The Other ◽

Functional Assay ◽

Liquid Chromatography Separation ◽

Fluorescent Derivative

ABSTRACT We have developed a novel assay specific to MraY, which catalyzes the first membrane step in the biosynthesis of bacterial cell wall peptidoglycan. This was accomplished by using UDP-MurNAc-Nε -dansylpentapeptide, a fluorescent derivative of the MraY nucleotide substrate, and a partially purified preparation of MraY solubilized from membranes of an Escherichia coli overproducing strain. Two versions of the assay were developed, one consisting of the high-pressure liquid chromatography separation of the substrate and product (dansylated lipid I) and the other, without separation and adapted to the high-throughput format, taking advantage of the different fluorescence properties of the nucleotide and lipid I in the reaction medium. The latter assay was validated with a set of natural and synthetic MraY inhibitors.

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An Escherichia coli Biosensor Strain for Amplified and High Throughput Detection of Antimicrobial Agents

CrossRef Listing of Deleted DOIs ◽

10.1177/108705710200700204 ◽

2002 ◽

Vol 7 (2) ◽

pp. 119-125 ◽

Cited By ~ 15

Author(s):

Minna-Liisa Anko ◽

Jussi Kurittu ◽

Matti Karp

Keyword(s):

Escherichia Coli ◽

High Throughput ◽

High Throughput Screening ◽

Antimicrobial Agents ◽

Test Organism ◽

Plasmid Copy Number ◽

Host Cells ◽

Reporter System ◽

Plasmid Copy ◽

K 12

We report here the construction of a bacterial reporter system for high-throughput screening of antimicrobial agents. The test organism is the Escherichia coli K-12 strain carrying luciferase genes luxC, luxD, luxA, luxB, and luxE from the bioluminescent bacterium Photorhabdus luminescens in a runaway replication plasmid. The replication of the plasmid can be induced, resulting in a change of the plasmid copy number from 1-2/cell to several hundreds per cell within tens of minutes. This increase in plasmid copies is independent of the replication of the host cells. The system will therefore amplify the effects of antibiotics inhibiting bacterial replication machinery, such as fluoroquinolones, and the inhibitory effects can be measured in real time by luminometry. The biosensor was compared with a strain engineered to emit light constitutively, and it was shown to be much more sensitive to various antibiotics than conventional overnight cultivation methods. The approach shows great potential for high-throughput screening of new compounds.

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