Development of a high-throughput fluorescence polarization assay to identify novel ligands of glutamate carboxypeptidase II

Glenda Alquicer, David Sedlák, Youngjoo Byun, Jiří Pavlíček, Marigo Stathis, Camilo Rojas, Barbara Slusher, Martin Gilbert Pomper, Petr Bartůněk, Cyril Bařinka

Research output: Contribution to journalArticle


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.

Original languageEnglish (US)
Pages (from-to)1030-1040
Number of pages11
JournalJournal of Biomolecular Screening
Issue number8
Publication statusPublished - Sep 2012



  • fluorescence polarization
  • glutamate carboxypeptidase II
  • high-throughput screening
  • metallopeptidase
  • prostate-specific membrane antigen

ASJC Scopus subject areas

  • Analytical Chemistry
  • Drug Discovery
  • Pharmacology
  • Biochemistry
  • Molecular Medicine
  • Biotechnology

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