Global kinome activity analysis of kinase signaling pathways using a pamgene-12® peptide array platform
Ser, Thr and Tyr kinases are responsible for the regulation of several cellular processes. When kinases are abnormally expressed or controlled, they promote adverse cancer hallmarks, including cell survival, metastasis and proliferation (1). Reciprocally, protein kinase inhibitor (PKI) drugs have recently joined the arsenal of anti-cancer drugs. In this respect, the fundamental characterization of hyper activated kinases in cancer or kinase inhibition profile following cancer therapy in patients is of utmost importance to optimize therapeutic interventions. Overcoming cancer therapy resistance remains a fundamental challenge for most treatments. Of special note, since the number of FDA approved PKI small molecule drugs has been increased from 1 to 26, peptide array based kinome activity profiling offers new perspectives for patient stratification and tailored cancer therapy design of FDA approved and/or (pre) clinical kinase inhibitors (2). Pamchip® peptide array technology allows parallel measurement of multiple kinase activities in cells as well patient tissues, by profiling peptide phosphorylation characteristics by a pharmacology-on-chip approach (3). Besides its well established anti-cancer applications, Pamchip® peptide array technology also offers innovative opportunities in the field of cardio metabolic health, neurodegenerative disease, as well as pharmacological structure-activity studies of preclinical kinase inhibitors. For more information on scientific publications and brief technology outline visit www.pamgene.com.
Key application areas
Fundamental and discovery research
Compound mode of action
Biomarker and clinical research
Readout of microarray data
Data analysis and biological interpretation
Real time measurements of Pamchip peptide phosphorylation intensities are illustrated in figure 3A and 3B. The median signal-noise background (fluorescence intensity) of each spotted peptide phosphorylation is recorded every 50ms, as summarized in figure 3C. Pamgene Bio-navigator® algorithms based on the phosphonet database (collection of 950,000 known phosphorylation sites in over 21,000 human proteins) allow to convert phosphopeptide intensities into a ranked list of hyper activated/repressed upstream kinases (kinome activity signature) (4). Briefly, fluorescence intensity data of phosphopeptide signals of control and treatment setups are uploaded to Bio-navigator® which implements statistical tests and algorithms for inferring and scoring upstream kinase analysis associated with the Pamchip phosphopeptide profile of interest (Fig. 4B) (5). Outputs of Bionavigator® are compatible with Ingenuity ® or Metacore® pathway analysis, to predict the downstream biological effects or calculation of pathway specific activation or inhibition Z-scores (figure 4A). Finally, the ranked kinome activity signature associated with a Pamchip phosphopeptide profile of interest can be projected onto the phylogenetic kinome tree (figure 4C).
- Hanahan D, Weinberg RA. Hallmarks of cancer: The next generation. Vol. 144, Cell. 2011. p. 646–74.
- Wu P, Nielsen TE, Clausen MH. FDA-approved small-molecule kinase inhibitors. Vol. 36, Trends in Pharmacological Sciences. 2015. p. 422–39.
- Hilhorst R, Houkes L, Mommersteeg M, Musch J, van den Berg A, Ruijtenbeek R. Peptide microarrays for profiling of serine/threonine kinase activity of recombinant kinases and lysates of cells and tissue samples. Methods Mol Biol. 2013 Jan;977:259– 71.
- Alex Dussaq, 1 Joshua C Anderson, 1 Christopher D Willey 1 and Jonas S Almeida2. Mechanistic Parameterization of the Kinomic Signal in Peptide Arrays. J Proteomics Bioinform. 2016;9(5):151–157.
- Naji F, de Wijn R MA. BioNavigator, software to interpret PamChip® measurements.