A kinase is a type of enzyme that transfers phosphate groups from high-energy donor molecules, such as adenosine triphosphate (ATP), to specific target molecules (substrates), causing activation or inactivation of the target protein; this process is called phosphorylation. Protein phosphorylation is one of the key signal transduction mechanisms in a host of biological pathways including cell growth, proliferation, apoptosis and metabolism. There are two main types of kinase, those that phosphorylate tyrosine residues and those that phosphorylate serine and/or threonine residues in target proteins. In recent years, kinases have emerged as a major source of interest for therapeutics, particularly for cancer related therapies. Relatively few kinase inhibitors have been identified to date and so a large amount of effort is being given to identifying these potential pathway modifying targets.
There are a large number of methods for assessing kinase function and activity. Several different classes of kinase assay technology have developed over recent years, each of which have their relative merits and drawbacks 1, 2 .
The traditional ‘gold-standard’ kinase assay is a radio-isotope binding assay in which the phosphorylation reaction of a peptide substrate is initiated by the addition of gamma 33P labelled ATP. The 33P labeled substrate is then bound to a membrane and any unbound radioisotope is washed away before detection. The incorporation of the radio-labeled phosphate into the kinase peptide substrate is directly proportional to the kinase activity. Although this assay is a highly sensitive method for measuring kinase activity, with very little background noise, it is now not routinely used for high-throughput screening due to the inherent problems with disposal of radioactive material and the numerous washing and detection steps involved.
Before fluorescence based assays became widely-used, ELISAs were commonly employed for kinase analysis. Biotin labeled peptides are bound on a plate or membrane and a kinase reaction is initiated by the addition of kinase and ATP. Phosphorylation is detected by a phospho-specific antibody and kinase function is assessed using a colorimetric read-out. The use of ELISA based assays has dropped in recent years as there is a lack of good quality phospho-specific antibodies and the technology is not suited to large drug-discovery screening programmes.
Fluorescence based kinase assays are now the most commonly used for high-throughput screening drug discovery due to the prevalence of fluorescence detection systems, allowing for automation of the assay and therefore relatively low cost.
Fluorescence polarization is a widely used method of detection to screen for kinase inhibitors. It is based on the principle that when in solution, smaller molecules rotate faster than larger molecules. A fluorescently labeled peptide substrate rotates quickly and so will have a low fluorescence polarization. When a phosphorylation reaction is initiated by addition of a kinase and ATP, any phosphorylated peptide substrates will be recognized by a phospho-antibody. The binding of the antibody will slow the rotation of the molecule (high fluorescence polarization) and this change in speed can be measured. As this is a one step assay with no additional wash steps, it is well suited to screening large numbers of kinase inhibitor candidates. However, one of the major drawbacks for this and other fluorescence based assays, is that there can be a high background signal and false positives from unused fluorescent compounds and labeled substrates.
Fluorescent resonance energy transfer assays use substrate peptides labeled with a fluorescent donor molecule and a non-fluorescent acceptor molecule. When the donor and acceptor are spatially close to each other, attached to either end of the short peptide, the acceptor molecule will quench any fluorescence emission from the donor molecule. The first step of a FRET kinase assay is the incubation of the FRET labeled substrate with ATP and kinase leading to the transfer of g-phosphate to the substrate peptide. The second step of the reaction involves adding a site specific protease to the samples. If the protease cleaves a non-phosphorylated peptide, the fluorescent donor and the acceptor moiety are sufficiently separated to disrupt the FRET and so a fluorescent signal is produced that can be measured as a ratio of donor emission:acceptor emission. FRET is another technology suited to automation for large scale screening projects. However, the peptide substrate design can be difficult to optimize as the donor and acceptor fluorophores need to be within a well defined distance in order for the energy to transfer.
Time resolved fluorescence uses fluorophores with a long decay time and measures levels of fluorescence over a period of time. Lanthanide fluorescent labels are often used for this purpose as they have relatively long emission times compared to standard fluorophores (microseconds compared to nanoseconds). The advantage of this technology is that contaminating fluorescent molecules which contribute to background noise will have a shorter decay time than the lanthanide ions and so the signal is much cleaner. There are several commercially available TRF assays, mainly based either on ELISA assays or combined with FRET assays.
The mobility shift assay works on the fact that phosphorylated peptide substrates are more negatively charged than unphosphorylated peptides. When peptides are subjected to gel electrophoresis, the phosphorylated and unphosphorylated substrates will have different mobility. However, as the difference in charge between the peptide and the substrate is key, the results are highly influenced by the size and sequence of the substrate. This assay is therefore unsuitable for larger peptides or whole protein analysis and works best when the peptide substrates contain only a specific phosphorylation site sequence. Ideally a combination of several methods will give the most comprehensive overview of kinase activity and function.
There are a large number of commercially available kinase assays that have been developed in recent years based on the technologies described above. Each technology has its strengths and weaknesses and the most suitable assay will depend on the aim of the project. For high-throughput screening drug discovery projects, methods that can be easily automated and miniaturized, such as the fluorescence based assays, are the most suitable. For projects requiring a more in depth profile of the kinase function, a more robust assay such as the radiometric assays will give more reliable results.
1Olive M. (2004) Quantitative methods for analysis of protein phosphorylation in drug development. Expert Rev Proteomics 1: 89-103 [PubMedID: 15966829]
2Ma H. et al (2008) The Challenge of selecting protein kinase assays for lead discovery optimization. Expert Opin. Drug Discovery. 3: 607-632 [PubMedID: 19662101]
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