A colorimetric assay for kinase activity based on the aggregation of gold nanoparticles.
UK researchers have developed a system for assessing the activity of a crucial class of enzymes involved in cellular signalling pathways.
The enzymes, kinases, are central to many metabolic processes, making them key drug targets. The system could for the first time lead to high-throughput screening of potential kinase inhibitors, say its developers.
The researchers, from the Centre for Nanoscale Science at the University of Liverpool, previously demonstrated that spherical gold nanoparticles could be stabilised in solution and coated with oligopeptides, giving them the appearance and properties of a protein.
Now, the team, led by Mathias Brust and David Fernig, has incorporated the nanoparticles into an assay for kinase inhibition.
Kinases phosphorylate proteins with the terminal phosphate of adenosine triphosphate(ATP).
The Liverpool team constructed peptide-coated nanoparticles that could be phosphorylated by kinases. They used ATP whose terminal phosphate is linked to a small organic molecule, biotin, which binds powerfully but non-covalently to a protein called avidin.
To test kinase activity, the kinase was presented with biotinylated ATP and the peptide-coated gold nanoparticles. When phosphorylation occurred, the terminal phosphate, together with its biotin moeity, was transferred to the nanoparticles.
These particles were mixed with a second population of gold nanoparticles - which was coated with avidin.
If biotin was present on the first set of particles, it bound to the avidin-coated particles, and the gold nanoparticles aggregated.
’There is a well-established optical phenomenon whereby when gold nanoparticles aggregate there is a colour shift from red to blue,’ Brust told Chemistry World. ’This colour change is immediately visible and immediately measurable.’
If no aggregation occurred - because no biotin was transferred to the nanoparticles due to a failure of phosphorylation - there was no colour shift.
’This is a simple and quick way to test potential inhibitors of kinase activity,’ said Brust.
The researchers have now tested the system on two different kinases and three inhibitors. ’This could provide a format for massive parallel testing on microplates for potential kinase inhibitors,’ said Brust. ’It may have important implications for the future use of nanoparticle-based technologies in drug discovery.’
J. Am. Chem. Soc, 2006 (DOI: 10.1021/ja058135y)
No comments yet