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Mar 1, 2009 (Vol. 29, No. 5)

TechNote: High-Throughput Kinase Selectivity Profiling

Bridging the Gulf between Compound Libraries and Rapid Discovery

  • Improving Solutions

    Click Image To Enlarge +
    Figure 1. The human kinome is comprised of 518 protein kinases grouped by sequence into eight families.

    Continued advances in high-throughput screening technologies, assay platforms, and kinase panel size have enabled researchers to make broader assessments of compound/ kinase selectivity earlier in the discovery and development process.  This offers the benefit of thorough library annotation of privileged kinase-focused compound libraries and the ability to quickly identify novel candidates. It can also reveal opportunities to expand therapeutic utility of existing compounds.

    Although a number of service providers and kit options have emerged to facilitate discovery efforts, most provide limited kinase assay breadth, and many utilize varying assay formats within the panel, making it difficult to compare results. 

    Moreover, assays are not performed under standardized assay conditions, which can reduce assay sensitivity while making it difficult to compare data across kinases.

    To bridge the gulf between small molecule compound libraries and quantitative kinase selectivity profiling, KINOMEscan™ (a division of Ambit Biosciences) has developed a high-throughput, active site-dependent competition-binding assay platform. Available as a custom service, this technology provides scientists with access to a large commercially available panel of robust kinase assays (Figure 1) and reproducible, high-quality data.

    This approach permits a large number of compounds to be run in parallel as a single experiment with highly standardized assay conditions and reagents. It also allows scientists to quantitatively evaluate and compare compounds against a large number of distinct kinases and important mutant forms to obtain necessary potency and selectivity data to support discovery programs.

  • Competitive-Binding Technology

    Click Image To Enlarge +
    Figure 2. Schematic overview of the assay

    In the KINOMEscan platform a test compound is combined with human kinases tagged with DNA and an active-site directed ligand that is immobilized on a solid support (Figure 2). 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. 

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