title: 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.
authors.affiliation: School of Pharmacy, Centre for Biomolecular Sciences, University of Nottingham, Nottingham, NG7 2RD, Nottinghamshire, UK.
authors.forename: Alexander J M
authors.initials: AJ
authors.lastname: Disney
authors.affiliation: School of Pharmacy, Centre for Biomolecular Sciences, University of Nottingham, Nottingham, NG7 2RD, Nottinghamshire, UK.
authors.forename: Barrie
authors.initials: B
authors.lastname: Kellam
authors.affiliation: School of Pharmacy, Centre for Biomolecular Sciences, University of Nottingham, Nottingham, NG7 2RD, Nottinghamshire, UK. lodewijk.dekker@nottingham.ac.uk.
authors.forename: Lodewijk V
authors.identifier: 0000-0001-5247-3297
authors.initials: LV
authors.lastname: Dekker
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000478:Alkylation; D001665:Binding Sites; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry*; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000378:metabolism*; D019311:Staurosporine / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D047428:Protein Kinase Inhibitors; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D019311:Staurosporine
keywords: alkylation; fluorescein; fluorescent probes; kinases; screening