Responsive microsecond-lifetime photoluminescent probes for analysis of protein kinases and their inhibitors

• Published in: Biochimica et biophysica acta 2013-07, Vol.1834 (7), p.1330-1335
• Main Authors: Kasari, Marje, Ligi, Kadri, Williams, J.A. Gareth, Vaasa, Angela, Enkvist, Erki, Viht, Kaido, Pålsson, Lars-Olof, Uri, Asko
• Format: Article
• Language: English
• Subjects: Algorithms; Anisotropy; Binding Sites; Binding, Competitive; Biocatalysis - drug effects; Bisubstrate inhibitor; fluorescence; fluorescent dyes; Fluorescent Dyes - chemistry; Fluorescent Dyes - metabolism; Kinetics; Live cell imaging; Luminescent probe; lysine; Lysine - chemistry; Lysine - metabolism; Models, Chemical; Molecular Structure; phosphorescence; photoluminescence; Protein Binding; Protein kinase; Protein Kinase C - antagonists & inhibitors; Protein Kinase C - chemistry; Protein Kinase C - metabolism; Protein Kinase Inhibitors - metabolism; Protein Kinase Inhibitors - pharmacology; protein kinases; Protein Kinases - chemistry; Protein Kinases - metabolism; Reporter displacement assay; Spectrophotometry; Staurosporine - metabolism; Staurosporine - pharmacology; thiophene; Thiophenes - chemistry; Thiophenes - metabolism; Time Factors; Time-gated luminescence; wavelengths
• ISSN: 1570-9639; 0006-3002; 1878-1454
• DOI: 10.1016/j.bbapap.2013.02.039

Responsive ARC-Lum probes were used for measurement of the concentration of active protein kinases (PKs) and determination of affinity of inhibitors of PKs. ARC-Lum probes incorporate thiophene or a selenophene heterocycle and a fluorophore conjugated to the lysine residue in the peptide fragment. In the complex with a PK, ARC-Lum probes emit long-lifetime (microsecond-scale) luminescence at the emission wavelengths of the fluorescent label if the complex is illuminated at the excitation wavelength of the thiophene- or selenophene-containing phosphorescence donors. Bisubstrate ARC-Lum probes bind with sub-nanomolar affinity with several PKs of the AGC group. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012). [Display omitted] •Responsive luminescent probes (ARC-Lum) for protein kinase (PK) research•Kinetic and thermodynamic characterization of PK inhibitors in homogeneous format•Characterization of binding properties of tight-binding inhibitors of PKs•Cell membrane permeable ARC-Lum probes for monitoring PK activity in live cells