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

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.

Abstract

Copyright © 2020 American Chemical Society. 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.

Citation

Moreira, B. P., Armstrong, T., Batista, I. C. A., Clemente Tavares, N., Pires, C. V., de Moraes Mourão, M., …Dekker, L. V. (2020). Use of BODIPY-Labeled ATP Analogues in the Development and Validation of a Fluorescence Polarization-Based Assay for Screening of Kinase Inhibitors. ACS Omega, 5(16), 9064-9070. https://doi.org/10.1021/acsomega.9b03344

• Journal Article Type: Article
• Acceptance Date: Mar 10, 2020
• Online Publication Date: Apr 16, 2020
• Publication Date: Apr 28, 2020
• Deposit Date: May 22, 2020
• Publicly Available Date: May 27, 2020
• Journal: ACS Omega
• Electronic ISSN: 2470-1343
• Publisher: American Chemical Society
• Peer Reviewed: Peer Reviewed
• Volume: 5
• Issue: 16
• Pages: 9064-9070
• DOI: https://doi.org/10.1021/acsomega.9b03344
• Public URL: https://nottingham-repository.worktribe.com/output/4488146
• Publisher URL: https://pubs.acs.org/doi/10.1021/acsomega.9b03344