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Development of a high-throughput fluorescence polarization assay to identify novel ligands of glutamate carboxypeptidase II
G. Alquicer, D. Sedlák, Y. Byun, J. Pavlícek, M. Stathis, C. Rojas, B. Slusher, MG. Pomper, P. Bartunek, C. Barinka,
Language English Country United States
Document type Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't
- MeSH
- Antigens, Surface genetics metabolism MeSH
- Fluorescent Dyes chemical synthesis MeSH
- Fluorescence Polarization methods MeSH
- Glutamate Carboxypeptidase II antagonists & inhibitors genetics metabolism MeSH
- Small Molecule Libraries pharmacology MeSH
- Humans MeSH
- Ligands MeSH
- High-Throughput Screening Assays methods MeSH
- Protein Binding MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
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.
References provided by Crossref.org
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