Journal articles: 'High-throughput screening assay'

1

ARAI, Koshi. "Assay in high throughput screening." Folia Pharmacologica Japonica 118, no. 2 (2001): 81–88. http://dx.doi.org/10.1254/fpj.118.81.

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Kolb, Alfred J., and Kenneth Neumann. "Luciferase Measurements in High Throughput Screening." Journal of Biomolecular Screening 1, no. 2 (March 1996): 85–88. http://dx.doi.org/10.1177/108705719600100207.

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Luminescence assays are becoming more popular in high throughput screening (HTS) laboratories with the luciferase reporter gene being the most common. As with other assays that are adapted to HTS, improvements have been made to the luciferase assay to make it better suited to the requirements of HTS. For the luciferase reporter gene, these improvements included stabilization of the enzyme, increasing the half-life of the luminescence signal to 5 h, and eliminating separation steps (centrifugation and aliquot transfer) after cell lysis. The improved assay, LucLite, is homogeneous and is measured directly in the cell culture media. In addition to reagent improvements, a temperature-controlled, multidetector microplate counter, TopCount, can quickly and accurately measure luminescence signals.

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Shumate, Chris, Scott Beckey, Peter Coassin, and Harry Stylli. "Ultra-High Throughput Screening." Laboratory Automation News 2, no. 4 (September 1997): 24–29. http://dx.doi.org/10.1177/221106829700200406.

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Aurora Biosciences Corporation designs and develops proprietary drug discovery systems, services and technologies to accelerate and enhance the discovery of new pharmaceuticals. Aurora is developing an integrated technology platform centered around two technologies; 1) a portfolio of proprietary fluorescent assay technologies and, 2) an ultra-high throughput screening (“UHTS”) system designed to allow assay miniaturization and to overcome many of the limitations associated with the traditional drug discovery process. This approach takes advantage of the opportunities created by recent advances in genomics and combinatorial chemistry that have generated many new therapeutic targets and an abundance of new small molecule compounds. Aurora believes its integrated platform will accelerate the drug discovery process by shortening the time required to identify high quality lead compounds and to optimize those compounds into drug development candidates.

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Sittampalam, G. Sitta, Steven D. Kahl, and William P. Janzen. "High-throughput screening: advances in assay technologies." Current Opinion in Chemical Biology 1, no. 3 (October 1997): 384–91. http://dx.doi.org/10.1016/s1367-5931(97)80078-6.

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Silverman, Lauren, Robert Campbell, and James R. Broach. "New assay technologies for high-throughput screening." Current Opinion in Chemical Biology 2, no. 3 (June 1998): 397–403. http://dx.doi.org/10.1016/s1367-5931(98)80015-x.

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Sivaraja, M., H. Giordano, and M. G. Peterson. "High-Throughput Screening Assay for Helicase Enzymes." Analytical Biochemistry 265, no. 1 (December 1998): 22–27. http://dx.doi.org/10.1006/abio.1998.2875.

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Parham, Fred, Chris Austin, Noel Southall, Ruili Huang, Raymond Tice, and Christopher Portier. "Dose-Response Modeling of High-Throughput Screening Data." Journal of Biomolecular Screening 14, no. 10 (October 14, 2009): 1216–27. http://dx.doi.org/10.1177/1087057109349355.

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The National Toxicology Program is developing a high-throughput screening (HTS) program to set testing priorities for compounds of interest, to identify mechanisms of action, and potentially to develop predictive models for human toxicity. This program will generate extensive data on the activity of large numbers of chemicals in a wide variety of biochemical- and cell-based assays. The first step in relating patterns of response among batteries of HTS assays to in vivo toxicity is to distinguish between positive and negative compounds in individual assays. Here, the authors report on a statistical approach developed to identify compounds positive or negative in an HTS cytotoxicity assay based on data collected from screening 1353 compounds for concentration-response effects in 9 human and 4 rodent cell types. In this approach, the authors develop methods to normalize the data (removing bias due to the location of the compound on the 1536-well plates used in the assay) and to analyze for concentration-response relationships. Various statistical tests for identifying significant concentration-response relationships and for addressing reproducibility are developed and presented.

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Poul, Emmanuel Le, Sunao Hisada, Yoshinori Mizuguchi, Vincent J. Dupriez, Emmanuel Burgeon, and Michel Detheux. "Adaptation of Aequorin Functional Assay to High Throughput Screening." Journal of Biomolecular Screening 7, no. 1 (February 2002): 57–65. http://dx.doi.org/10.1177/108705710200700108.

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AequoScreen™, a cellular aequorin-based functional assay, has been optimized for luminescent high-throughput screening (HTS) of G protein-coupled receptor (GPCRs). AequoScreen is a homogeneous assay in which the cells are loaded with the apoaequorin cofactor coelenterazine, diluted in assay buffer, and injected into plates containing the samples to be tested. A flash of light is emitted following the calcium increase resulting from the activation of the GPCR by the sample. Here we have validated a new plate reader, the Hamamatsu Photonics FDSS6000, for HTS in 96- and 384-well plates with CHO-K1 cells stably coexpressing mitochondrial apoaequorin and different GPCRs (AequoScreen cell lines). The acquisition time, plate type, and cell number per well have been optimized to obtain concentration-response curves with 4000 cells/well in 384-well plates and a high signal: background ratio. The FDSS6000 and AequoScreen cell lines allow reading of twenty 96- or 384-well plates in 1 h with Z’ values of 0.71 and 0.78, respectively. These results bring new insights to functional assays, and therefore reinforce the interest in aequorin-based assays in a HTS environment.

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Zander Balderud, Linda, David Murray, Niklas Larsson, Uma Vempati, Stephan C. Schürer, Marcus Bjäreland, and Ola Engkvist. "Using the BioAssay Ontology for Analyzing High-Throughput Screening Data." Journal of Biomolecular Screening 20, no. 3 (December 15, 2014): 402–15. http://dx.doi.org/10.1177/1087057114563493.

