The KinAffinity® service efficiently profiles kinase inhibitor targets across the native kinome of any given cell line or tissue. It simultaneously determines an inhibitor’s affinity to kinases expressed in a cell line or tissue. A ready-made matrix captures a cell’s or tissue’s expressed kinome, for rapid, precise and effective profiling of kinase inhibitors.
KinAffinity® collects detailed information about a kinase inhibitor’s selectivity, which is essential in the drug discovery process. Inhibition of a single off-target kinase can induce toxicity that might undermine an entire clinical trial. Thus, the question of how to design selective as well as multi-specific inhibitors without causing widespread toxicity effects is becoming more and more important.
KinAffinity® service combines chemical proteomics with high-end quantitative mass spectrometry to deliver a complete picture of your kinase inhibitor's interactions with the cellular kinome:
KinAffinity® can profile a compound against endogenously expressed, full-length proteins in the presence of cellular co-factors and native complex partners a major advantage over traditional biochemical panel screening using recombinantly expressed proteins or only protein domains.
The data obtained by KinAffinity® allow comprehensive risk assessment in the drug discovery process and significantly support decision-making during drug development, e.g. for analyzing kinase inhibitor selectivity, optimizing lead compounds, or selecting clinical candidates.
Figure 1 (left): Endogenously expressed kinases are captured and enriched by KinAffinity, eluted, then identified and quantified by mass spectrometry.
Figure 2 (right): An equilibrium binding reaction is established between the cell/tissue lysate, the compound bound to KinAffinity and the 'free' kinase inhibitor. The kinase targets that remain bound to KinAffinity are eluted, then identified and quantified by mass spectrometry.
KinAffinity® was developed for fast, reliable profiling of kinase inhibitors. Several broad-band kinase inhibitors bound to a ready-to-use matrix capture and enrich the expressed kinome of a cell line or tissue.
The binding affinity of the free kinase inhibitor (Kd, free values) are derived by a two step process.
Firstly, the affinity of each of the enriched protein kinases for KinAffinity® is determined (Figure 1). Secondly, in competition experiments the kinase inhibitor displaces targets bound to KinAffinity® (Figure 2). The final Kd, free values are derived by applying algorithms to these two data sets.
SILAC (Stable Isotope Labeling With Amino Acids In Cell Culture), TMT (Tandem Mass Tags) or iTRAQ (Isobaric Tags for Relative and Absolute Quantification) proteome labeling are used for quantitative mass spectrometry analysis of cell or tissue samples. Combined with state-of-the-art proprietary affinity-based separation and data processing methods, this technology can determine a kinase inhibitor’s affinities to the expressed kinases of a cell line or tissue.
Every customer project includes a dedicated series of control experiments in order to obtain the highest quality results and appropriately annotated data. The controls ensure that any background binding signal is excluded, bead saturation is avoided and the binding equilibrium status is defined for each target/compound interaction.
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KinAffinity® is a highly validated, reproducible technology that has been used to profile several small molecules. To read more about its applicability please also see AN4.