Hormesis in high-throughput screening of antibacterial compounds in E coli

2010 ◽  
Vol 29 (8) ◽  
pp. 667-677 ◽  
Author(s):  
Edward J Calabrese ◽  
George R Hoffmann ◽  
Edward J Stanek ◽  
Marc A Nascarella
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Antibacterial Agents ◽  
Threshold Model ◽  
Concentration Effects ◽  
Data Set ◽  
E Coli ◽  
Model Predictions ◽  
High Throughput Study ◽  
Repeat Measure

This article assesses the response below a toxicological threshold for 1888 antibacterial agents in Escherichia coli, using 11 concentrations with twofold concentration spacing in a high-throughput study. The data set had important strengths such as low variability in the control (2%—3% SD), a repeat measure of all wells, and a built-in replication. Bacterial growth at concentrations below the toxic threshold is significantly greater than that in the controls, consistent with a hormetic concentration response. These findings, along with analyses of published literature and complementary evaluations of concentration-response model predictions of low-concentration effects in yeast, indicate a lack of support for the broadly and historically accepted threshold model for responses to concentrations below the toxic threshold.

Large-scale High-throughput Screening Revealed 5'-(carbonylamino)-2,3'- bithiophene-4'-carboxylate as Novel Template for Antibacterial Agents

2020 ◽  
Vol 17 (5) ◽  
pp. 716-724
Author(s):  
Yan A. Ivanenkov ◽  
Renat S. Yamidanov ◽  
Ilya A. Osterman ◽  
Petr V. Sergiev ◽  
Vladimir A. Aladinskiy ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Antibacterial Agents ◽  
Sos Response ◽  
Luciferase Assay ◽  
Translational Machinery ◽  
E Coli ◽  
New Class

Background: The key issue in the development of novel antimicrobials is a rapid expansion of new bacterial strains resistant to current antibiotics. Indeed, World Health Organization has reported that bacteria commonly causing infections in hospitals and in the community, e.g. E. Coli, K. pneumoniae and S. aureus, have high resistance vs the last generations of cephalosporins, carbapenems and fluoroquinolones. During the past decades, only few successful efforts to develop and launch new antibacterial medications have been performed. This study aims to identify new class of antibacterial agents using novel high-throughput screening technique. Methods: We have designed library containing 125K compounds not similar in structure (Tanimoto coeff.< 0.7) to that published previously as antibiotics. The HTS platform based on double reporter system pDualrep2 was used to distinguish between molecules able to block translational machinery or induce SOS-response in a model E. coli system. MICs for most active chemicals in LB and M9 medium were determined using broth microdilution assay. Results: In an attempt to discover novel classes of antibacterials, we performed HTS of a large-scale small molecule library using our unique screening platform. This approach permitted us to quickly and robustly evaluate a lot of compounds as well as to determine the mechanism of action in the case of compounds being either translational machinery inhibitors or DNA-damaging agents/replication blockers. HTS has resulted in several new structural classes of molecules exhibiting an attractive antibacterial activity. Herein, we report as promising antibacterials. Two most active compounds from this series showed MIC value of 1.2 (5) and 1.8 μg/mL (6) and good selectivity index. Compound 6 caused RFP induction and low SOS response. In vitro luciferase assay has revealed that it is able to slightly inhibit protein biosynthesis. Compound 5 was tested on several archival strains and exhibited slight activity against gram-negative bacteria and outstanding activity against S. aureus. The key structural requirements for antibacterial potency were also explored. We found, that the unsubstituted carboxylic group is crucial for antibacterial activity as well as the presence of bulky hydrophobic substituents at phenyl fragment. Conclusion: The obtained results provide a solid background for further characterization of the 5'- (carbonylamino)-2,3'-bithiophene-4'-carboxylate derivatives discussed herein as new class of antibacterials and their optimization campaign.

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.

Retrospective Analysis of an Experimental High-Throughput Screening Data Set by Recursive Partitioning

2001 ◽  
Vol 3 (3) ◽  
pp. 267-277 ◽  
Author(s):  
A. Michiel van Rhee ◽  
Jon Stocker ◽  
David Printzenhoff ◽  
Chris Creech ◽  
P. Kay Wagoner ◽  
...  
Keyword(s):  
Retrospective Analysis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Recursive Partitioning ◽  
Data Set

scholarly journals Using Weighted Entropy to Rank Chemicals in Quantitative High-Throughput Screening Experiments

2013 ◽  
Vol 19 (3) ◽  
pp. 344-353 ◽  
Author(s):  
Keith R. Shockley
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Nonlinear Models ◽  
Hill Equation ◽  
Parameter Estimates ◽  
Chemical Library ◽  
Data Set ◽  
Screening Experiments ◽  
The Hill ◽  
Weighted Entropy

Quantitative high-throughput screening (qHTS) experiments can simultaneously produce concentration-response profiles for thousands of chemicals. In a typical qHTS study, a large chemical library is subjected to a primary screen to identify candidate hits for secondary screening, validation studies, or prediction modeling. Different algorithms, usually based on the Hill equation logistic model, have been used to classify compounds as active or inactive (or inconclusive). However, observed concentration-response activity relationships may not adequately fit a sigmoidal curve. Furthermore, it is unclear how to prioritize chemicals for follow-up studies given the large uncertainties that often accompany parameter estimates from nonlinear models. Weighted Shannon entropy can address these concerns by ranking compounds according to profile-specific statistics derived from estimates of the probability mass distribution of response at the tested concentration levels. This strategy can be used to rank all tested chemicals in the absence of a prespecified model structure, or the approach can complement existing activity call algorithms by ranking the returned candidate hits. The weighted entropy approach was evaluated here using data simulated from the Hill equation model. The procedure was then applied to a chemical genomics profiling data set interrogating compounds for androgen receptor agonist activity.

