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Kinase-Inhibitor Activity Profiling (KICP) Service


The Kinase Inhibitor Compound Profiling (KICP) Services are a convenient and cost-effective solution to assist scientists in ascertaining of the specificity of lead compounds and their mechanisms of action for drug discovery. This information is critical for the selection of better drug candidates for clinical testing. Approximately a third of all pharmaceutical R&D is now focused on protein kinases as drug targets. Kinexus currently has over 265 human protein kinases available for screening with our KICP Service. This number will continue to increase in the near future. At least 515 human protein kinases target the phosphorylation of apparently more than 500,000 phosphorylation sites in the proteome. In view of this, it is critical to establish the specificity of any kinase drug candidate for clinical studies. 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. This 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. This in vitro service from Kinexus compliments the in vivo compound profiling analyses that are offered with our Kinex™ antibody microarray and Kinetworks™ multi-immunoblotting services. With these proteomics services, it is feasible to determine the effects of compounds and other treatments of animals and isolated cells on the expression levels of over 193 known protein kinases and 176 other proteins as well as the status of at least 270 different phosphosites to monitor indirectly the activities of their upstream kinases.

The 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. You can also download an MS-Excel spreadsheet with very detailed information on each protein kinase presently available with our KICP Service along with active hyperlinks to other websites from the previous webpage.

We provide a wide range of options to our clients with the 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.

Quantity of Compound Required

The amount of each compound required for the KICP Service depends on how many kinase activity measures are to be performed and the concentrations at which each compound will be tested. The final volume of the KICP assays are 25 µl, and the stock concentration of the compound to be tested should be at least 10-times the final concentration of the highest dose desired for KICP analysis. For example, if 100 µM is the single concentration of a compound to be tested against one kinase in triplicate, then (3 x 2.5 µl =) 7.5 µl of a 1 mM compound solution would be the minimum amount required. However, we recommend that a minimum volume of 50 µl of 10X concentrated compound stock solution in water or 2% DMSO is provided in a 1.5 ml Eppendorf vial; please Paraffin wax wrap the closed lid for further protection. If the compound is supplied in powder form, please provide sufficient material so that the compound can be prepared at as 10X concentrated solution with a volume of at least 500 µl.

Kinase Assay Conditions

Due to the distinct protein/peptide substrate and other assay conditions for the different protein kinases, the components of the various assays are optimized for each enzyme and are not described here. If left unspecified by the client, most assays are performed for 15 minutes duration, at 30°C, with 50 µM [γ-33P]ATP in a final volume of 25 µl. The assays are typically terminated by spotting 20 µl of the reaction mixture onto a phosphocellulose P81 plate. The phosphocellulose P81 plate is washed 3 times for approximately 15 minutes each in a 1% phosphoric acid solution to remove unreacted [γ-33P]ATP. The radioactivity in the captured 33P-labeled peptide/protein substrate on the P81 plate is quantified in a scintillation counter.