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High-throughput screening (HTS) is the main starting point for hit identification in drug discovery programs. This has led to a rapid increase of available screening data both within pharmaceutical companies and the public domain. We have used the BioAssay Ontology (BAO) 2.0 for assay annotation within AstraZeneca to enable comparison with external HTS methods. The annotated assays have been analyzed to identify technology gaps, evaluate new methods, verify active hits, and compare compound activity between in-house and PubChem assays. As an example, the binding of a fluorescent ligand to formyl peptide receptor 1 (FPR1, involved in inflammation, for example) in an in-house HTS was measured by fluorescence intensity. In total, 155 active compounds were also tested in an external ligand binding flow cytometry assay, a method not used for in-house HTS detection. Twelve percent of the 155 compounds were found active in both assays. By the annotation of assay protocols using BAO terms, internal and external assays can easily be identified and method comparison facilitated. They can be used to evaluate the effectiveness of different assay methods, design appropriate confirmatory and counterassays, and analyze the activity of compounds for identification of technology artifacts.

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10

Skehan, Philip. "Dealing with the data deluge in high throughput screening." Journal of Automated Methods and Management in Chemistry 22, no. 5 (2000): 145–48. http://dx.doi.org/10.1155/s1463924600000237.

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Numerical taxonomy and pattern recognition analysis offer powerful tools that can greatly reduce the information burden of multiple-assay screening programs. These methods can be used to rationally design prescreens, identify assays that have similar chemical response patterns, select reporter assays for chemical response groups, evaluate drug selectivity, and predict a drug's likely mechanism of action. When combined with assays designed to identify lead compounds that have characteristics likely to cause failure at a later and more expensive stage of development, a simple three-stage primary discovery process consisting of a rational prescreen, reporters, and clinical failure assay can reduce the number of required culture wells by more than 20-fold and can eliminate all but 1–2 drugs per 1000 tested as leads for further evaluation and development.

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Lee, Sang-Yun, Il Doh, and Dong Woo Lee. "A High Throughput Apoptosis Assay using 3D Cultured Cells." Molecules 24, no. 18 (September 16, 2019): 3362. http://dx.doi.org/10.3390/molecules24183362.

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A high throughput apoptosis assay using 3D cultured cells was developed with a micropillar/microwell chip platform. Live cell apoptosis assays based on fluorescence detection have been useful in high content screening. To check the autofluorescence of drugs, controls (no caspase-3/7 reagent in the assay) for the drugs are necessary which require twice the test space. Thus, a high throughput capability and highly miniaturized format for reducing reagent usage are necessary in live cell apoptosis assays. Especially, the expensive caspase-3/7 reagent should be reduced in a high throughput screening system. To solve this issue, we developed a miniaturized apoptosis assay using micropillar/microwell chips for which we tested seventy drugs (six replicates) per chip and reduced the assay volume to 1 µL. This reduced assay volume can decrease the assay costs compared to the 10–40 µL assay volumes used in 384 well plates. In our experiments, among the seventy drugs, four drugs (Cediranib, Cabozatinib, Panobinostat, and Carfilzomib) induced cell death by apoptosis. Those results were confirmed with western blot assays and proved that the chip platform could be used to identify high potency apoptosis-inducing drugs in 3D cultured cells with alginate.

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McGraphery, Kate, and Wilfried Schwab. "Comparative Analysis of High-Throughput Assays of Family-1 Plant Glycosyltransferases." International Journal of Molecular Sciences 21, no. 6 (March 23, 2020): 2208. http://dx.doi.org/10.3390/ijms21062208.

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The ability of glycosyltransferases (GTs) to reduce volatility, increase solubility, and thus alter the bioavailability of small molecules through glycosylation has attracted immense attention in pharmaceutical, nutraceutical, and cosmeceutical industries. The lack of GTs known and the scarcity of high-throughput (HTP) available methods, hinders the extrapolation of further novel applications. In this study, the applicability of new GT-assays suitable for HTP screening was tested and compared with regard to harmlessness, robustness, cost-effectiveness and reproducibility. The UDP-Glo GT-assay, Phosphate GT Activity assay, pH-sensitive GT-assay, and UDP2-TR-FRET assay were applied and tailored to plant UDP GTs (UGTs). Vitis vinifera (UGT72B27) GT was subjected to glycosylation reaction with various phenolics. Substrate screening and kinetic parameters were evaluated. The pH-sensitive assay and the UDP2-TR-FRET assay were incomparable and unsuitable for HTP plant GT-1 family UGT screening. Furthermore, the UDP-Glo GT-assay and the Phosphate GT Activity assay yielded closely similar and reproducible KM, vmax, and kcat values. Therefore, with the easy experimental set-up and rapid readout, the two assays are suitable for HTP screening and quantitative kinetic analysis of plant UGTs. This research sheds light on new and emerging HTP assays, which will allow for analysis of novel family-1 plant GTs and will uncover further applications.

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Harris, S. Richard, Russell K. Garlick, Joseph J. Miller, Harry N. Harney, and Philip J. Monroe. "Complement C5a Receptor Assay for High Throughput Screening." Journal of Receptor Research 11, no. 1-4 (January 1991): 115–28. http://dx.doi.org/10.3109/10799899109066393.

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14

Schrader, Paul S., Elizabeth H. Burrows, and Roger L. Ely. "High-Throughput Screening Assay for Biological Hydrogen Production." Analytical Chemistry 80, no. 11 (June 2008): 4014–19. http://dx.doi.org/10.1021/ac702633q.

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Wyatt, Brittney N., Leggy A. Arnold, and Martin St. Maurice. "A high-throughput screening assay for pyruvate carboxylase." Analytical Biochemistry 550 (June 2018): 90–98. http://dx.doi.org/10.1016/j.ab.2018.04.012.

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Sui, Yunxia, and Zhijin Wu. "Alternative Statistical Parameter for High-Throughput Screening Assay Quality Assessment." Journal of Biomolecular Screening 12, no. 2 (January 11, 2007): 229–34. http://dx.doi.org/10.1177/1087057106296498.

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High-throughput screening is an essential process in drug discovery. The ability to identify true active compounds depends on the high quality of assays and proper analysis of data. The Z factor, presented by Zhang et al. in 1999, provides an easy and useful summary of assay quality and has been a widely accepted standard. However, as data analysis has undergone much improvement recently, the assessment of assay quality has not evolved in parallel. In this article, the authors study the implications of Z factor values under different conditions and link the Z factor with the power of discovering true active compounds. They discuss the different interpretations of Z factor depending on error distributions and advocate direct analysis of power as assay quality assessment. They also propose that in estimating assay quality parameters, adjustments in data analysis should be taken into account. Studying the power of identifying true “hits” gives a more direct interpretation of assay quality and may provide guidance in assay optimization on some occasions.

