scholarly journals High-Throughput Microplate Phosphorylation Assays Based on DevR-DevS/Rv2027c 2-Component Signal Transduction Pathway to Screen for Novel Antitubercular Compounds

2005 ◽  
Vol 10 (3) ◽  
pp. 215-224 ◽  
Author(s):  
Deepak Kumar Saini ◽  
Jaya Sivaswami Tyagi
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Response Regulator ◽  
Signal Transduction Pathway ◽  
Cellulose Ester ◽  
Reaction Conditions ◽  
Component Systems ◽  
Novel Target ◽  
Radioactivity Retention ◽  
Transfer Properties

DevR-DevS (Rv3133c-Rv3132c) and DevR-Rv2027c have been established through their autophosphorylation and phospho-transfer properties to constitute bonafide regulatory 2-component systems of Mycobacterium tuberculosis. DevR has also been shown by others to play a key regulatory role in the expression of M. tuberculosis genes comprising the dormancy regulon. The authors describe high-throughput phosphorylation assays in a microplate format using DevS and Rv2027c histidine kinases and DevR response regulator proteins from M. tuberculosis. The assays were designed to measure [γ-32P]ATP-dependent autophosphorylation of DevS/Rv2027c and also the phosphotransfer reaction to DevR. First, the optimal reaction conditions were established using the conventional method of radiolabeling the 2-component proteins by [γ-32P]ATP and followed by gel electrophoresis-based analysis. Next, the assays were converted to a high-throughput format in which the radiolabeled protein retained on a filter using mixed cellulose ester-based 96-well filter plates was analyzed for radioactivity retention by scintillation counting. The utility of these assays to screen for inhibitors is illustrated using 2-mercaptobenzimidazole, ethidium bromide, and EDTA. The high quality and flexibility of these assays will enable their use in high-throughput screening for new antitubercular compounds directed against 2-component systems that comprise a novel target in dormant mycobacteria.

scholarly journals A rapid, inexpensive yeast-based dual-fluorescence assay of programmed—1 ribosomal frameshifting for high-throughput screening

2011 ◽  
Vol 39 (14) ◽  
pp. e97-e97 ◽  
Author(s):  
Rasa Rakauskaitė ◽  
Pei-Yu Liao ◽  
Michael H. J. Rhodin ◽  
Kelvin Lee ◽  
Jonathan D. Dinman
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Fluorescence Assay ◽  
Dual Fluorescence ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ribosomal Frameshifting ◽  
Reading Frame ◽  
Endogenous Viruses ◽  
Efficient Replication ◽  
Novel Target

Abstract Programmed −1 ribosomal frameshifting (−1 PRF) is a mechanism that directs elongating ribosomes to shift-reading frame by 1 base in the 5′ direction that is utilized by many RNA viruses. Importantly, rates of −1 PRF are fine-tuned by viruses, including Retroviruses, Coronaviruses, Flavivriuses and in two endogenous viruses of the yeast Saccharomyces cerevisiae, to deliver the correct ratios of different viral proteins for efficient replication. Thus, −1 PRF presents a novel target for antiviral therapeutics. The underlying molecular mechanism of −1 PRF is conserved from yeast to mammals, enabling yeast to be used as a logical platform for high-throughput screens. Our understanding of the strengths and pitfalls of assays to monitor −1 PRF have evolved since the initial discovery of −1 PRF. These include controlling for the effects of drugs on protein expression and mRNA stability, as well as minimizing costs and the requirement for multiple processing steps. Here we describe the development of an automated yeast-based dual fluorescence assay of −1 PRF that provides a rapid, inexpensive automated pipeline to screen for compounds that alter rates of −1 PRF which will help to pave the way toward the discovery and development of novel antiviral therapeutics.

scholarly journals Preparation of Metal Oxides Containing ppm Levels of Pd as Catalysts for the Reduction of Nitroarene and Evaluation of Their Catalytic Activity by the Fluorescence-Based High-Throughput Screening Method

Catalysts ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 542
Author(s):  
Taeho Lim ◽  
Min Su Han
Keyword(s):  
Catalytic Activity ◽  
Metal Oxides ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Reduction Reaction ◽  
Precipitation Method ◽  
Reaction Conditions ◽  
One Pot ◽  
Co Precipitation

Herein, an easily accessible and efficient green method for the reduction of nitroarene compounds was developed using metal oxide catalysts. Heterogeneous metal oxides with or without Pd were prepared by a simple and scalable co-precipitation method and used for the reduction of nitroarenes. A fluorescence-based high-throughput screening (HTS) method was also developed for the rapid analysis of the reaction conditions. The catalytic activity of the metal oxides and reaction conditions were rapidly screened by the fluorescence-based HTS method, and Pd/CuO showed the highest catalytic activity under mild reaction conditions. After identifying the optimal reaction conditions, various nitroarenes were reduced to the corresponding aniline derivatives by Pd/CuO (0.005 mol% of Pd) under these conditions. Furthermore, the Pd/CuO catalyst was used for the one-pot Suzuki–Miyaura cross-coupling/reduction reaction. A gram-scale reaction (20 mmol) was successfully performed using the present method, and Pd/CuO showed high reusability without a loss of catalytic activity for five cycles.

