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,Naiara Clemente Tavares,Camilla Valente Pires,Marina De Moraes Mourão,Lodewijk V Dekker,Franco H Falcone,Izabella Cristina Andrade Batista,Tom Armstrong

doi:10.1021/acsomega.9b03344

Bernardo Pereira Moreira, Naiara Clemente Tavares + Show 6 more

Open Access

https://doi.org/10.1021/acsomega.9b03344

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Journal: ACS omega	Publication Date: Apr 16, 2020
Citations: 4	License type: cc-by

Affiliation: Fundação Oswaldo Cruz, University of Nottingham

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.

Highlights

A very first step on the path for the discovery of novel therapeutics is the screening of compound libraries in the search for new small-molecule modulators of biological targets
We performed an in silico docking prediction of the ATP and the BODIPY FL ATP-γ-S (adenosine 5′-O-(3-thiotriphosphate), BODIPY FL) probe into the nucleotide-binding pocket (G-loop) of the protein kinase A (PKA)
To optimize the probe concentration to be used in the assay, fluorescence polarization (FP) was measured for several concentrations of the probe, ranging from 10−13 to 10−7 M in three different conditions (Figure 1B)

Summary

Introduction

A very first step on the path for the discovery of novel therapeutics is the screening of compound libraries in the search for new small-molecule modulators of biological targets. A wide range of robust assay technologies are currently available and, no single technology is broad enough to address all of the needs in the drug discovery field, most of them are suitable for high-throughput screening (HTS). Fluorescence polarization (FP) is a powerful approach by which alterations in the apparent molecular weight of a fluorescent probe in solution are indicated by changes in the polarization of the sample’s emitted light.[1] Since FP was first applied to screening, newly advanced methods have substantially boosted this technology in the field. Advantages of FP assays include the use of an allin-one (homogeneous) format fitted to study molecular processes in solution, comparatively low cost, availability of time-course analysis, and relatively insensitivity to some type of assay interferences such as inner filter effects.[1−3]

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