Use of BODIPY-Labeled ATP Analogues in the Development and Validation of a Fluorescence Polarization-Based Assay for Screening of Kinase Inhibitors

Bernardo Pereira Moreira; Tom Armstrong; Izabella Cristina Andrade Batista; Naiara Clemente Tavares; Camilla Valente Pires; Marina de Moraes Mourão; Franco H Falcone; Lodewijk V Dekker

doi:10.1021/acsomega.9b03344

Use of BODIPY-Labeled ATP Analogues in the Development and Validation of a Fluorescence Polarization-Based Assay for Screening of Kinase Inhibitors

ACS Omega. 2020 Apr 16;5(16):9064-9070. doi: 10.1021/acsomega.9b03344. eCollection 2020 Apr 28.

Authors

Affiliations

¹ Biomedizinisches Forschungszentrum Seltersberg, Institut für Parasitologie, Justus-Liebig-Universität Gießen, 35392 Gießen, Germany.
² Instituto de Pesquisas René Rachou, Fundação Oswaldo Cruz-FIOCRUZ, Belo Horizonte 30190-002, Minas Gerais, Brazil.
³ School of Pharmacy, Division of Molecular Therapeutics and Formulation, University of Nottingham, Nottingham NG7 2RD, United Kingdom.
⁴ School of Chemistry, University of Nottingham, Nottingham NG7 2RD, United Kingdom.
⁵ School of Pharmacy, Division of Biomolecular Science and Medicinal Chemistry, University of Nottingham, Nottingham NG7 2RD, United Kingdom.

Abstract

The screening of compound libraries to identify small-molecule modulators of specific biological targets is crucial in the process for the discovery of novel therapeutics and molecular probes. Considering the need for simple single-tool assay technologies with which one could monitor "all" kinases, we developed a fluorescence polarization (FP)-based assay to monitor the binding capabilities of protein kinases to ATP. We used BODIPY ATP-y-S as a probe to measure the shift in the polarization of a light beam when passed through the sample. We were able to optimize the assay using commercial Protein Kinase A (PKA) and H7 efficiently inhibited the binding of the probe when added to the reaction. Furthermore, we were able to employ the assay in a high-throughput fashion and validate the screening of a set of small molecules predicted to dock into the ATP-binding site of PKA. This will be useful to screen larger libraries of compounds that may target protein kinases by blocking ATP binding.