High Throughput Screening Identifies Novel Inhibitors of Escherichia coli Dihydrofolate Reductase that Are Competitive with Dihydrofolate.

ChemInform ◽  
2003 ◽  
Vol 34 (43) ◽  
Author(s):  
Michela Zolli-Juran ◽  
Jonathan D. Cechetto ◽  
Rebecca Hartlen ◽  
Denis M. Daigle ◽  
Eric D. Brown
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
Dihydrofolate Reductase ◽  
High Throughput Screening

scholarly journals Experimental Screening of Dihydrofolate Reductase Yields a “Test Set” of 50,000 Small Molecules for a Computational Data-Mining and Docking Competition

2005 ◽  
Vol 10 (7) ◽  
pp. 653-657 ◽  
Author(s):  
Nadine H. Elowe ◽  
Jan E. Blanchard ◽  
Jonathan D. Cechetto ◽  
Eric D. Brown
Keyword(s):  
Escherichia Coli ◽  
Data Mining ◽  
Small Molecules ◽  
High Throughput ◽  
Dihydrofolate Reductase ◽  
High Throughput Screening ◽  
Valuable Resource ◽  
Training Set ◽  
Test Set ◽  
Computational Data

High-throughput screening (HTS) generates an abundance of data that are a valuable resource to be mined. Dockers and data miners can use “real-world” HTS data to test and further develop their tools. A screen of 50,000 diverse small molecules was carried out against Escherichia coli dihydrofolate reductase (DHFR) and compared with a previous screen of 50,000 compounds against the same target. Identical assays and conditions were maintained for both studies. Prior to the completion of the second screen, the original screening data were publicly released for use as a “training set,” and computational chemists and data analysts were challenged to predict the activity of compounds in this second “test set.” Upon completion, the primary screen of the test set generated no potent inhibitors of DHFR activity.

High throughput screening identifies novel inhibitors of Escherichia coli dihydrofolate reductase that are competitive with dihydrofolate

2003 ◽  
Vol 13 (15) ◽  
pp. 2493-2496 ◽  
Author(s):  
Michela Zolli-Juran ◽  
Jonathan D. Cechetto ◽  
Rebecca Hartlen ◽  
Denis M. Daigle ◽  
Eric D. Brown
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
Dihydrofolate Reductase ◽  
High Throughput Screening

scholarly journals A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli

2016 ◽  
Vol 60 (10) ◽  
pp. 5995-6002 ◽  
Author(s):  
Kristin R. Baker ◽  
Bimal Jana ◽  
Henrik Franzyk ◽  
Luca Guardabassi
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
High Throughput Screening ◽  
Gram Negative Bacteria ◽  
Chromogenic Substrate ◽  
Intrinsic Resistance ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Screening Platform

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.

scholarly journals High-throughput Screening and Sensitized Bacteria Identify an M. tuberculosis Dihydrofolate Reductase Inhibitor with Whole Cell Activity

PLoS ONE ◽  
2012 ◽  
Vol 7 (6) ◽  
pp. e39961 ◽  
Author(s):  
Anuradha Kumar ◽  
Meng Zhang ◽  
Linyun Zhu ◽  
Reiling P. Liao ◽  
Charles Mutai ◽  
...  
Keyword(s):  
High Throughput ◽  
Dihydrofolate Reductase ◽  
High Throughput Screening ◽  
Reductase Inhibitor ◽  
Cell Activity ◽  
Whole Cell

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

2005 ◽  
Vol 10 (7) ◽  
pp. 725-729 ◽  
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.

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

2004 ◽  
Vol 48 (3) ◽  
pp. 897-902 ◽  
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.

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

2002 ◽  
Vol 7 (2) ◽  
pp. 119-125 ◽  
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.

scholarly journals A High-Throughput Screen of the GTPase Activity of Escherichia coli EngA to Find an Inhibitor of Bacterial Ribosome Biogenesis

2013 ◽  
Vol 18 (7) ◽  
pp. 830-836 ◽  
Author(s):  
Amrita Bharat ◽  
Jan E. Blanchard ◽  
Eric D. Brown
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
High Throughput Screening ◽  
Ribosome Biogenesis ◽  
Signal Amplitude ◽  
Low Noise ◽  
High Throughput Screen ◽  
Reaction Conditions ◽  
Screening Process ◽  
Bacterial Ribosome

The synthesis of ribosomes is an essential process, which is aided by a variety of trans-acting factors in bacteria. Among these is a group of GTPases essential for bacterial viability and emerging as promising targets for new antibacterial agents. Herein, we describe a robust high-throughput screening process for inhibitors of one such GTPase, the Escherichia coli EngA protein. The primary screen employed an assay of phosphate production in a 384-well density. Reaction conditions were chosen to maximize sensitivity for the discovery of competitive inhibitors while maintaining a strong signal amplitude and low noise. In a pilot screen of 31,800 chemical compounds, 44 active compounds were identified. Furthermore, we describe the elimination of nonspecific inhibitors that were detergent sensitive or reactive as well as those that interfered with the high-throughput phosphate assay. Four inhibitors survived these common counterscreens for nonspecificity, but these chemicals were also inhibitors of the unrelated enzyme dihydrofolate reductase, suggesting that they too were promiscuously active. The high-throughput screen of the EngA protein described here provides a meticulous pilot study in the search for specific inhibitors of GTPases involved in ribosome biogenesis.

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