Abstract
RNA triphosphatases are attractive and mostly unexplored therapeutic targets for the development of broad spectrum antiprotozoal, antiviral and antifungal agents. The use of malachite green as a readout for phosphatases is well characterized and widely employed. However, the reaction depends on high quantities of inorganic phosphate to be generated, which makes this assay not easily amenable to screening in 1536-well format. The overly long reading times required also prohibit its use to screen large chemical libraries. To overcome these limitations, we sought to develop a fluorescence polarization (FP) -based assay for triphosphatases, compatible with miniaturization and fast readouts. For this purpose, we took advantage of the nucleoside triphosphatase activity of this class of enzyme to successfully adapt the Transcreener™ ADP assay based on the detection of generated ADP by immunocompetition fluorescence polarization to the RNA triphosphatase TbCet1 in 1536-well format. We also tested the performance of this newly developed assay in a pilot screen of 3,000 compounds and we confirmed the activity of the obtained hits. We present and discuss our findings and their importance for the discovery of novel drugs by high throughput screening.
Keywords: Triphosphatase, drug discovery, high throughput screening, fluorescence polarization
Combinatorial Chemistry & High Throughput Screening
Title: Development and Validation of a High-Density Fluorescence Polarization-Based Assay for the Trypanosoma RNA Triphosphatase TbCet1
Volume: 12 Issue: 3
Author(s): Christophe Antczak, David Shum, Constantin Radu, Venkatraman E. Seshan and Hakim Djaballah
Affiliation:
Keywords: Triphosphatase, drug discovery, high throughput screening, fluorescence polarization
Abstract: RNA triphosphatases are attractive and mostly unexplored therapeutic targets for the development of broad spectrum antiprotozoal, antiviral and antifungal agents. The use of malachite green as a readout for phosphatases is well characterized and widely employed. However, the reaction depends on high quantities of inorganic phosphate to be generated, which makes this assay not easily amenable to screening in 1536-well format. The overly long reading times required also prohibit its use to screen large chemical libraries. To overcome these limitations, we sought to develop a fluorescence polarization (FP) -based assay for triphosphatases, compatible with miniaturization and fast readouts. For this purpose, we took advantage of the nucleoside triphosphatase activity of this class of enzyme to successfully adapt the Transcreener™ ADP assay based on the detection of generated ADP by immunocompetition fluorescence polarization to the RNA triphosphatase TbCet1 in 1536-well format. We also tested the performance of this newly developed assay in a pilot screen of 3,000 compounds and we confirmed the activity of the obtained hits. We present and discuss our findings and their importance for the discovery of novel drugs by high throughput screening.
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Cite this article as:
Antczak Christophe, Shum David, Radu Constantin, Seshan E. Venkatraman and Djaballah Hakim, Development and Validation of a High-Density Fluorescence Polarization-Based Assay for the Trypanosoma RNA Triphosphatase TbCet1, Combinatorial Chemistry & High Throughput Screening 2009; 12 (3) . https://dx.doi.org/10.2174/138620709787581729
DOI https://dx.doi.org/10.2174/138620709787581729 |
Print ISSN 1386-2073 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5402 |
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