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Patwardhan, Chaitanya A., Eyad Alfa, Su Lu, and Ahmed Chadli. "Progesterone Receptor Chaperone Complex–Based High-Throughput Screening Assay." Journal of Biomolecular Screening 20, no. 2 (September 2, 2014): 223–29. http://dx.doi.org/10.1177/1087057114549147.

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Hsp90 and its co-chaperones are known to be important for cancer cell survival. The N-terminal inhibitors of Hsp90 that are in ongoing clinical trials as antitumor agents have unfortunately shown disappointing efficacies in the clinic. Thus, novel inhibitors of the Hsp90 machine with a different mechanism of action are urgently needed. We report here the development of a novel high-throughput screening assay platform to identify small-molecule inhibitors of Hsp90 and its co-chaperones. This assay quantitatively measures the ability of Hsp90 and its co-chaperones to refold/protect the progesterone receptor, a physiological client of Hsp90, in a 96-well plate format. We screened the National Institutes of Health clinical collection drug library and identified capsaicin as a hit molecule. Capsaicin is a Food and Drug Administration–approved drug for topical use in pain management. Cell survival assays showed that capsaicin selectively kills cancer cells and destabilizes several Hsp90 client proteins. Thus, our data may explain the seemingly pleotropic effect of capsaicin.

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Greenwalt, Dale E., Janet Szabo, and Ilana Manchel. "High Throughput Cell-Based Assay of Hematopoietic Progenitor Differentiation." Journal of Biomolecular Screening 6, no. 6 (December 2001): 383–92. http://dx.doi.org/10.1177/108705710100600604.

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The in vitro efficacy of drug candidates relative to hematopoietic stem cell proliferation and differentiation is currently assayed through use of the clonogenic "colony assay." The extremely low throughput of this assay precludes its use in library screening and much drug discovery work. A rapid-throughput assay of progenitor cell differentiation based on the quantification of hematopoietic lineage-specific markers has been developed. The CELISA assay employs a single incubation with a lanthanide-conjugated primary antibody and subsequent time-resolved fluorescence spectroscopy. The rapid-throughput nature of this assay is enhanced by the use of cell culture-compatible filter plates to reduce the number of manipulations as compared to currently available cell-based assays. The culture and assay are done in 96-well plates, and the quantitation process requires approximately 1 hour. The myeloid, erythroid, and megakaryocytic lineages can be objectively quantified; data from the assay correlate extremely well with data generated through use of the traditional colony assay. This assay makes possible both rapid-throughput drug discovery and toxicity screening in the area of hematopoiesis.

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Lavery, Patrick, Murray J. B. Brown, and Andrew J. Pope. "Simple Absorbance-Based Assays for Ultra-High Throughput Screening." Journal of Biomolecular Screening 6, no. 1 (February 2001): 3–9. http://dx.doi.org/10.1177/108705710100600102.

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In order to accommodate the predicted increase in screening required of successful pharmaceutical companies, miniaturized, high-speed HTS formats are necessary. Much emphasis has been placed on sensitive fluorescence techniques, but some systems, particularly enzymes interconverting small substrates, are likely to be refractory to such approaches. We show here that simple absorbance-based assays can be miniaturized to 10-,.d volumes in 1536- well microplates compatible with the requirements for ultra-high throughput screening. We demonstrate that, with low-cost hardware, assay performance is wholly predictable from the 2-fold decrease in pathlength for fully filled 1536-well plates compared to 96- and 384-well microplates. A number of enzyme systems are shown to work in this high-density format, and the inhibition parameters determined are comparable with those in standard assay formats. We also demonstrate the utility of kinetics measurements in miniaturized format with improvements in assay quality and the ability to extract detailed mechanistic information about inhibitors.

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Siebring-van Olst, Ellen, Christie Vermeulen, Renee X. de Menezes, Michael Howell, Egbert F. Smit, and Victor W. van Beusechem. "Affordable Luciferase Reporter Assay for Cell-Based High-Throughput Screening." Journal of Biomolecular Screening 18, no. 4 (October 30, 2012): 453–61. http://dx.doi.org/10.1177/1087057112465184.

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The firefly luciferase gene is commonly used in cell-based reporter assays. Convenient luciferase assay reagents for use in high-throughput screening (HTS) are commercially available. However, the high cost of these reagents is not within the means of some academic laboratories. Therefore, we set out to develop an affordable luciferase assay reagent applicable in an HTS format using simple liquid-handling steps. The reagent was homemade from individual chemical components and optimized for luminescence intensity and stability. We determined the minimal concentrations of the most expensive components, dithiothreitol (DTT) and D-luciferin, resulting in a total assay reagent cost of less than 1 cent per sample. Signal stability was maximized by omission of coenzyme A and reduction of DTT concentration. The assay was validated in a high-throughput setting using two cancer cell lines carrying a p53-dependent luciferase reporter construct and siRNAs modulating p53 transcriptional activity. Induction of p53 activity by silencing PPM1D or SYVN1 and reduction of p53 activity by silencing p53 remained constant over a 2-h measurement period, with good assay quality (Z′ factors mostly above 0.5). Hence, the luciferase assay described herein can be used for affordable reporter readout in cell-based HTS.

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Kevorkov, Dmytro, and Vladimir Makarenkov. "Statistical Analysis of Systematic Errors in High-Throughput Screening." Journal of Biomolecular Screening 10, no. 6 (September 2005): 557–67. http://dx.doi.org/10.1177/1087057105276989.

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High-throughput screening (HTS) is an efficient technology for drug discovery. It allows for screening of more than 100,000 compounds a day per screen and requires effective procedures for quality control. The authors have developed a method for evaluating a background surface of an HTS assay; it can be used to correct raw HTS data. This correction is necessary to take into account systematic errors that may affect the procedure of hit selection. The described method allows one to analyze experimental HTS data and determine trends and local fluctuations of the corresponding background surfaces. For an assay with a large number of plates, the deviations of the background surface from a plane are caused by systematic errors. Their influence can be minimized by the subtraction of the systematic background from the raw data. Two experimental HTS assays from the ChemBank database are examined in this article. The systematic error present in these data was estimated and removed from them. It enabled the authors to correct the hit selection procedure for both assays.

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Patel, Dhara A., Anand C. Patel, William C. Nolan, Guangming Huang, Arthur G. Romero, Nichole Charlton, Eugene Agapov, Yong Zhang, and Michael J. Holtzman. "High-Throughput Screening Normalized to Biological Response." Journal of Biomolecular Screening 19, no. 1 (July 16, 2013): 119–30. http://dx.doi.org/10.1177/1087057113496848.

