title: Development of a high-throughput fluorescence polarization assay to identify novel ligands of glutamate carboxypeptidase II.
abstract: 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.
authors: Institute of Biotechnology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors: Glenda
authors: G
authors: Alquicer
authors: David
authors: D
authors: Sedlák
authors: Youngjoo
authors: Y
authors: Byun
authors: Jirí
authors: J
authors: Pavlícek
authors: Marigo
authors: M
authors: Stathis
authors: Camilo
authors: C
authors: Rojas
authors: Barbara
authors: B
authors: Slusher
authors: Martin G
authors: MG
authors: Pomper
authors: Petr
authors: P
authors: Bartunek
authors: Cyril
authors: C
authors: Barinka
mesh_terms: D000954:Antigens, Surface / Q000235:genetics / Q000378:metabolism; D005454:Fluorescence Polarization / Q000379:methods*; D005456:Fluorescent Dyes / Q000138:chemical synthesis; D043425:Glutamate Carboxypeptidase II / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008024:Ligands; D011485:Protein Binding; D054852:Small Molecule Libraries / Q000494:pharmacology
chemical_list: D000954:Antigens, Surface; D005456:Fluorescent Dyes; D008024:Ligands; D054852:Small Molecule Libraries; C473848:FOLH1 protein, human; D043425:Glutamate Carboxypeptidase II