Rapid, Low-Cost Multiplexed Assay of Kinase Inhibition

Robert L. Stevenson, Ph.D.

Protein kinases phosphorylate serine, threonine, and tyrosine amino acid residues in proteins. Upregulated kinase activity is found in diabetes, rheumatoid arthritis, and some cancers. Thus, kinase inhibitors are a popular class of drugs.¹ Protein kinases differ in their propensity to phosphorylate specific amino acids and particular sites. Traditional assays of protein kinase activity and inhibition focus on single protein or peptide assays, which makes screening slow and tedious, and may mask the relative preferences.

A report by Qanquian Zhang and colleagues at the Institute of Organic Chemistry in Shanghai, China, describes a general protocol for screening kinase inhibitors using capillary electrophoresis with laser-induced fluorescence detection² (P/ACE™ CE-LIF, Beckman Coulter, Brea, CA).

The work flow is simple, fast, and low cost. Target peptides labeled with 5-carboxyfluorescein were incubated with cell lysate in the presence of 0.5 mmol/L adenosine triphosphate (ATP) for 25 min at 37 ºC. The phosphorylated peptides were analyzed by capillary electrophoresis-laser induced fluorescence (CE-LIF). Corrected peak areas were calculated by dividing the area of each product peak by the area for the internal standard. Inhibition activity was measured by the reduction of corrected peak area in the presence of controlled amounts of inhibitor. This provided the usual % inhibition vs concentration curve and IC₅₀ values. IC₅₀ values obtained with this assay are in the range of values reported in the open literature usually for single substrate–enzyme assays. The authors attribute the high speed and sensitivity to the choice of CE-LIF.

Another key advantage is using the lysate as the enzyme source. They expect that the lysate may be closer to in vivo conditions since the protein kinases are still in their native environment. Perhaps this will make the results more useful in predicting in vivo activity. The authors are also quick to point out that CE-LIF avoids the necessity of enrichment and desalting as required by mass spectrometry detection.

References

1. http://en.wikipedia.org/wiki/Protein_kinase_inhibitor; accessed Sept 15, 2013.
2. Zhang, Q. et al. Inhibitor screening and selectivity assessment against multiple cellular protein kinases by capillary electrophoresis with laser-induced fluorescence detection. SEPU2013, 31(7), 646–55.

Robert L. Stevenson, Ph.D., is a Consultant and Editor of Separation Science for American Laboratory/Labcompare; e-mail: [email protected].