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The process of conducting cell-based phenotypic screens can result in data sets from small libraries or portions of large libraries, making accurate hit picking from multiple data sets important for efficient drug discovery. Here, we describe a screen design and data analysis approach that allow for normalization not only between quadrants and plates but also between screens or batches in a robust, quantitative fashion, enabling hit selection from multiple data sets. We independently screened the MicroSource Spectrum and NCI Diversity Set II libraries using a cell-based phenotypic high-throughput screening (HTS) assay that uses an interferon-stimulated response element (ISRE)–driven luciferase-reporter assay to identify interferon (IFN) signal enhancers. Inclusion of a per-plate, per-quadrant IFN dose-response standard curve enabled conversion of ISRE activity to effective IFN concentrations. We identified 45 hits based on a combined z score ≥2.5 from the two libraries, and 25 of 35 available hits were validated in a compound concentration-response assay when tested using fresh compound. The results provide a basis for further analysis of chemical structure in relation to biological function. Together, the results establish an HTS method that can be extended to screening for any class of compounds that influence a quantifiable biological response for which a standard is available.

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Gosnell, Paul A., Amy D. Hilton, Lynette M. Anderson, Lyman Wilkins, and William P. Janzen. "Compound Library Management in High Throughput Screening." Journal of Biomolecular Screening 2, no. 2 (March 1997): 99–102. http://dx.doi.org/10.1177/108705719700200208.

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Compound library management is inherent to the success of a high throughput screen. The library management system developed at Sphinx Pharmaceuticals accomplishes this through a series of Paradox and Orade database applications. This suite of applications allows compounds to be grouped as libraries, inventoried, and scheduled for testing in a screen. Compound libraries can be defied using any combination of source, structure, and target, and may be divided into sublibraries, allowing a flexible mix of compounds to be scheduled across several targets. Test requests can then be made for an entire library, sublibrary, or individual mother plates. The priority of requests is set by a combination of assay and compound source, allowing management of sources that require special handling. An inventory module tracks both physical volumes and the volume available for requests by plate and well. Positive sample tracking is maintained throughout the screening process from the time of request through the compound preparation process, to the date of test, and subsequent re-test if necessary. Furthermore, assay results can be tracked to the exact well on the assay plate. The tracking of this data allows a flexible diversity set for each target, tracks compound preparation to date, and facilitates the scheduling of preparation.

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Devkota, Ashwini K., Mangalika Warthaka, Ramakrishna Edupuganti, Clint D. J. Tavares, William H. Johnson, Bulent Ozpolat, Eun Jeong Cho, and Kevin N. Dalby. "High-Throughput Screens for eEF-2 Kinase." Journal of Biomolecular Screening 19, no. 3 (September 27, 2013): 445–52. http://dx.doi.org/10.1177/1087057113505204.

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eEF-2 kinase is a potential therapeutic target for breast cancer, gliomas, and depression. No potent inhibitors of eEF-2K have been reported, and thus development of high-throughput assay systems may expedite the process. Two high-throughput assays are described for eEF-2K using recombinant, tag-free enzyme purified from bacteria. The first is a fluorescence-based assay that uses the phosphorylation of a Sox-based peptide substrate by eEF-2K, which results in a 5-fold increase in fluorescence emission, allowing for continuous monitoring of the kinase activity. The second is a luminescence-based assay that produces a luminescence signal, which correlates with the amount of adenosine triphosphate remaining in the kinase reaction. Both assays have been optimized and miniaturized for a 384-well plate format and validated in screens. In conclusion, we demonstrated that a traditional radiolabeled assay can be readily transferred to universal spectroscopic assays that are robust and will facilitate high-throughput screening of larger size libraries for the identification of small-molecule inhibitors and significantly contribute to the development of therapies for targeting eEF2K.

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Astle, Thomas W. "MicroTape-A 384-Well Ultra High Throughput Screening System." JALA: Journal of the Association for Laboratory Automation 4, no. 2 (May 1999): 31–34. http://dx.doi.org/10.1177/221106829900400207.

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The use of a sprocket driven carrier tape provides a unique solution for Ultra High Throughput Screening. 10-microliter wells in the 384 well pattern are embossed in a polypropylene or polycarbonate carrier tape. For compound handling, 100,000 compounds in 5 microliter aliquots may be stored, sealed and frozen in a roll 16 inches in diameter and 4 inches wide. The sprocket drive provides recall to any given compound. At time of use, the peelable seal is removed for access to the compound aliquots. Two levels of assay automation are provided. In Phase I the compound MicroTape is used to support assays in the standard 384 well plate format. In Phase II the compounds are transferred directly to the assay MicroTape for Ultra High Throughput Screening at the rate of 100,000 per day.

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Harrill, Joshua A., Logan J. Everett, Derik E. Haggard, Thomas Sheffield, Joseph L. Bundy, Clinton M. Willis, Russell S. Thomas, Imran Shah, and Richard S. Judson. "High-Throughput Transcriptomics Platform for Screening Environmental Chemicals." Toxicological Sciences 181, no. 1 (February 4, 2021): 68–89. http://dx.doi.org/10.1093/toxsci/kfab009.

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Abstract New approach methodologies (NAMs) that efficiently provide information about chemical hazard without using whole animals are needed to accelerate the pace of chemical risk assessments. Technological advancements in gene expression assays have made in vitro high-throughput transcriptomics (HTTr) a feasible option for NAMs-based hazard characterization of environmental chemicals. In this study, we evaluated the Templated Oligo with Sequencing Readout (TempO-Seq) assay for HTTr concentration-response screening of a small set of chemicals in the human-derived MCF7 cell model. Our experimental design included a variety of reference samples and reference chemical treatments in order to objectively evaluate TempO-Seq assay performance. To facilitate analysis of these data, we developed a robust and scalable bioinformatics pipeline using open-source tools. We also developed a novel gene expression signature-based concentration-response modeling approach and compared the results to a previously implemented workflow for concentration-response analysis of transcriptomics data using BMDExpress. Analysis of reference samples and reference chemical treatments demonstrated highly reproducible differential gene expression signatures. In addition, we found that aggregating signals from individual genes into gene signatures prior to concentration-response modeling yielded in vitro transcriptional biological pathway altering concentrations (BPACs) that were closely aligned with previous ToxCast high-throughput screening assays. Often these identified signatures were associated with the known molecular target of the chemicals in our test set as the most sensitive components of the overall transcriptional response. This work has resulted in a novel and scalable in vitro HTTr workflow that is suitable for high-throughput hazard evaluation of environmental chemicals.

