ABSTRACT
Peptide microarrays can be used to measure the activity of multiple protein kinases (PKs), which can be used to elucidate kinomics for drug discovery and diagnosis. Here we demonstrated a new microarray for ratiometric detection of the activity of PKs using peptide nucleic acid (PNA)-tagged peptides labeled with two different fluorophores, Cy3 and Cy5. We successfully detected cellular PK activities based on ratiometry, and applied the system for evaluation of an inhibitory drug.
Subject(s)
Peptide Nucleic Acids/chemistry , Protein Array Analysis/methods , Protein Kinases/metabolism , Amino Acid Sequence , Base Sequence , DNA/chemistry , Fluorescent Dyes/chemistry , Molecular Sequence Data , PhosphorylationABSTRACT
Here we developed a simple set-and-mix assay to perform high-throughput screening of protein kinase A (PKA) inhibitors from the LOPAC 1280 compound library. This assay is based on the color change of gold nanoparticles on aggregation induced by a cationic substrate peptide as coagulant. In spite of the simplicity of this assay system, this assay can be applied to drug screening based on cellular kinases. We successfully found several highly active inhibitors, including compounds that have not been reported before.
