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Development of a High-Throughput Fluorescence Polarization Assay to Identify Novel Ligands of Glutamate Carboxypeptidase II
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SYSNO ASEP 0385319 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title Development of a High-Throughput Fluorescence Polarization Assay to Identify Novel Ligands of Glutamate Carboxypeptidase II Author(s) Alquicer, Glenda (BTO-N)
Sedlák, David (UMG-J) RID
Byun, Y. (KR)
Pavlíček, Jiří (BTO-N) RID
Stathis, M. (US)
Rojas, C. (US)
Slusher, B. (US)
Pomper, M.G. (US)
Bartůněk, Petr (UMG-J) RID
Bařinka, Cyril (BTO-N) RID, ORCIDSource Title Journal of Biomolecular Screening - ISSN 1087-0571
Roč. 17, č. 8 (2012), s. 1030-1040Number of pages 11 s. Language eng - English Country US - United States Keywords fluorescence polarization ; glutamate carboxypeptidase II ; high-throughput screening Subject RIV EB - Genetics ; Molecular Biology R&D Projects ME10031 GA MŠMT - Ministry of Education, Youth and Sports (MEYS) LC06077 GA MŠMT - Ministry of Education, Youth and Sports (MEYS) Institutional support UMG-J - RVO:68378050 CEZ AV0Z50520701 - BTO-N (2007-2013) UT WOS 000307543000003 DOI 10.1177/1087057112451924 Annotation Glutamate carboxypeptidase II (GCPII) is an important target for therapeutic and diagnostic interventions aimed at prostate cancer and neurologic disorders. Here we describe the development and optimization of a high-throughput screening (HTS) assay based on fluorescence polarization (FP) that facilitates the identification of novel scaffolds inhibiting GCPII. First, we designed and synthesized a fluorescence probe based on a urea-based inhibitory scaffold covalently linked to a Bodipy TMR fluorophore (TMRGlu). Next, we established and optimized conditions suitable for HTS and evaluated the assay robustness by testing the influence of a variety of physicochemical parameters (e.g., pH, temperature, time) and additives. Using known GCPII inhibitors, the FP assay was shown to be comparable to benchmark assays established in the field. Finally, we evaluated the FP assay by HTS of a 20 000-compound library. The novel assay presented here is robust, highly reproducible (Z' = 0.82), inexpensive, and suitable for automation, thus providing an excellent platform for HTS of small-molecule libraries targeting GCPII. Workplace Institute of Biotechnology Contact Monika Kopřivová, Monika.Koprivova@ibt.cas.cz, Tel.: 325 873 700 Year of Publishing 2013
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