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Sachsenmeier, Kris F., Carl Hay, Erin Brand, Lori Clarke, Kim Rosenthal, Sandrine Guillard, Steven Rust, Ralph Minter, and Robert Hollingsworth. "Development of a Novel Ectonucleotidase Assay Suitable for High-Throughput Screening." Journal of Biomolecular Screening 17, no. 7 (April 20, 2012): 993–98. http://dx.doi.org/10.1177/1087057112443987.

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5′-Ectonucleotidase (NT5E) catalyzes the conversion of adenosine monophosphate to adenosine and free phosphate. The role of this ectonucleotidase and its production of adenosine are linked with immune function, angiogenesis, and cancer. NT5E activity is typically assayed either by chromatographic quantification of substrates and products using high-performance liquid chromatography (HPLC) or by quantification of free phosphate using malachite green. These methods are not suitable for robust screening assays of NT5E activity. HPLC is not readily suitable for the rapid and efficient assay of multiple samples and malachite green is highly sensitive to the phosphate-containing buffers common in various media and sample buffers. Here the development and validation of a novel high-throughput ectonucleotidase screening assay are described, which makes use of a luciferase-based assay reagent, the Promega CellTiter-Glo kit, to measure the catabolism of AMP by NT5E. This multiwell plate-based assay facilitates the screening of potential ectonucleotidase antagonists and is unaffected by the presence of contaminating phosphate molecules present in screening samples.

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Marlowe, Timothy, Carlos Alvarado, Andrew Rivera, Felicia Lenzo, Rohini Nott, Dena Bondugji, Justin Montoya, et al. "Development of a High-Throughput Fluorescence Polarization Assay to Detect Inhibitors of the FAK–Paxillin Interaction." SLAS DISCOVERY: Advancing the Science of Drug Discovery 25, no. 1 (September 12, 2019): 21–32. http://dx.doi.org/10.1177/2472555219874313.

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Focal adhesion kinase (FAK) is a promising cancer drug target due to its massive overexpression in multiple solid tumors and its critical role in the integration of signals that control proliferation, invasion, apoptosis, and metastasis. Previous FAK drug discovery and high-throughput screening have exclusively focused on the identification of inhibitors that target the kinase domain of FAK. Because FAK is both a kinase and scaffolding protein, the development of novel screening assays that detect inhibitors of FAK protein–protein interactions remains a critical need. In this report, we describe the development of a high-throughput fluorescence polarization (FP) screening assay that measures the interactions between FAK and paxillin, a focal adhesion–associated protein. We designed a tetramethylrhodamine (TAMRA)-tagged paxillin peptide based on the paxillin LD2 motif that binds to the focal adhesion targeting (FAT) domain with significant dynamic range, specificity, variability, stability, and a Z’-factor suitable for high-throughput screening. In addition, we performed a pilot screen of 1593 compounds using this FP assay, showing its feasibility for high-throughput drug screening. Finally, we identified three compounds that show dose-dependent competition of FAT–paxillin binding. This assay represents the first described high-throughput screening assay for FAK scaffold inhibitors and can accelerate drug discovery efforts for this promising drug target.

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Moorwood, Catherine, Neha Soni, Gopal Patel, Steve D. Wilton, and Tejvir S. Khurana. "A Cell-Based High-Throughput Screening Assay for Posttranscriptional Utrophin Upregulation." Journal of Biomolecular Screening 18, no. 4 (October 30, 2012): 400–406. http://dx.doi.org/10.1177/1087057112465648.

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Duchenne muscular dystrophy (DMD) is a devastating muscle-wasting disease caused by mutations in the dystrophin gene. Utrophin is a homologue of dystrophin that can compensate for its absence when overexpressed in DMD animal models. Utrophin upregulation is therefore a promising therapeutic approach for DMD. Utrophin is regulated at both transcriptional and posttranscriptional levels. Transcriptional regulation has been studied extensively, and assays have been described for the identification of utrophin promoter-targeting molecules. However, despite the profound impact that posttranscriptional regulation has on utrophin expression, screening assays have not yet been described that could be used to discover pharmaceuticals targeting this key phase of regulation. We describe the development and validation of a muscle cell line–based assay in which a stably expressed luciferase coding sequence is flanked by the utrophin 5′- and 3′-untranslated regions (UTRs). The assay was validated using the posttranscriptional regulation of utrophin by miR-206. The assay has a Z′ of 0.7, indicating robust performance in high-throughput format. This assay can be used to study utrophin regulatory mechanisms or to screen chemical libraries for compounds that upregulate utrophin posttranscriptionally via its UTRs. Compounds identified via this assay, used alone or in a synergistic combination with utrophin promoter-targeting molecules, would be predicted to have therapeutic potential for DMD.

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Card, Amy, Chris Caldwell, Hyunsuk Min, Bina Lokchander, Hualin Xi, Simone Sciabola, Ajith V. Kamath, et al. "High-Throughput Biochemical Kinase Selectivity Assays: Panel Development and Screening Applications." Journal of Biomolecular Screening 14, no. 1 (November 21, 2008): 31–42. http://dx.doi.org/10.1177/1087057108326663.

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Kinases represent attractive targets for drug discovery. Eight small-molecule kinase inhibitors are currently marketed in the area of oncology, and numerous others are in clinical trials. Characterization of the selectivity profiles of these compounds is important to target appropriate patient populations and to reduce the potential of toxicity due to off-target effects. The authors describe the development, validation, and utilization of a biochemical kinase assay panel for the selectivity profiling of inhibitors. The panel was developed as 29 radiometric Flashplate™ assays, and then an initial 13 were transitioned to a nonradiometric Caliper mobility shift assay format. Generation of high-quality data from the panel is detailed along with a comparison of the assay formats. Both assay technologies were found to be suitable for panel screening, but mobility shift assays yielded higher data quality. The selectivity data generated here should be useful in computational modeling and help facilitate, in conjunction with sequence and structural information, the rational design of inhibitors with well-defined selectivity profiles. ( Journal of Biomolecular Screening 2009:31-42)

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Luu, Kim Yen, and Thomas D. Duensing. "High Throughput Flow Screening Assays to Profile Cell-Mediated Killing." Blood 126, no. 23 (December 3, 2015): 4629. http://dx.doi.org/10.1182/blood.v126.23.4629.4629.

