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

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.

Functionalised Cofactor Mimics for Interactome Discovery and Beyond

“…In a recent example, a fluorescence polarization-based assay employing ATP-γ-S BODIPY FL was used to screen a small set of predicted inhibitors of the Protein Kinase A ATPpocket. [96] Fluorescence polarisation was also recently applied to identify competitive inhibitors of KATs, using a fluorescein-functionalised Ac-CoA probe (4.1, Figure 4), illustrating its versatility for inhibitor discovery. [97] Fluorescent cofactor analogues often make use of linkers to minimise disruption to protein binding; indeed, inclusion of a linker was recently found to be necessary to generate rhodamine-SAM mimics recognised by the methyltransferase M.TaqI and thus capable of labelling the enzyme's DNA targets.…”

“…These ATP analogues not only serve to investigate ATP binding but also can be used for high‐throughput‐screening approaches. In a recent example, a fluorescence polarization‐based assay employing ATP‐γ‐S BODIPY FL was used to screen a small set of predicted inhibitors of the Protein Kinase A ATP‐pocket [96] . Fluorescence polarisation was also recently applied to identify competitive inhibitors of KATs, using a fluorescein‐functionalised Ac‐CoA probe ( 4.1 , Figure 4), illustrating its versatility for inhibitor discovery [97] …”

Funktionalisierte Cofaktor‐Analoga für die Erforschung von Interaktomen und darüber hinaus

“…In einem aktuellen Beispiel wurde ein auf Fluoreszenzpolarisation basierender Assay unter Verwendung von ATP-γ-S BODIPY FL verwendet, um eine kleine Bibliothek von mutmaßlichen Inhibitoren der ATP-Bindungstasche der Proteinkinase A zu untersuchen. [96] Fluoreszenzpolarisation wurde zudem auch zur Identifizierung kompetitiver KAT-Inhibitoren eingesetzt, wobei eine mit Fluorescein funktionalisierte Ac-CoA-Sonde (4.1, Abbildung 4) zum Einsatz kam. Dies verdeutlicht die Vielseitigkeit der Methodik bei der Suche nach neuen Inhibitoren.…”

“…Diese ATP‐Analoga dienen nicht nur der Untersuchung der ATP‐Bindung, sondern können auch für Screening‐Verfahren mit hohen Durchsätzen verwendet werden. In einem aktuellen Beispiel wurde ein auf Fluoreszenzpolarisation basierender Assay unter Verwendung von ATP‐γ‐S BODIPY FL verwendet, um eine kleine Bibliothek von mutmaßlichen Inhibitoren der ATP‐Bindungstasche der Proteinkinase A zu untersuchen [96] . Fluoreszenzpolarisation wurde zudem auch zur Identifizierung kompetitiver KAT‐Inhibitoren eingesetzt, wobei eine mit Fluorescein funktionalisierte Ac‐CoA‐Sonde ( 4.1 , Abbildung 4) zum Einsatz kam.…”