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.
authors.affiliation: High Throughput Screening Core Facility, Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA.
authors.forename: Christophe
authors.initials: C
authors.lastname: Antczak
authors.forename: David
authors.initials: D
authors.lastname: Shum
authors.forename: Constantin
authors.initials: C
authors.lastname: Radu
authors.forename: Venkatraman E
authors.initials: VE
authors.lastname: Seshan
authors.forename: Hakim
authors.initials: H
authors.lastname: Djaballah
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000187:drug effects* / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000032:analysis; D000818:Animals; D000981:Antiprotozoal Agents; D055808:Drug Discovery; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005454:Fluorescence Polarization / Q000379:methods*; D054852:Small Molecule Libraries; D014345:Trypanosoma / Q000201:enzymology*
chemical_list: D000981:Antiprotozoal Agents; D054852:Small Molecule Libraries; D000244:Adenosine Diphosphate; D017766:Acid Anhydride Hydrolases; C023090:RNA triphosphatase