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.

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

2009 ◽  
Vol 14 (9) ◽  
pp. 1092-1101 ◽  
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)

scholarly journals Use of a Novel Homogeneous Fluorescent Technology in High Throughput Screening

1996 ◽  
Vol 1 (4) ◽  
pp. 203-210 ◽  
Author(s):  
Janet M. Kolb ◽  
Gregory Yamanaka ◽  
Susan P. Manly
Keyword(s):  
Time Delay ◽  
High Throughput ◽  
High Throughput Screening ◽  
Time Resolved Fluorescence ◽  
Reaction Conditions ◽  
Time Resolved ◽  
Attractive Candidate ◽  
Endogenous Fluorescence ◽  
High Throughput Screens ◽  
Fluorescent Compounds

A new fluorescent technology called homogeneous time-resolved fluorescence (HTRF) is sensitive, homogeneous, and quite tolerant to extremes in reaction conditions. These characteristics make this technique an attractive candidate for use in high throughput screens. The assay system uses a pair of fluorescent compounds to label biomolecules. The long-lived nature of the fluorescence of one of them, europium cryptate, facilitates the homogeneous nature of the assay. Furthermore, the introduction of a time delay in reading the signal eliminates the principal difficulty in applying fluorescence to screening formats, that of endogenous fluorescence of samples tested (especially natural products). This technique is robust and sensitive, and we report here its utility in a high throughput screening format.

scholarly journals Optimization of Spectrophotometric and Fluorometric Assays Using Alternative Substrates for the High-Throughput Screening of Lipase Activity

2021 ◽  
Vol 2021 ◽  
pp. 1-10
Author(s):  
Jun-Young Park ◽  
Jisu Ha ◽  
Yoonseok Choi ◽  
Pahn-Shick Chang ◽  
Kyung-Min Park
Keyword(s):  
High Throughput ◽  
Lipase Activity ◽  
High Throughput Screening ◽  
Agricultural Products ◽  
Reaction Conditions ◽  
Alternative Substrates ◽  
Standard Reaction ◽  
Ph Conditions ◽  
Chromobacterium Viscosum ◽  
Optimized Conditions

The effects of reaction conditions on the spectrophotometric and fluorometric assays using alternative substrates (p-nitrophenyl palmitate and 4-methylumbelliferyl oleate) were investigated to optimize them for the high-throughput screening of lipase activity from agricultural products. Four model lipases from Chromobacterium viscosum, Pseudomonas fluorescens, Sus scrofa pancreas, and wheat germ (Triticum aestivum) were allowed to hydrolyze the alternative substrates at different substrate concentrations (1–5 mM), operating pH (5.0–8.0), and operating temperatures (25–55°C). The results show that both the spectrophotometric and fluorometric assays worked well at the standard reaction conditions (pH 7.0 and 30°C) for finding a typical lipase, although pH conditions should be considered to detect the catalytic activity of lipases, which are applicable to more acidic or alkaline pH circumstances. To validate the optimized conditions, the high-throughput screening of lipase activity was conducted using 17 domestic agricultural products. A pileus of Pleurotus eryngii showed the highest activity in both the spectrophotometric (633.42 μU/mg) and fluorometric (101.77 μU/mg) assays. The results of this research provide practical information for the high-throughput screening of lipases using alternative substrates on microplates.

scholarly journals The Response Regulator BfmR Is a Potential Drug Target for Acinetobacter baumannii

mSphere ◽  
2016 ◽  
Vol 1 (3) ◽  
Author(s):  
Thomas A. Russo ◽  
Akshay Manohar ◽  
Janet M. Beanan ◽  
Ruth Olson ◽  
Ulrike MacDonald ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
High Throughput ◽  
Bactericidal Activity ◽  
High Throughput Screening ◽  
Response Regulator ◽  
Gram Negative ◽  
Content Type ◽  
New Antibiotics ◽  
Increased Sensitivity