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Abstract Cell-mediated killing is an immune response that involves the activation of cells such as phagocytes, natural killer cells (NK), or antigen-specific cytotoxic T-lymphocytes to induce death to pathogenic cells. Enhancing T-cell mediated killing of tumor cells is emerging as a successful therapeutic approach for a variety of cancers. A number of exciting immuno-oncology technologies are being developed, including adoptive cell transfer approaches involving chimeric antigen receptor T- cells (CAR-T), tumor infiltrating lymphocytes (TIL) and natural killer (NK) cells. Biologics and small molecule approaches are also being developed to increase T-cell mediated killing of tumor cells by modulating molecular interactions among immune checkpoints such as PD-1 and CTLA4. While showing great promise, improvements to these therapies are actively being sought, and novel assay technologies aimed at identifying better and safer treatments are needed. Traditional assays for monitoring cell-mediated killing are only capable of homogenous live/dead readouts for an entire sample. Additionally, the gold-standard chromium release assay involves a laborious protocol and radioactive reagents. As an alternative platform for cell-mediated killing studies, IntelliCyt's iQue Screener is a high throughput suspension screening platform based on flow cytometry. The system can identify multiple cell types in suspension and report multiple cell killing readouts, including cell viability and apoptosis in streamlined no wash assay formats. Here we demonstrate two example high throughput flow assays for cell-mediated killing in assay models using NK cells and chimeric antigen receptor (CAR) T-cells. For the NK cell assay, the NK92 cell line was utilized as an effector cell and Jurkat cells were utilized as target cells. By labeling either target or effector cells with a cell encoder dye, both cell populations can be simultaneously monitored. Viability was determined by cell membrane integrity and Caspase 3 activation for both Jurkat and NK92 cells, and compounds that were generally cytotoxic to both NK92 and Jurkat cells could be identified. The specificity of the cell-mediated killing response was demonstrated using known signal transduction inhibitors including sunitinib, U73122, pp2, and wortmannin that would attenuate the NK cell killing activity, at a fixed target to effector cell ratio. In the second assay model using CAR T-cells, the efficacy of different CARs at targeting and killing a B-cell line (NALM-6) was profiled using multiplex assays for both cell endpoints and secreted cytokine endpoints. In addition to cell encoding and live/dead analysis, the secretion of multiple cytokines including inflammatory markers and Granzyme B were evaluated using bead-based ELISA on the same analysis platform. These application examples highlight the robustness and flexibility of the iQue Screener for performing multiplexed screening assays with cells and beads to provide greater contextual value for cell mediated killing studies. Disclosures Luu: IntelliCyt Corp: Employment. Duensing:IntelliCyt Corp: Employment.

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Andraos, Charlene, Il Je Yu, and Mary Gulumian. "Interference: A Much-Neglected Aspect in High-Throughput Screening of Nanoparticles." International Journal of Toxicology 39, no. 5 (July 16, 2020): 397–421. http://dx.doi.org/10.1177/1091581820938335.

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Despite several studies addressing nanoparticle (NP) interference with conventional toxicity assay systems, it appears that researchers still rely heavily on these assays, particularly for high-throughput screening (HTS) applications in order to generate “big” data for predictive toxicity approaches. Moreover, researchers often overlook investigating the different types of interference mechanisms as the type is evidently dependent on the type of assay system implemented. The approaches implemented in the literature appear to be not adequate as it often addresses only one type of interference mechanism with the exclusion of others. For example, interference of NPs that have entered cells would require intracellular assessment of their interference with fluorescent dyes, which has so far been neglected. The present study investigated the mechanisms of interference of gold NPs and silver NPs in assay systems implemented in HTS including optical interference as well as adsorption or catalysis. The conventional assays selected cover all optical read-out systems, that is, absorbance (XTT toxicity assay), fluorescence (CytoTox-ONE Homogeneous membrane integrity assay), and luminescence (CellTiter Glo luminescent assay). Furthermore, this study demonstrated NP quenching of fluorescent dyes also used in HTS (2′,7′-dichlorofluorescein, propidium iodide, and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzamidazolocarbocyanin iodide). To conclude, NP interference is, as such, not a novel concept, however, ignoring this aspect in HTS may jeopardize attempts in predictive toxicology. It should be mandatory to report the assessment of all mechanisms of interference within HTS, as well as to confirm results with label-free methodologies to ensure reliable big data generation for predictive toxicology.

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Plant, Helen, Clare Stacey, Choi-Lai Tiong-Yip, Jarrod Walsh, Qin Yu, and Kirsty Rich. "High-Throughput Hit Screening Cascade to Identify Respiratory Syncytial Virus (RSV) Inhibitors." Journal of Biomolecular Screening 20, no. 5 (February 5, 2015): 597–605. http://dx.doi.org/10.1177/1087057115569428.

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Respiratory syncytial virus (RSV) infects 99% of children by age 2 years and is a leading cause of serious lower respiratory tract infection (LRTI) and infant hospitalization in the United Kingdom. Identification of efficacious RSV therapeutics has been hindered by the lack of a robust and appropriate primary assay for high-throughput screening (HTS). Here we report an HTS cascade that identified inhibitors of RSV replication using a robust RSV replicon luminescence-reporter assay for the primary campaign. The performance of the assay was consistent and reliable at scale, with Z′ of 0.55 ± 0.08 across 150 assay plates and signal-to-background ratios >40. The HTS assay was used to screen the AstraZeneca compound library of 1 million compounds at a single concentration of 10 µM. Hits specifically targeting the RSV replicon were determined using a series of hit generation assays. Compounds nonspecifically causing cell toxicity were removed, and hits were confirmed in live viral inhibition assays exhibiting greater physiological relevance than the primary assay. In summary, we developed a robust screening cascade that identified hit molecules that specifically targeted RSV replication.

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Jang, Dae Song, Narsimha R. Penthala, Eugene O. Apostolov, Xiaoying Wang, Tariq Fahmi, Peter A. Crooks, and Alexei G. Basnakian. "Novel High-Throughput Deoxyribonuclease 1 Assay." Journal of Biomolecular Screening 20, no. 2 (October 17, 2014): 202–11. http://dx.doi.org/10.1177/1087057114555828.

