Development and validation of a high-density fluorescence polarization- based assay for the trypanosoma RNA triphosphatase TbCet1 Journal Article
| Authors: | Antczak, C.; Shum, D.; Radu, C.; Seshan, V. E.; Djaballah, H. |
| Article Title: | Development and validation of a high-density fluorescence polarization- based assay for the trypanosoma RNA triphosphatase TbCet1 |
| 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: | unclassified drug; dose response; nonhuman; drug targeting; methodology; protein analysis; animal; metabolism; animals; drug structure; high throughput screening; drug effect; drug screening; enzymology; drug discovery; fluorescence polarization; triphosphatase; enzyme inhibitor; malachite green; microbial enzyme; nucleoside triphosphatase; rna triphosphatase tbcet 1; rna triphosphatase tbcet 1 inhibitor; ski 210381; ski 210394; ski 210479; ski 211052; ski 211166; ski 211300; ski 218027; ski 218300; acid anhydride hydrolase; adenosine diphosphate; antiprotozoal agent; rna triphosphatase; enzyme activity; transcreener adp fluorescence polarization assay; trypanosoma brucei; drug development; molecular library; trypanosoma; validation study; acid anhydride hydrolases; antiprotozoal agents; drug evaluation, preclinical; small molecule libraries |
| Journal Title: | Combinatorial Chemistry and High Throughput Screening |
| Volume: | 12 |
| Issue: | 3 |
| ISSN: | 1386-2073 |
| Publisher: | Bentham Science Publishers |
| Date Published: | 2009-03-01 |
| Start Page: | 258 |
| End Page: | 268 |
| Language: | English |
| DOI: | 10.2174/138620709787581729 |
| PUBMED: | 19275531 |
| PROVIDER: | scopus |
| PMCID: | PMC3626118 |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 4" - "Export Date: 30 November 2010" - "CODEN: CCHSF" - "Source: Scopus" |
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