The developing zebrafish (Danio rerio): A vertebrate model for high-throughput screening of chemical libraries

2011 ◽  
Vol 93 (3) ◽  
pp. 268-280 ◽  
Author(s):  
Charles A. Lessman
Keyword(s):  
High Throughput ◽  
Danio Rerio ◽  
High Throughput Screening ◽  
Chemical Libraries ◽  
Vertebrate Model

scholarly journals High-Throughput Fluorescence Assay for Small-Molecule Inhibitors of Autophagins/Atg4

2011 ◽  
Vol 16 (2) ◽  
pp. 174-182 ◽  
Author(s):  
Chih-Wen Shu ◽  
Charitha Madiraju ◽  
Dayong Zhai ◽  
Kate Welsh ◽  
Paul Diaz ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Caspase 3 ◽  
Bioactive Molecules ◽  
Dependent Manner ◽  
Chemical Libraries ◽  
Evolutionarily Conserved ◽  
Identification And Characterization

Autophagy is an evolutionarily conserved process for catabolizing damaged proteins and organelles in a lysosome-dependent manner. Dysregulation of autophagy may cause various diseases, such as cancer and neurodegeneration. However, the relevance of autophagy to diseases remains controversial because of the limited availability of chemical modulators. Herein, the authors developed a fluorescence-based assay for measuring activity of the autophagy protease, autophagin-1(Atg4B). The assay employs a novel reporter substrate of Atg4B composed of a natural substrate (LC3B) fused to an assayable enzyme (PLA2) that becomes active upon cleavage by this cysteine protease. A high-throughput screening (HTS) assay was validated with excellent Z′ factor (>0.7), remaining robust for more than 5 h and suitable for screening of large chemical libraries. The HTS assay was validated by performing pilot screens with 2 small collections of compounds enriched in bioactive molecules ( n = 1280 for Lopac™ and 2000 for Spectrum™ library), yielding confirmed hit rates of 0.23% and 0.70%, respectively. As counterscreens, PLA2 and caspase-3 assays were employed to eliminate nonspecific inhibitors. In conclusion, the LC3B-PLA2 reporter assay provides a platform for compound library screening for identification and characterization of Atg4B-specific inhibitors that may be useful as tools for interrogating the role of autophagy in disease models.

scholarly journals High-Throughput Identification of Inhibitors of Human Mitochondrial Peptide Deformylase

2007 ◽  
Vol 12 (4) ◽  
pp. 521-535 ◽  
Author(s):  
Christophe Antczak ◽  
David Shum ◽  
Sindy Escobar ◽  
Bhramdeo Bassit ◽  
Earl Kim ◽  
...  
Keyword(s):  
High Throughput ◽  
Antiproliferative Activity ◽  
High Throughput Screening ◽  
Peptide Deformylase ◽  
Chemical Libraries ◽  
New Class ◽  
Cytotoxicity Studies ◽  
Antiproliferative Agent ◽  
And Performance

The human mitochondrial peptide deformylase (HsPDF) provides a potential new target for broadly acting antiproliferative agents. To identify novel nonpeptidomimetic and nonhydroxamic acid—based inhibitors of HsPDF, the authors have developed a high-throughput screening (HTS) strategy using a fluorescence polarization (FP)—based binding assay as the primary assay for screening chemical libraries, followed by an enzymatic-based assay to confirm hits, prior to characterization of their antiproliferative activity against established tumor cell lines. The authors present the results and performance of the established strategy tested in a pilot screen of 2880 compounds and the identification of the 1st inhibitors. Two common scaffolds were identified within the hits. Furthermore, cytotoxicity studies revealed that most of the confirmed hits have antiproliferative activity. These findings demonstrate that the designed strategy can identify novel functional inhibitors and provide a powerful alternative to the use of functional assays in HTS and support the hypothesis that HsPDF inhibitors may constitute a new class of antiproliferative agent. ( Journal of Biomolecular Screening 2007:521-535)

scholarly journals A Thallium-Sensitive, Fluorescence-Based Assay for Detecting and Characterizing Potassium Channel Modulators in Mammalian Cells

2004 ◽  
Vol 9 (8) ◽  
pp. 671-677 ◽  
Author(s):  
C. David Weaver ◽  
David Harden ◽  
Steven I. Dworetzky ◽  
Barbara Robertson ◽  
Ronald J. Knox
Keyword(s):  
Potassium Channels ◽  
High Throughput ◽  
High Throughput Screening ◽  
Mammalian Cells ◽  
Ease Of Use ◽  
Functional Assay ◽  
Chemical Libraries ◽  
Potassium Channel Modulators ◽  
Small Molecule Modulators