Data Analysis of High-Throughput Screening Results:  Application of Multidomain Clustering to the NCI Anti-HIV Data Set

2002 ◽  
Vol 45 (14) ◽  
pp. 3082-3093 ◽  
Author(s):  
Susan Y. Tamura ◽  
Patricia A. Bacha ◽  
Heather S. Gruver ◽  
Ruth F. Nutt
Keyword(s):  
Data Analysis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Data Set ◽  
Anti Hiv

scholarly journals A Homogeneous, High-Throughput Fluorescence Anisotropy-Based DNA Supercoiling Assay

2010 ◽  
Vol 15 (9) ◽  
pp. 1088-1098 ◽  
Author(s):  
Adam Shapiro ◽  
Haris Jahic ◽  
Swati Prasad ◽  
David Ehmann ◽  
Jason Thresher ◽  
...  
Keyword(s):  
High Throughput ◽  
Fluorescence Anisotropy ◽  
High Throughput Screening ◽  
Topoisomerase I ◽  
Dna Supercoiling ◽  
New Drugs ◽  
E Coli ◽  
Cellular Processes ◽  
The Difference ◽  
Cellular Replication

The degree of supercoiling of DNA is vital for cellular processes, such as replication and transcription. DNA topology is controlled by the action of DNA topoisomerase enzymes. Topoisomerases, because of their importance in cellular replication, are the targets of several anticancer and antibacterial drugs. In the search for new drugs targeting topoisomerases, a biochemical assay compatible with automated high-throughput screening (HTS) would be valuable. Gel electrophoresis is the standard method for measuring changes in the extent of supercoiling of plasmid DNA when acted upon by topoisomerases, but this is a low-throughput and laborious method. A medium-throughput method was described previously that quantitatively distinguishes relaxed and supercoiled plasmids by the difference in their abilities to form triplex structures with an immobilized oligonucleotide. In this article, the authors describe a homogeneous supercoiling assay based on triplex formation in which the oligonucleotide strand is labeled with a fluorescent dye and the readout is fluorescence anisotropy. The new assay requires no immobilization, filtration, or plate washing steps and is therefore well suited to HTS for inhibitors of topoisomerases. The utility of this assay is demonstrated with relaxation of supercoiled plasmid by Escherichia coli topoisomerase I, supercoiling of relaxed plasmid by E. coli DNA gyrase, and inhibition of gyrase by fluoroquinolones and nalidixic acid.

scholarly journals High-Throughput Screen for Inhibitors of Transglycosylase and/or Transpeptidase Activities of Escherichia coli Penicillin Binding Protein 1b

2004 ◽  
Vol 48 (1) ◽  
pp. 30-40 ◽  
Author(s):  
B. Chandrakala ◽  
Radha K. Shandil ◽  
Upasana Mehra ◽  
Sudha Ravishankar ◽  
Parvinder Kaur ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
Antibacterial Agents ◽  
Binding Protein ◽  
Type A ◽  
The Other ◽  
Penicillin Binding Protein ◽  
E Coli ◽  
Comparison Of The Results ◽  
High Throughput Assay

ABSTRACT Penicillin binding protein (PBP) 1b of Escherichia coli has both transglycosylase and transpeptidase activities, which are attractive targets for the discovery of new antibacterial agents. A high-throughput assay that detects inhibitors of the PBPs was described previously, but it cannot distinguish them from inhibitors of the MraY, MurG, and lipid pyrophosphorylase. We report on a method that distinguishes inhibitors of both activities of the PBPs from those of the other three enzymes. Radioactive peptidoglycan was synthesized by using E. coli membranes. Following termination of the reaction the products were analyzed in three ways. Wheat germ agglutinin (WGA)-coated scintillation proximity assay (SPA) beads were added to one set, and the same beads together with a detergent were added to a second set. Type A polyethylenimine-coated WGA-coated SPA beads were added to a third set. By comparison of the results of assays run in parallel under the first two conditions, inhibitors of the transpeptidase and transglycosylase could be distinguished from inhibitors of the other enzymes, as the inhibitors of the other enzymes showed similar inhibitory concentrations (IC50s) under both conditions but the inhibitors of the PBPs showed insignificant inhibition in the absence of detergent. Furthermore, comparison of the results of assays run under conditions two and three enabled the distinction of transpeptidase inhibitors. Penicillin and other β-lactams showed insignificant inhibition with type A beads compared with that shown with WGA-coated SPA beads plus detergent. However, inhibitors of the other four enzymes (tunicamycin, nisin, bacitracin, and moenomycin) showed similar IC50s under both conditions. We show that the main PBP being measured under these conditions is PBP 1b. This screen can be used to find novel transglycosylase or transpeptidase inhibitors.

scholarly journals Plasmodium palmitoylation machinery engineered in E. coli for high‐throughput screening of palmitoyl acyl‐transferase inhibitors

FEBS Open Bio ◽  
2019 ◽  
Vol 9 (2) ◽  
pp. 248-264 ◽  
Author(s):  
Preeti Yadav ◽  
R Ayana ◽  
Swati Garg ◽  
Ravi Jain ◽  
Raj Sah ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Acyl Transferase ◽  
E Coli
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