The KINOMEscan platform utilizes a simple high-throughput competitive binding assay technology. A test compound is combined with human kinases tagged with DNA and an active-site directed ligand which is immobilized on a solid support. During equilibration, if the test compound binds to the kinase and either directly or indirectly competes with the ligand, fewer kinase molecules are able to interact with the active site directed ligand. Conversely, if the test compound does not compete, kinase molecules are free to bind to the immobilized ligand. The results are then read out by quantifying the amount of tagged kinase bound to the solid support using highly sensitive quantitative PCR. For more information, watch our technology tutorial or read an in-depth technology description.

KINOMEscan affords benefits to investigators at all stages of the drug discovery and development continuum. A few of these include a comprehensive understanding of compound selectivity against the largest commercial panel of kinase assays, detection of non-type I inhibitors, rapid data turnaround, accurate, precise and reproducible data, flexible business models and screening options plus more. To learn more about the benefits of working with KINOMEscan, please visit our benefits page.

Does profiling with KINOMEscan generate the expected results for known kinase inhibitors?

How are KINOMEscan's assays validated?

All assays undergo a rigorous validation process to ensure data accuracy, consistency and integrity prior to entering production.  These include benchmark analysis against a panel of reference compounds, comparison against published values and statistical analysis of relevant assay parameters to confirm data accuracy, precision and reproducibility.  Once in production, assay performance is continually monitored throughout each experiment and must pass a rigorous multi-step quality control process. Multiple positive and negative controls are employed for each production run and all assays are performed in duplicate to ensure high quality reproducible data.  Assay results that fall outside of established parameters are invalidated and repeated until data are approved for release.

Can allosteric inhibitors be detected using KINOMEscan technology?

Do you have assays for binding to sites other than ATP-binding site?

How do you characterize phosphorylation/activation status of your kinases?

The phosphorylation/activation status of assays that comprise the scanMODE panel is characterized by two different mechanisms. Kinases are probed by Western blot analysis using pan- and phosphospecific antibodies to confirm phosphorylation status. Kinase phosphorylation status is also confirmed using a panel of known type I and type II kinase inhibitors and results are assessed against internal benchmarks and published/expected values. 

What is the concentration of ATP in your assays?

What is the concentration of test compound in the assays?

Compounds can be screened at any desired concentration. The upper concentration limit is determined only by compound solubility. Typically, primary screens are performed at 10 uM or 1 uM but can be adjusted based on specific client requirements.

No. Binding assays are performed under conditions in which binding constants measured for the interaction between kinases and test compounds are independent of the affinity of the immobilized ligand for the kinase. This has been experimentally verified by measuring binding constants for several kinase/test compound combinations using different immobilized ligands (for some of the kinases, more than one immobilized ligand can be used to build an assay). Test compound binding affinity did not change with the immobilized ligand used and confirms that results are independent of the affinity of the immobilized ligand for the kinase. Additional details regarding compound binding constant relative to affinity of bait can be found on p.335, of Fabian, et al. (2005) and supplementary details of Wodicka et al. (2010)

KINOMEscan uses quantitative PCR to quantitate binding affinity in each of our assays. This extremely sensitive readout enables a wide dynamic range and accurate affinity determination values from less than 100pM to greater than 10uM.

 

Scores for primary screens (single concentration) are reported as ‘Percent of DMSO Control’ (POC) and are calculated in the following manner:

test compound = client supplied compound

positive control = control compound (0% control)

Although data derived from competition binding assays (thermodynamic constant) is fundamentally different from that obtained in activity assays, there is generally good agreement between the data sets. This data comparisons and assessments to be made between KINOMEscan binding data (POC) and inhibition data derived from other sources. If desired, POC values may be converted to percent competition by taking the inverse of the POC value:

(100 – POC) = Percent Competition

Eg: 30 POC = (100- 30) or 70 Percent Competition

What is the relationship between the data obtained from single concentration primary screens and Kd determinations?

Relationship between Binding Constant Distribution (Kds)

& Single Concentration 10uM Primary Screen Values

What if I don't see my assay in your panel?  Are custom assay development services available?

How are data reported? What is the standard data turnaround?

What is the importance of profiling against mutant kinases?  What diseases are associated with mutant kinases?

Mutated kinases have been implicated as causative agents in a diverse range of diseases, including many forms of cancers. These same mechanisms can also confer resistance to therapeutic agents thereby rendering them ineffective or lead to relapse after initial positive response. The availability of disease relevant mutant kinases affords investigators an important tool to elucidate the mechanisms of action for their compounds, reveal novel therapeutic opportunities, or as starting points for next generation drugs. For example, BIRB-786 a potent inhibitor of human p38-alpha kinase also binds ABL1(T315I) with much higher affinity than wild type ABL1. Thus, by profiling compounds against a panel of disease relevant mutant kinase assays investigators are afforded not only a broad understanding of compound potential but also a strategy by which novel inhibitors against important mutant kinases may be identified and advanced in the clinic.

What concentration should I use to screen my compound?

In general, we hesitate to provide specific screening recommendations to our clients as our response may depend on the specific questions being asked and may be further influenced by other factors. However, based on our experience, a 10uM screen will provide robust POC data for all assays with Kds < 1uM. Likewise, a 1uM screen will provide robust POC data for all assays with Kds < 100nM.  Another approach that many of our clients take is to screen at two or more concentrations - for example 10uM and 1uM, or 1uM and 0.1uM. This will provide additional information in which to make a more quantitative assessment of your compound's potency and selectivity while minimizing the possibility of missing important interactions.  Additional information on this is available here.

Are detailed data on phosphorylation/activation status of kinases available?

The phosphorylation status has not been ascertained for all assays. However, KINOMEscan offers scanMODE, a panel of ABL1 assays (wild type and clinically-relevant mutants) in which the phosphorylation/activation status has been characterized and may be useful for compound binding mode elucidation). For more information on the scanMODE, click here.

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