Potassium channels have been identified as targets for a large number of therapeutic indications. The ability to use a high-throughput functional assay for the detection and characterization of small-molecule modulators of potassium channels is very desirable. However, present techniques capable of screening very large chemical libraries are limited in terms of data quality, temporal resolution, ease of use, and requirements for specialized instrumentation. To address these issues, the authors have developed a fluorescence-based thalliumflux assay. This assay is capable of detectingmodulators of both voltageand ligand-gated potassium channels expressed inmammalian cells. The thalliumflux assay can use instruments standard to most high-throughput screening laboratories, and using such equipment has been successfully employed to screen large chemical libraries consisting of hundreds of thousands of compounds.

scholarly journals Development and Application of a High-Throughput Screening Method to Evaluate Antifungal Activity against Trichophyton tonsurans

2015 ◽  
Vol 20 (9) ◽  
pp. 1171-1177 ◽  
Author(s):  
Barry Preuett ◽  
J. Steven Leeder ◽  
Susan Abdel-Rahman
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Inhibitory Concentration ◽  
Screening Method ◽  
Trichophyton Tonsurans ◽  
Chemical Libraries ◽  
Drug Classes ◽  
Positive Growth ◽  
Z Scores ◽  
Growth Controls

There exist relatively few drug classes on the market to treat dermatophyte infections. This investigation was designed to develop and validate high-throughput methodology for screening and confirmation of chemicals for activity against Trichophyton tonsurans. Growth characteristics were examined on two platforms (96- and 384-well) in three media at eight spore concentrations over a period of up to 120 h. Microspectrophotometry was used to automate plate reads. The 384-well platform was used to screen more than 7000 compounds from six chemical libraries. Z-scores for optical density relative to positive growth controls were used to flag compounds of interest and activity confirmed in separate assays. The final conditions selected for both screening and confirmation with minimum inhibitory concentration (MIC) determination were growth for 48 h at 32 °C in SabDex with 1 × 104 spores per reaction. Sensitivity and specificity averaged 99.2% (range, 95.2%−100%) and 99.8% (range, 99.1%−100%), respectively. MICs for known antifungals were similar to those reported by others using Clinical and Laboratory Standards Institute methods. Several novel compound classes were identified to have activity against T. tonsurans with potency comparable to known antifungals. A robust, reproducible assay is described that permits high-throughput screening in T. tonsurans.

scholarly journals High throughput screening using acoustic droplet ejection to combine protein crystals and chemical libraries on crystallization plates at high density

2015 ◽  
Vol 191 (1) ◽  
pp. 49-58 ◽  
Author(s):  
Ella Teplitsky ◽  
Karan Joshi ◽  
Daniel L. Ericson ◽  
Alexander Scalia ◽  
Jeffrey D. Mullen ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
High Density ◽  
Protein Crystals ◽  
Chemical Libraries ◽  
Droplet Ejection

scholarly journals High-Throughput Screening for Protein Synthesis Inhibitors Targeting Aminoacyl-tRNA Synthetases

2017 ◽  
Vol 23 (2) ◽  
pp. 174-182 ◽  
Author(s):  
Jiwon Kong ◽  
Pengfei Fang ◽  
Franck Madoux ◽  
Timothy P. Spicer ◽  
Louis Scampavia ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Small Molecules ◽  
High Throughput ◽  
High Throughput Screening ◽  
Protein Synthesis Inhibitors ◽  
Chemical Libraries ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Reticulocyte Lysate ◽  
The Individual

Aminoacylation has been implicated in a wide variety of cancers. Aminoacyl-tRNA synthetases (ARSs) exist in large excess in tumor cells due to their increased demand for translation, whereas most other protein-synthesis apparatuses are quantitatively limited. Among other components that constitute the translation machinery—namely, tRNA, amino acid, ATP, and ARS—ARS is the only target that can be blocked by small molecules. No constitutively active ARSs have been reported, and mutations of ARS can cause inaccurate substrate recognition and malformation of the multi-ARS complex (MSC). Hence, interference of the activity is expected to be independent of genotype without developing resistance. Here, we report a high-throughput screening (HTS) system to find mammalian ARS inhibitors. The rabbit–reticulocyte lysate we used closely resembles both the individual and complexed structures of human ARSs, and it may predispose active compounds that are readily applicable for humankind. This assay was further validated because it identified familiar translational inhibitors from a pilot screen, such as emetine, proving its suitability for our purpose. The assay demonstrated excellent quality control (QC) parameters and reproducibility, and is proven ready for further HTS campaigns with large chemical libraries.

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