Kinase selectivity assays

Card, A., Caldwell, C., Min, H., et al. (2009) High-throughput biochemical kinase selectivity assays panel development and screening applications. J Biomol Screen 14(1), 31-42. 

Caliper Life Sciences also provides a CE based kinase panel. The Caliper Life Sciences panel consists of 177 wt kinases (including some lipid kinases) and 12 mutant kinases. This company provides kinases and substrates on assay plates and is not strictly a screening service, but it could be a good way forward for labs that have access to a Caliper CE instrument. The company does also provide a smaller panel as a screening service. For more information about both of these, see http //www.caliperls.com/products/reagents/kinase-profiling/ profilerpro-kinase-selectivity-assay-kits.htm. 

Table 2. Kinase selectivity assays and cell-based ELISA IC50s for antiphosphorylation of kinases.

Additional selectivity assays for a wider range of molecular targets will also be introduced. These could include, for example, a panel of kinases representing the various classes of kinases or a panel of GPCRs. 

GMCSF)-stimulated STAT5 phosphorylation assay in TF1 cells. The kinase selectivity of these compounds was reproduced functionally in a JAK3-dri-ven, IL-2-stimulated STAT5 phosphorylation assay in HT2 cells. Both examples demonstrated favorable iv pharmacokinetics in Sprague-Dawley rats [42]. 

Two rounds of targeted libraries (2x44 and 2x88) were designed, synthesized, and assayed within 6 months. This effort led to a new lead matter 1819-1 with improved potency ( 10 folds), kinase selectivity (>100 folds), and better solubility ( 1 log unit or 10 folds) when compared with the original HTS hit Cpd-1. The extensive SAR information spanned by those 264... 

In mammalian cells, there are multiple forms of enzymes within the same family, e.g., protein kinase C, phospholipase C, and caspase. A selective assay system would be instrumental in such cases to advance understanding of the respective role for each form within the same enzyme family. An example for design of a selective assay system for the superfamily of phospholipase A2 is provided in this chapter. Such a selective assay system may play a signihcant enabling role for PLA2 and other enzyme families in the discovery of inhibitors relevant to the treatment of pathologies involving those enzymes. 

S.2.4 Value of Sensitive and Selective Assays for Kinases and Phosphatases... 

I. Yasuda, A. Kishimoto, S. Tanaka, M. Tominga, A. Sakurai, and Y. Nishizuka, A Synthetic Peptide Substrate for Selective Assay of Protein Kinase C. Biochem. Biophys. Res. Commun., 166,1220-1227,1990. 

In order to determine the selectivity of each peptide sensor substrate, assays with a panel of recombinantly expressed, activated kinases can be performed (Figure 1.7b). In this case, the concentration of substrate peptide is held constant at two to three times the determined K. For example, the p38 sensor depicted in Figure 1.6 was incubated with various MAP (mitogen-activated protein) and non-MAP kinases and fluorescence emission was monitored. Figure 1.7b demonstrates the selectivity of the p38 sensor for the target kinase [23]. Following this initial screen, kinase selectivity can be addressed directly in cell lysates. Cells can be stimulated to activate the kinase of interest and kinase activity can be determined in the presence and absence of a selective inhibitor of the kinase of interest. Residual activity in the presence of inhibitor would indicate sensor cross talk with other kinases. 

Endosperm halves were collected, homogenized, fractionated and enzymes assayed at selected times following germination of castor beans in moist vermiculite in the dark at 30°C. For the ethanolamine kinase (EK) assay the reaction mixture contained in a final volume of 50 i 100 mM Tris-HCl (pH8.3), 5 mM ATP, 10 mM MgCb, 563 [iM [2- C]ethanolamine. The reaction was carried out at 37" C for 1 h, terminated with 80 al of 60% trichloroacetic acid, and phosphoethanolamine separated from ethanolamine by ascending paper chromatography. Ethanolamine-phosphate cytidylyltransferase (ECT) was prepared from germinated castor bean endosperm and assayed as described by... 

Devarajan, K, Ma, H, and Peterson, J.R. (2011) Comprehensive assay of kinase catalytic activity reveals features of kinase selectivity. Nature Biotechnology, 29, 1039-1045. 

Kunkel, E. J., Plavec, L, Nguyen, D., Melrose, J., Rosier, E. S., Kao, L. T., Wang, Y., Hytopoulos, E., Bishop, A. C., Bateman, R., et al. (2004). Rapid structure-activity and selectivity analysis of kinase inhibitors by Bio Map analysis in complex human primary cell-based models. ASSAY Drug Dev. Technol. 2 431-441. 

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