ABSTRACT Increasing antibiotic resistance in bacteria, particularly Gram-negative bacilli, has significantly affected the ability of physicians to treat infections, with resultant increased morbidity, mortality, and health care costs. In fact, some strains of bacteria are resistant to all available antibiotics, such as Acinetobacter baumannii, which is the focus of this report. Therefore, the development of new antibiotics active against these resistant strains is urgently needed. In this study, BfmR is further validated as an intriguing target for a novel class of antibiotics. Successful inactivation of BfmR would confer the multiple benefits of a decreased ability of A. baumannii to survive in human body fluids, increased sensitivity to complement-mediated bactericidal activity and, importantly, increased sensitivity to other antibiotics. Structural studies support the potential for this “druggable” target, as they identify the potential for small-molecule binding at functionally relevant sites. Next-phase high-throughput screening studies utilizing BfmR are warranted. Identification and validation is the first phase of target-based antimicrobial development. BfmR (RstA), a response regulator in a two-component signal transduction system (TCS) in Acinetobacter baumannii, is an intriguing potential antimicrobial target. A unique characteristic of BfmR is that its inhibition would have the dual benefit of significantly decreasing in vivo survival and increasing sensitivity to selected antimicrobials. Studies on the clinically relevant strain AB307-0294 have shown BfmR to be essential in vivo. Here, we demonstrate that this phenotype in strains AB307-0294 and AB908 is mediated, in part, by enabling growth in human ascites fluid and serum. Further, BfmR conferred resistance to complement-mediated bactericidal activity that was independent of capsular polysaccharide. Importantly, BfmR also increased resistance to the clinically important antimicrobials meropenem and colistin. BfmR was highly conserved among A. baumannii strains. The crystal structure of the receiver domain of BfmR was determined, lending insight into putative ligand binding sites. This enabled an in silico ligand binding analysis and a blind docking strategy to assess use as a potential druggable target. Predicted binding hot spots exist at the homodimer interface and the phosphorylation site. These data support pursuing the next step in the development process, which includes determining the degree of inhibition needed to impact growth/survival and the development a BfmR activity assay amenable to high-throughput screening for the identification of inhibitors. Such agents would represent a new class of antimicrobials active against A. baumannii which could be active against other Gram-negative bacilli that possess a TCS with shared homology. IMPORTANCE Increasing antibiotic resistance in bacteria, particularly Gram-negative bacilli, has significantly affected the ability of physicians to treat infections, with resultant increased morbidity, mortality, and health care costs. In fact, some strains of bacteria are resistant to all available antibiotics, such as Acinetobacter baumannii, which is the focus of this report. Therefore, the development of new antibiotics active against these resistant strains is urgently needed. In this study, BfmR is further validated as an intriguing target for a novel class of antibiotics. Successful inactivation of BfmR would confer the multiple benefits of a decreased ability of A. baumannii to survive in human body fluids, increased sensitivity to complement-mediated bactericidal activity and, importantly, increased sensitivity to other antibiotics. Structural studies support the potential for this “druggable” target, as they identify the potential for small-molecule binding at functionally relevant sites. Next-phase high-throughput screening studies utilizing BfmR are warranted.

High Throughput Autophosphorylation Assay for Bacterial Protein Histidine Kinases

1997 ◽  
Vol 2 (2) ◽  
pp. 85-90 ◽  
Author(s):  
Siddhartha Roychoudhury ◽  
Susan M. Collins ◽  
Barbara A. Hynd ◽  
Christian N. Parker
Keyword(s):  
High Throughput ◽  
Bacterial Chemotaxis ◽  
Bacterial Protein ◽  
Reaction Conditions ◽  
Histidine Kinases ◽  
Peptide Substrates ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems ◽  
High Throughput Assay

Protein histidine kinases play a major role in bacteria as sensor components in the so-called "two-component" systems involved in signal transduction. We describe a high throughput assay for these kinases using CheA, the bacterial chemotaxis-regulating kinase from Escherichia coil. The purpose of the assay was to monitor ATP-dependent autophosphorylation of the kinase resulting in the phosphorylation of the conserved histidine residue. Unlike most eukaryotic protein kinases, "two-component" kinases are not known to phosphorylate histones and small peptide substrates. This limits the level of phosphorylation and consequently the signal generated in these assays. We, therefore, established the desirable reaction conditions first using the conventional method involving the radio labeling of CheA by [y-32P]ATP followed by gel electrophoresis-based analysis. Next, we converted the assay to a high throughput format in which CheA, autophosphorylated and radiolabeled with [y-33P]ATP, was trapped in a filter via anionic or hydrophobic interaction using diethyl aminoethyl or nitrocellulose-based 96-well filter plates, respectively. Free [y-33P]ATP was removed by washing the wells with high salt buffers. The dried plates were then analyzed for radioactivity associated with the wells by scintillation counting. Finally, we performed and validated the assay in a partially automated format using nitrocellulose-based filter plates.

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