Development of a High-Throughput Fluorescence Polarization Assay to Identify Novel Ligands of Glutamate Carboxypeptidase II

0385319 - BTÚ 2013 RIV US eng J - Journal Article
Alquicer, Glenda - Sedlák, David - Byun, Y. - Pavlíček, Jiří - Stathis, M. - Rojas, C. - Slusher, B. - Pomper, M.G. - Bartůněk, Petr - Bařinka, Cyril
Development of a High-Throughput Fluorescence Polarization Assay to Identify Novel Ligands of Glutamate Carboxypeptidase II.
Journal of Biomolecular Screening. Roč. 17, č. 8 (2012), s. 1030-1040. ISSN 1087-0571
R&D Projects: GA MŠMT(CZ) ME10031; GA MŠMT(CZ) LC06077
Institutional research plan: CEZ:AV0Z50520701
Institutional support: RVO:68378050
Keywords : fluorescence polarization * glutamate carboxypeptidase II * high-throughput screening
Subject RIV: EB - Genetics ; Molecular Biology
Impact factor: 2.207, year: 2012

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.
Permanent Link: http://hdl.handle.net/11104/0214606