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Deoxyribonuclease I (DNase I), the most active and abundant apoptotic endonuclease in mammals, is known to mediate toxic, hypoxic, and radiation injuries to the cell. Neither inhibitors of DNase I nor high-throughput methods for screening of high-volume chemical libraries in search of DNase I inhibitors are, however, available. To overcome this problem, we developed a high-throughput DNase I assay. The assay is optimized for a 96-well plate format and based on the increase of fluorescence intensity when fluorophore-labeled oligonucleotide is degraded by the DNase. The assay is highly sensitive to DNase I compared to other endonucleases, reliable (Z’ ≥ 0.5), and operationally simple, and it has low operator, intraassay, and interassay variability. The assay was used to screen a chemical library, and several potential DNase I inhibitors were identified. After comparison, 2 hit compounds were selected and shown to protect against cisplatin-induced kidney cell death in vitro. This assay will be suitable for identifying inhibitors of DNase I and, potentially, other endonucleases.

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Wagstaff, Ryan, Michael Hedrick, Jun Fan, Paul D. Crowe, and Daniel DiSepio. "High-Throughput Screening for Norepinephrine Transporter Inhibitors Using the FLIPRTetra." Journal of Biomolecular Screening 12, no. 3 (January 26, 2007): 436–41. http://dx.doi.org/10.1177/1087057106297994.

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Monoamine transporters regulate the concentration of neurotransmitters in the synapse following neurotransmission and are very important drug targets in the pharmaceutical industry. Because of the labor-intensive nature of functional uptake assays using radioactive substrates, high-throughput screening for monoamine transporter inhibitors has been limited to radioligand binding assays. In this article, the authors describe the development of a 384-well, high-throughput functional screening assay for norepinephrine transporter inhibitors using the FLIPRTetra and a recently identified fluorescent substrate, 4-(4-dimethylaminostyryl)- N-methyl-pyridinium (ASP+). ( Journal of Biomolecular Screening 2007:436-441)

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Roy, Anuradha, and Mohammad A. Mir. "Development of High-Throughput Screening Assay for Antihantaviral Therapeutics." SLAS DISCOVERY: Advancing the Science of Drug Discovery 22, no. 6 (March 24, 2017): 767–74. http://dx.doi.org/10.1177/2472555217699942.

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Humans acquire hantavirus infection by the inhalation of aerosolized excreta of infected rodent hosts. There is no treatment for hantavirus diseases at present. Therapeutic intervention during early stages of viral infection can improve the outcome of this zoonotic viral illness. The interaction between an evolutionary conserved sequence at the 5′ terminus of hantaviral genomic RNA and hantavirus nucleocapsid protein plays a critical role in the hantavirus replication cycle. This unique interaction is a novel target for therapeutic intervention of hantavirus disease. We developed a very sensitive, tractable, and cost-effective fluorescence-based assay to monitor the interaction between the nucleocapsid protein and the target RNA sequence. The assay was optimized for high-throughput screening of chemical libraries to identify molecules that interrupt this RNA–protein interaction. The assay was validated using a library of 6880 chemical compounds. This validation screen demonstrated the reproducibility and validity of required statistical criteria for high-throughput screening. The assay is ready to use for high-throughput screening of large chemical libraries to identify antihantaviral therapeutic molecules and can be amenable to similar targets in other viruses.

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Rogers, Mark V. "Light on high-throughput screening: fluorescence-based assay technologies." Drug Discovery Today 2, no. 4 (April 1997): 156–60. http://dx.doi.org/10.1016/s1359-6446(97)01016-7.

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Andexer, Jennifer, Jan-Karl Guterl, Martina Pohl, and Thorsten Eggert. "A high-throughput screening assay for hydroxynitrile lyase activity." Chemical Communications, no. 40 (2006): 4201. http://dx.doi.org/10.1039/b607863j.

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Lim, Changwon, Pranab K. Sen, and Shyamal D. Peddada. "Robust Analysis of High Throughput Screening (HTS) Assay Data." Technometrics 55, no. 2 (May 2013): 150–60. http://dx.doi.org/10.1080/00401706.2012.749166.

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Wu, Yu-Wei, and Yu-Hui Tsai. "A Rapid Transglutaminase Assay for High-Throughput Screening Applications." Journal of Biomolecular Screening 11, no. 7 (September 14, 2006): 836–43. http://dx.doi.org/10.1177/1087057106291585.

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Transglutaminases (TGs) are widely distributed enzymes that catalyze posttranslational modification of proteins by Ca2+-dependent cross-linking reactions. The family members of TGs participate in many significant processes of biological functions such as tissue regeneration, cell differentiation, apoptosis, and certain pathologies. A novel technique for TG activity assay was developed in this study. It was based on the rapid capturing, fluorescence quenching, and fast separation of the unreacted fluorescent molecules from the macromolecular product with magnetic dextran-coated charcoal. As few as 3 ng of guinea pig liver transglutaminase (gpTG) could be detected by the method; activities of 96 TG samples could be measured within an hour. The Km of gpTG determined by this method for monodansylcadaverine (dansyl-CAD) and N, N-dimethylcasein was 14 and 5 μM, respectively. A typical competitive inhibition pattern of cystamine on dansyl-CAD for gpTG activity was also demonstrated. The application of this technique is not limited to the use of dansyl-CAD as the fluorescent substrate of TG; other small fluor-labeled TG substrates may substitute dansyl-CAD. Finally, this method is rapid, highly sensitive, and inexpensive. It is suitable not only for high-throughput screening of enzymes or enzyme inhibitors but also for enzyme kinetic analysis.

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Falk, Shaun P., Andrew T. Ulijasz, and Bernard Weisblum. "Differential Assay for High-Throughput Screening of Antibacterial Compounds." Journal of Biomolecular Screening 12, no. 8 (December 2007): 1102–8. http://dx.doi.org/10.1177/1087057107308161.

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The previously described Bacillus subtilis reporter strain BAU-102 is capable of detecting cell wall synthesis inhibitors that act at all stages of the cell wall synthesis pathway. In addition, this strain is capable of detecting compounds with hydrophobic/ surfactant activity and alternative mechanisms of cell wall disruption. BAU-102 sequesters preformed β-gal in the periplasm, suggesting leakage of β-gal as the means by which this assay detects compound activities. A model is proposed according to which β-gal release by BAU-102 reflects activation of pathways leading to autolysis. The authors also report a simplified high-throughput assay using BAU-102 combined with the fluorogenic substrate N-methylumbelliferyl-β-D-galactoside as a single reagent. Cell wall inhibitors release β-gal consistently only after 60 min of incubation, whereas compounds with surfactant activity show an almost immediate release. A high-throughput screen of a 480-compound library of known bioactives yielded 8 compounds that cause β-gal release. These results validate the BAU-102 assay as an effective tool in antimicrobial drug discovery. ( Journal of Biomolecular Screening 2007:1102-1108)

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Abarca, Carla, M. Monsur Ali, Songtao Yang, Xiaofei Dong, and Robert H. Pelton. "A Colloidal Stability Assay Suitable for High-Throughput Screening." Analytical Chemistry 88, no. 5 (February 18, 2016): 2929–36. http://dx.doi.org/10.1021/acs.analchem.5b04915.

