High-throughput kinase assays with protein substrates using fluorescent polymer superquenching

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Protein phosphorylation is crucial in diseases like cancer, making kinases key drug targets.
• Current screening methods often use synthetic peptides, potentially missing inhibitors that bind outside the catalytic site.
• Previous work developed a fluorescence quench assay for synthetic peptides.

Purpose of the Study:

• To develop a novel assay for detecting phosphorylation of chemically unmodified proteins.
• To adapt a polymer superquenching platform for protein substrate analysis.
• To enable screening for kinase inhibitors using natural protein substrates.

Main Methods:

• A modified QTL Lightspeed assay was employed.
• Phosphorylation was quantified by protein interaction with metal-ion coordinating groups on fluorescent microspheres.
• A "turn on" fluorescence signal correlates with substrate phosphorylation.

Main Results:

• The assay successfully detected phosphorylation of Myelin Basic Protein (MBP), Histone H1, and Phosphorylated heat- and acid-stable protein (PHAS-1).
• Kinase inhibition studies using Protein Kinase Calpha (PKCalpha) and Interleukin-1 Receptor-associated Kinase 4 (IRAK4) yielded IC50 values comparable to peptide-based assays.
• High Z -values indicated assay robustness suitable for high-throughput screening (HTS).

Conclusions:

• The QTL Lightspeed system is a simple, mix-and-measure "turn on" assay for kinase activity detection.
• It effectively utilizes natural protein substrates.
• The platform is robust and suitable for identifying kinase inhibitors in drug discovery.