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May 15, 2013 (Vol. 33, No. 10)

Harnessing the Power of Kinase Inhibitors

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    The assay development and high-throughput screening system at the European ScreeningPort, Hamburg, Germany.

    The ~600 kinases that make up the human kinome comprise a group of exciting druggable targets that can be modulated using small molecule or monoclonal antibody approaches.

    There are currently 15 FDA-approved kinase inhibitor drugs, yet there remains much uncapped potential, as the biological function of ~90% of the kinome is unknown.

    Methods have been designed for improving small molecule type I kinase inhibitors, which target the ATP binding pocket common to all kinases in their active form, as well as type II inhibitors, which bind to the same area as type I molecules, but also to an additional allosteric binding pocket in the inactive DFG motif conformation of the kinase. Novel assays have identified kinase targets, improved the function of certain kinase inhibitors, and led to the discovery of new inhibitor classes to accelerate drug discovery.

    Sheraz Gul, Ph.D., vp and head of biology at European ScreeningPort, indicates that he has had a long-standing interest in kinases, particularly NF-κB inducing kinase (NIK). NIK is known to phosphorylate IKK-α at Ser-176, the same site where IKK-α autophosphorylates.

    Dr. Gul has noticed a few discrepancies over the years with regard to reported NIK biochemical assays: various suppliers who sell recombinantly expressed and purified NIK have shown that it is catalytically active in biochemical assays when using a number of substrates (peptide and the generic kinase substrate myelin basic protein), however not when using IKK-α protein or a peptide derived from it as substrates.

    Using such non-IKK-α derived substrates is somewhat of a concern, as myelin basic protein will undergo phosphorylation by most kinases. Additionally, it is possible that a highly active kinase partner bound to NIK may be responsible for the observed phosphorylation of non-IKK-α-derived substrates in biochemical assays.

    In light of the intriguing aspects of how physiologically relevant NIK activity arises, Dr. Gul concludes that, rather than developing a biochemical assay, a cell-based assay to search for NIK inhibitors that prevent IKK-α phosphorylation would more effective. However, cell-based assays do have their disadvantages, primarily that most hits often exhibit cytotoxicity.

    His research team began by performing a dual transfection assay of catalytically inactive full-length IKK-α and full-length NIK in insect cells, and have translated this to a HEK293 cell-based system and performed proof-of-concept screens against compound libraries with both assays. The screen that utilized the former assay yielded a sizeable number of hits, some of which were shown to prevent p52 translocation into the nucleus from the cytoplasm, as this would be the downstream effect of inhibiting NIK.

    As an alternative to the dual transfection assay, Dr. Gul’s research team made the surprising finding that batches of cells that were separately transfected with NIK and catalytically inactive IKK-α, which were mixed together upon cell lysis, did not result in the catalytically inactive IKK-α undergoing phosphorylation, indicating that its NIK- mediated phosphorylation can only occur in a native cellular environment. This was all the more reason to prioritize cell-based assays over biochemical assays when searching for NIK inhibitors that prevent IKK-α phosphorylation.

    “NIK is clearly an unusual kinase, as there have been no reports of a biochemical assay being developed that specifically makes use of its well-known substrate, IKK-α (or relevant kinase inactive mutant),” Dr. Gul says.

    “It is not necessary that every kinase assay be cell-based, but for those that have characteristics like NIK, you will get many hits in a biochemical assay whose activities will not translate to a cell-based assay. Therefore, in order to mitigate the risk of this all-too-common scenario, it should be best practice to use a panel of both biochemical and cell-based assays as early as possible.”

  • Phenotypic Screening

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    Array BioPharma has invented a cell-based phenotypic screening platform that it says enables discovery of novel drug targets. An overview of kinase-directed phenotypic screening is shown.

    “Over the past 15 years, Array BioPharma has synthesized over 10,000 kinase inhibitors, which together inhibit about 95% of the entire kinome,” says David Chantry, Ph.D., senior director of translational and cellular biology. “By applying this state-of-the-art collection of kinase inhibitors, we have invented a cell-based phenotypic screening platform that enables discovery of novel drug targets.”

    Dr. Chantry also notes that the alternative global approaches of molecular-driven target discovery, such as using an shRNA knockdown approach or systematic knockouts in animal models, have not been very fruitful when it comes to identifying targets for small molecule drug discovery.

    Array BioPharma’s phenotypic screening platform has multiple differentiating features from other platforms, most notably that Array researchers are able to reverse engineer from the screen back to the molecular targets in a chemoinformatic approach. All of the inhibitors in the curated collection have demonstrated cell activity at 1 µM or less, and data has been collected regarding in vitro properties of microsomal stability, permeability, and solubility.

    In total, Array BioPharma has collected ~1 million data points outlining the in vivo physical properties of their kinase inhibitor collection. Its library of diverse kinase inhibitors covers the vast majority of kinases, says Dr. Chantry, most of which have completely unknown functions.

    “Our approach ends up working like a Venn diagram: we obtain an overlapping set of information for each group of inhibitors that exhibit the same cellular phenotype in our assays,” adds Jim Winkler, Ph.D., vp of discovery biology and translational medicine.

    “There might be one inhibitor that inhibits 20 kinases across the kinome, and another that inhibits 15, most of them different from the first inhibitor; but if you do this for enough inhibitors, they will ideally only have one kinase as a common target. Using all of the selectivity data we have collected for each inhibitor, we end up combining target validation and target identification in the same process.”

    Depending on the therapeutic application, each phenotypic screen will be unique in terms of the number of hits and the nature of the intersection of shared kinases that are inhibited, but the end result is a manageable list of potential targets, which can then be further analyzed by traditional approaches like RNAi knockdown.

    A subset of the inhibitor library was recently used to demonstrate the strength of the platform. The researchers were interested in identifying a single kinase target that would affect cytokine production by both innate and adaptive immunity, as there are currently targets that affect each type of immunity separately but no single approach for both.

    In addition to the clinical relevance of this phenotype, Array BioPharma already had inhibitors of the p38 and MEK pathway in its collection to provide useful internal controls. Using a reverse-engineering approach, Dr. Chantry and colleagues discovered and validated a novel kinase target that regulates cytokine production by cells of both the innate and adaptive immune system.

    “We are interested in using this phenotypic screening platform to drive collaborations with other companies, to discover novel targets, and develop clinical candidates,” Dr. Winkler adds. “We would like to work with collaborators that have in-depth biology expertise in their particular therapeutic area, and an understanding of which are the most relevant phenotypic screens that can lead to novel therapies for patients with serious unmet medical needs.”


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