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Le Poul, Emmanuel, Sunao Hisada, Yoshinori Mizuguchi, Vincent J. Dupriez, Emmanuel Burgeon, and Michel Detheux. "Adaptation of Aequorin Functional Assay to High Throughput Screening." Journal of Biomolecular Screening 7, no. 1 (February 1, 2002): 57–65. http://dx.doi.org/10.1089/108705702753520341.

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Cook, Neil D. "Scintillation proximity assay: a versatile high-throughput screening technology." Drug Discovery Today 1, no. 7 (July 1996): 287–94. http://dx.doi.org/10.1016/1359-6446(96)10026-x.

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Ryu, Jihye, Min Sik Eom, Wooseok Ko, Min Su Han, and Hyun Soo Lee. "A fluorescence-based glycosyltransferase assay for high-throughput screening." Bioorganic & Medicinal Chemistry 22, no. 8 (April 2014): 2571–75. http://dx.doi.org/10.1016/j.bmc.2014.02.027.

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Ahsen, Oliver von, Ulrike Voigtmann, Monika Klotz, Nikolay Nifantiev, Arndt Schottelius, Alexander Ernst, Beate Müller-Tiemann, and Karsten Parczyk. "A miniaturized high-throughput screening assay for fucosyltransferase VII." Analytical Biochemistry 372, no. 1 (January 2008): 96–105. http://dx.doi.org/10.1016/j.ab.2007.08.029.

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Khoronenkova, Svetlana V., and Vladimir I. Tishkov. "High-throughput screening assay for d-amino acid oxidase." Analytical Biochemistry 374, no. 2 (March 2008): 405–10. http://dx.doi.org/10.1016/j.ab.2007.12.021.

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Kempf, Mirjam-Colette, Jennifer Jones, Marintha L. Heil, and Olaf Kutsch. "A High-Throughput Drug Screening System for HIV-1 Transcription Inhibitors." Journal of Biomolecular Screening 11, no. 7 (September 14, 2006): 807–15. http://dx.doi.org/10.1177/1087057106290292.

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Identification of HIV-1 transcription inhibitors was previously performed using infectivity assays. As de novo HIV-1 infection is highly sensitive to even minor compound toxicities, these assays are plagued by extremely high levels of false-positive hits. Hit identification is further complicated because infectivity assays lack target specificity. The authors demonstrate that it is possible to overcome these limitations by establishing a stable, chronically actively HIV-1-infected reporter cell line that exclusively identifies HIV-1 transcription inhibitors. In the reporter cell line, 2 spectrally separated fluorescence proteins serve as simultaneously accessible quantitative markers of HIV-1 expression and drug toxicity. The combined analysis of these markers drastically reduces the level of false-positive hits. As determination of fluorescence intensity in a plate-based format can be performed in a noninvasive manner, repeated measurements of fluorescence levels over several days after compound addition can be used to define the kinetic and dynamic characteristics of inhibitory drug effects. In addition, because of the stable nature of the reporter cell line, the assay requires no cell manipulation during assay preparation or assay analysis, rendering the system extremely cost-effective and reliable.

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Walsh, James C. "High Throughput, Mechanism-Based Screening Techniques for Discovering Novel Agrochemicals." Journal of Biomolecular Screening 3, no. 3 (April 1998): 175–81. http://dx.doi.org/10.1177/108705719800300303.

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A variety of screening strategies can be employed for discovering novel agrochemicals such as fungicides, insecticides, herbicides and antiparasitics. Traditionally, random evaluation of chemical and natural products samples has been used in assay systems ranging from greenhouse testing down to in vitro microplate screening. This task can be formidable depending on the size of sample libraries and personnel resources available. One important tool in the overall discovery process is the application of highly specific and sensitive mechanism-based assays for the purpose of identifying novel leads. This article describes high throughput, agar-based screening techniques successfully implemented in a decentralized screening environment where laboratory space and staffing are limited. Through the prudent use of automated and manual high-density agar-based techniques, multiple sources of samples can now be processed and evaluated against multiple targets in a timely fashion. Assay validation can be streamlined. The advantages and disadvantages of an agar-based screening approach are discussed.

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De Rycker, Manu, Irene Hallyburton, John Thomas, Lorna Campbell, Susan Wyllie, Dhananjay Joshi, Scott Cameron, et al. "Comparison of a High-Throughput High-Content Intracellular Leishmania donovani Assay with an Axenic Amastigote Assay." Antimicrobial Agents and Chemotherapy 57, no. 7 (April 9, 2013): 2913–22. http://dx.doi.org/10.1128/aac.02398-12.

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ABSTRACTVisceral leishmaniasis is a neglected tropical disease with significant health impact. The current treatments are poor, and there is an urgent need to develop new drugs. Primary screening assays used for drug discovery campaigns have typically used free-living forms of theLeishmaniaparasite to allow for high-throughput screening. Such screens do not necessarily reflect the physiological situation, as the disease-causing stage of the parasite resides inside human host cells. Assessing the drug sensitivity of intracellular parasites on scale has recently become feasible with the advent of high-content screening methods. We describe here a 384-well microscopy-based intramacrophageLeishmania donovaniassay and compare it to an axenic amastigote system. A panel of eight reference compounds was tested in both systems, as well as a human counterscreen cell line, and our findings show that for most clinically used compounds both axenic and intramacrophage assays report very similar results. A set of 15,659 diverse compounds was also screened using both systems. This resulted in the identification of seven new antileishmanial compounds and revealed a high false-positive rate for the axenic assay. We conclude that the intramacrophage assay is more suited as a primary hit-discovery platform than the current form of axenic assay, and we discuss how modifications to the axenic assay may render it more suitable for hit-discovery.

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Journal articles on the topic 'High-throughput screening assay'

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