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

• Published in: ACS omega 2020-04, Vol.5 (16), p.9064-9070
• Main Authors: Moreira, Bernardo Pereira, Armstrong, Tom, Batista, Izabella Cristina Andrade, Clemente Tavares, Naiara, Pires, Camilla Valente, de Moraes Mourão, Marina, Falcone, Franco H, Dekker, Lodewijk V
• Format: Article
• Language: English
• ISSN: 2470-1343; 2470-1343
• DOI: 10.1021/acsomega.9b03344

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.