High throughput screening of potential GCPII inhibitors by fluorescence polarization

Glenda Alquicer¹, David Sedlák², Youngjoo Byun^{3, 4}, Jiri

Pavlicek¹, Marigo Statis⁵, Camilo Rojas⁵, Barbara Slusher⁵, Martin G.

Pomper³, Petr Bartunek² and Cyril Barinka¹

¹Institute of Biotechnology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 14220 Prague 4, Czech Republic

²Center for Chemical Genetics & CZ-OPENSCREEN, Institute of Molecular Genetics, v.v.i., Academy of Sciences of the Czech Republic, Vídeňská 1083, 14220 Prague, Czech Republic

³Department of Radiology, Johns Hopkins Medical Institutions, Baltimore, MD 21231, USA

⁴College of Pharmacy, Korea University, Sejong-ro, Jochiwon-eup, Yeongi-gun, Chungnam 339-700, South Korea

⁵Brain Science Institute, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA

Glutamate Carboxypeptidase II (GCPII) inhibition is an important aim for current research on diagnostic and therapeutic interventions inprostate cancer and neurologic disorders. Her we describe the development and optimization of a High Throughput Screening (HTS) assay, based on fluorescence polarization (FP), to identify new scaffolds inhibiting GCPII. The synthesis of a fluorescence probe was accomplished by covalently attaching a Bodipy TMR fluorophore to a urea-based GCPII inhibitor. The conditions for HTS and robustness of a FP based assay were optimized considering factors as pH, temperature, time and additives. The approach showed suitability to detect both competitive and non-competitive GCPII inhibitors and the results are comparable to those provided by benchmark assays. Thereafter, the assay was used to screen a library of 20,000 pharmacologically active compounds. The determination of a Z' factor 0.82 validates the performance of the assay in a high-throughput format. It additionally represents a non-hazardous, inexpensive and robust methodology to identify novel GCPII inhibitors.