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

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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Inhibitors of RecA Activity Discovered by High-Throughput Screening: Cell-Permeable Small Molecules Attenuate the SOS Response in Escherichia coli

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057109342126 ◽

2009 ◽

Vol 14 (9) ◽

pp. 1092-1101 ◽

Cited By ~ 40

Author(s):

Tim J. Wigle ◽

Jonathan Z. Sexton ◽

Anna V. Gromova ◽

Mallinath B. Hadimani ◽

Mark A. Hughes ◽

...

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

Small Molecules ◽

High Throughput ◽

High Throughput Screening ◽

Bacterial Infections ◽

Sos Response ◽

Cellular Targets ◽

Novel Target

The phenomenon of antibiotic resistance has created a need for the development of novel antibiotic classes with nonclassical cellular targets. Unfortunately, target-based drug discovery against proteins considered essential for in vitro bacterial viability has yielded few new therapeutic classes of antibiotics. Targeting the large proportion of genes considered nonessential that have yet to be explored by high-throughput screening, for example, RecA, can complement these efforts. Recent evidence suggests that RecA-controlled processes are responsible for tolerance to antibiotic chemotherapy and are involved in pathways that ultimately lead to full-fledged antibiotic resistance. Therefore inhibitors of RecA may serve as therapeutic adjuvants in combination chemotherapy of bacterial infectious diseases. Toward the goal of validating RecA as a novel target in the chemotherapy of bacterial infections, the authors have screened 35,780 small molecules against RecA. In total, 80 small molecules were identified as primary hits and could be clustered in 6 distinct chemotype clades. The most potent class of hits was further examined, and 1 member compound was found to inhibit RecA-mediated strand exchange and prevent ciprofloxacin-induced SOS expression in Escherichia coli. This compound represents the first small molecule demonstrating an ability to inhibit the bacterial SOS response in live bacterial cell cultures. ( Journal of Biomolecular Screening 2009:1092-1101)

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

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057105281173 ◽

2005 ◽

Vol 10 (7) ◽

pp. 653-657 ◽

Cited By ~ 22

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.

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Development of a Simple High-Throughput Screening Protocol Based on Biosynthetic Activity of Mycobacterium tuberculosis Glutamine Synthetase for the Identification of Novel Inhibitors

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057106292798 ◽

2006 ◽

Vol 11 (8) ◽

pp. 1035-1042 ◽

Cited By ~ 6

Author(s):

Upasana Singh ◽

Dhiman Sarkar

Keyword(s):

Enzyme Activity ◽

Mycobacterium Tuberculosis ◽

Glutamine Synthetase ◽

High Throughput ◽

High Throughput Screening ◽

Quantitative Estimation ◽

Signal To Noise ◽

Chemical Library ◽

Validation Data ◽

Screening Protocol

A high-throughput screening protocol has been developed for Mycobacterium tuberculosis glutamine synthetase by quantitative estimation of inorganic phosphate. The Km values determined at pH 6.8 are 22 mM for L-glutamic acid, 0.75 mM for NH4Cl, 3.25 mM for MgCl2, and 2.5 mM for adenosine triphosphate. The Km value for glutamine is affected significantly by the increase in pH of assay buffer. At the saturating level of the substrate, the enzyme activity at pH 6.8 and 25° C is found to be linear up to 3 h. The reduction of enzyme activity is negligible even in presence of 10% DMSO. The Z′ factor and signal-to-noise ratio are found to be 0.75 and 6.18, respectively, when the enzyme is used at 62.5 μg/ml concentration. The IC50 values obtained at pH 6.8 for both L-methionine S-sulfoximine and DL-phosphothriacin are 500 μM and 30 μM, respectively, which is lowest compared to the values obtained at other pH levels. The Beckman Coulter high-throughput screening platform was found to take 5 h 9 min to complete the screening of 60 plates. For each assay plate, a replica plate is used to normalize the data. Screening of 1164 natural product fractions/extracts and synthetic molecules from an in-house library was able to identify 12 samples as confirmed hits. Altogether, the validation data from screening of a small set of an in-house library coupled with Z′ and signal-to-noise values indicate that the protocol is robust for high-throughput screening of a diverse chemical library.

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A high-throughput screening assay based on automated microscopy for monitoring antibiotic susceptibility of Mycobacterium tuberculosis phenotypes

BMC Microbiology ◽

10.1186/s12866-021-02212-3 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Sadaf Kalsum ◽

Blanka Andersson ◽

Jyotirmoy Das ◽

Thomas Schön ◽

Maria Lerm

Keyword(s):

Mycobacterium Tuberculosis ◽

High Throughput ◽

High Throughput Screening ◽

Live Cell Imaging ◽

Cell Imaging ◽

Imaging System ◽

Aggregate Size ◽

Live Cell ◽

Screening Assay ◽

High Throughput Screening Assay

Abstract Background Efficient high-throughput drug screening assays are necessary to enable the discovery of new anti-mycobacterial drugs. The purpose of our work was to develop and validate an assay based on live-cell imaging which can monitor the growth of two distinct phenotypes of Mycobacterium tuberculosis and to test their susceptibility to commonly used TB drugs. Results Both planktonic and cording phenotypes were successfully monitored as fluorescent objects using the live-cell imaging system IncuCyte S3, allowing collection of data describing distinct characteristics of aggregate size and growth. The quantification of changes in total area of aggregates was used to define IC50 and MIC values of selected TB drugs which revealed that the cording phenotype grew more rapidly and displayed a higher susceptibility to rifampicin. In checkerboard approach, testing pair-wise combinations of sub-inhibitory concentrations of drugs, rifampicin, linezolid and pretomanid demonstrated superior growth inhibition of cording phenotype. Conclusions Our results emphasize the efficiency of using automated live-cell imaging and its potential in high-throughput whole-cell screening to evaluate existing and search for novel antimycobacterial drugs.

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A High Throughput Screening Assay for Anti-Mycobacterial Small Molecules Based on Adenylate Kinase Release as a Reporter of Cell Lysis

PLoS ONE ◽

10.1371/journal.pone.0129234 ◽

2015 ◽

Vol 10 (6) ◽

pp. e0129234 ◽

Cited By ~ 11

Author(s):

Lauren Forbes ◽

Katherine Ebsworth-Mojica ◽

Louis DiDone ◽

Shao-Gang Li ◽

Joel S. Freundlich ◽

...

Keyword(s):

Small Molecules ◽

High Throughput ◽

Adenylate Kinase ◽

High Throughput Screening ◽

Cell Lysis ◽

Screening Assay ◽

High Throughput Screening Assay ◽

Adenylate Kinase Release

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00537-16 ◽

2016 ◽

Vol 60 (10) ◽

pp. 5995-6002 ◽

Cited By ~ 7

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.

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