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Kinase-Inhibitor Activity Profiling (KICP) Services

The selection of the most specific and potent drug candidates is critical for successful clinical testing. Approximately a third of all pharmaceutical R&D is now focused on protein kinases as drug targets. At least 516 human protein kinases target the phosphorylation of apparently more than 650,000 phosphorylation sites in the proteome. In view of this, it is critical to establish the specificity of any kinase drug candidate for clinical studies. On the one hand, the more specific the kinase inhibitors, the lower the chances of off targets that could compromise on the utility of the drug from toxicity and other undesired side-effects. On the other hand, compounds that more potently inhibit an off target protein kinase could have useful therapeutic applications in diseases that are distinct from those that were originally intended.

Kinexus offers two complementary strategies to establish the specificity of putative protein kinase inhibitors. We recommend initial use of our Kinex™ Protein Kinase Microarray to evaluate the ability of test compounds to inhibit the binding of an ATP analogue to over 150 different human protein kinases. The hits from this cost-effective services can then be selectively validated with our Kinase-Inhibitor Activity Profiling (KICP) Service. Kinexus currently has over 265 human protein kinases available for screening with our KICP Service. This enzyme activity-based service relies on the use of gamma phosphate-radiolabeled ATP to phosphorylate peptide and recombinants protein substrates with purified and active preparations of human protein kinases. Kinexus performs the KICP Service under strict confidentiality, and all materials, information and results are used as directed by the client.

Our KICP Service uses the most reliable direct assay of protein kinase phosphotransferase activity that is known. The methodology is based on the direct quantification of radio-labeled phosphate from ATP (gamma-32P or gamma-33P) on to a peptide or protein substrate of a target protein kinase. This provides for a high signal to noise detection of phosphorylation, high reproducibility, and reduces the opportunity for artifacts inherent in other methods, such as the measurement of the production of ADP or disappearance of ATP.
Furthermore, the assay provides a direct measure of the effect of a compound on the enzymatic phosphotransferase activity of a target protein kinase, rather than a measure of the ability of a compound to bind near the active site of the kinase, as is performed with some other approaches to compound screening.

The preparations of recombinant protein kinases that we use in our KICP Service possess high specific activities, and generally represent full-length human clones. In some instances, we use kinases that feature activating mutations that may occur in vivo. But generally, the kinases are activated by endogenous phosphorylation in the baculovirus-infected insect cells or by the addition of the purified and activate upstream protein kinase. For each kinase used in the KCIP Service, the assay conditions have been carefully optimized to ensure the highest levels of phosphotransferase activity. The MS-Excel spreadsheet that can be downloaded below features detailed information on each of the protein kinases available with our service along with active hyperlinks to other websites.

We provide a wide range of options to our clients with our KICP Service. Individual compounds may be profiled against a panel of protein kinase targets to establish the specificity of the compound. Alternatively, a panel of compounds may be tested against a single kinase target to identify a lead compound with the highest potency. Compounds may be tested either using a single dose or at multiple concentrations in order to allow in-depth IC determinations. In addition, the protein kinase assays can be performed under varying ATP concentrations to evaluate competition with respect to ATP. Compounds can be supplied by the client as DMSO stocks of known concentration, as solid material in vials, or in 96-well plates. Turnaround with our KICP Service is within 3 weeks of receipt of compounds for testing.