Alkylation of Staurosporine to Derive a Kinase Probe for Fluorescence Applications

Abstract

The natural product staurosporine is a high‐affinity inhibitor of nearly all mammalian protein kinases. The labelling of staurosporine has proven effective as a means of generating protein kinase research tools. Most tools have been generated by acylation of the 4′‐methylamine of the sugar moiety of staurosporine. Herein we describe the alkylation of this group as a first step to generate a fluorescently labelled staurosporine. Following alkylation, a polyethylene glycol linker was installed, allowing subsequent attachment of fluorescein. We report that this fluorescein–staurosporine conjugate binds to cAMP‐dependent protein kinase in the nanomolar range. Furthermore, its binding can be antagonised with unmodified staurosporine as well as ATP, indicating it targets the ATP binding site in a similar fashion to native staurosporine. This reagent has potential application as a screening tool for protein kinases of interest.

Natural-product-based fluorescent probes: recent advances and applications

“…126–129 In addition, fluorescently labeled derivatives 129 (ref. 130) and 130 (ref. 131) were generated by alkylation of the 4′-methylamine, followed by conjugation with fluorescein and TAMRA moieties via polyethylene glycol linkers.…”

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

“…We recently described a fluorescent tool based on the nonspecific kinase inhibitor staurosporine. The tool was highly suitable for FP applications and allowed monitoring the ATP-binding site of a large number of kinases and in this way enabled identification of inhibitory substances . Although the FP technique is easily adapted for HTS applications, a significant number of kinases could not be measured using this tool.…”

“…The tool was highly suitable for FP applications and allowed monitoring the ATP-binding site of a large number of kinases and in this way enabled identification of inhibitory substances. 4 Although the FP technique is easily adapted for HTS applications, a significant number of kinases could not be measured using this tool. With the emergence of the new therapeutic areas for kinase drug discovery and considering the still considerably large orphan kinase family in, for example, oncology applications, the need arises for simple universal assay technologies with which one could monitor most kinases.…”