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May 15, 2010 (Vol. 30, No. 10)

R&D in the Protein Kinase Field Blossoms

With Over 500 Inhibitors in Various Stages of Development, Effective Assays Are Critical

  • B-Cell Mediated Diseases

    Click Image To Enlarge +
    Binding mode of Avila Therapeutics’ Btk inhibitor (blue) to the Btk kinase catalytic domain (green): The company’s drug binds specifically to the Btk catalytic binding site then binds uniquely with a Cys (yellow) in the binding site, leading to selective silencing.

    Avila Therapeutics engineers molecules that it says are able to selectively silence drug targets. “We generate covalent drugs that strongly and resiliently bond to specific disease-causing proteins, thereby silencing them and eradicating the corresponding disease,” explains Juswinder Singh, Ph.D., CSO and co-founder.

    Avila is focused on Bruton’s tyrosine kinase (Btk), which is expressed in B cells and thus “represents an exciting target for the treatment of autoimmune diseases and hematological cancers.” This has been a difficult target for pharma to create selective inhibitors. “We designed AVL-292, a highly selective, covalent Btk inhibitor that potently inhibits Btk enzymatic activity in vitro.”

    According to Dr. Singh, AVL-292 demonstrates impressive efficacy in several rodent arthritis disease models. “And, because AVL-292 binds to Btk irreversibly, we have developed a covalent probe assay that enables direct measurement of Btk occupancy and can correlate target occupancy with pharmacodynamic response.”

    Erythropoietin-producing hepatocellular carcinoma (Eph) receptors are highly conserved transmembrane tyrosine kinases made up of multiple domains that participate in an array of complex cell signaling pathways. Marcie Glicksman, Ph.D., co-director of the laboratory for drug discovery at the Harvard NeuroDiscovery Center, and her group are studying the EphB3 receptor subtype, which is expressed during embryonic development and in discrete areas of the adult brain, including the cerebellum and hippocampus. It co-localizes to brain regions with high levels of ephrin B ligand expression.

    “We identified this as a kinase for stroke, and it is an important kinase for neuroprotection,” she says. “It’s an interesting case of kinase and ligand bi-directional signaling. A lot of the biology is not yet well understood.

    “Figuring out cell selectivity is a challenge, more selective is better. People do these large panels, but you don’t know what’s going to be relevant in the cell and in the animal. Kinase activity in the cell and animal is sometimes more relevant than at the isolated protein.”

  • RTK Inhibitor Discovery

    Zhao Ren, M.D., Ph.D., staff scientist at Elan Pharmaceuticals, and his co-workers developed a novel cellular assay in-house. Receptor tyrosine kinases (RTKs) “are an important class of drug targets because of the critical roles they play in cell proliferation and differentiation,” notes Dr. Ren. RTK inhibitors have been aggressively pursued in many different therapeutic areas, including oncology and inflammatory diseases, “and some of these are potential drug candidates.”

    According to Dr. Ren, homogenous biochemical assays using recombinant kinase domains of RTKs have been widely adopted to drive inhibitor discovery and structure-activity relationship (SAR) studies. However, there is a lack of robust cellular assays that can measure RTK activity in a high-throughput fashion.

    “Interestingly, people examine this class of target using biochemical assays, expressing the kinase domains in bacteria and insect cells, purifying them. We, however, want to explore a different strategy. Cellular assays that can measure RTK activity in their natural environment might be a better approach. A much higher hurdle is developing an assay that works in cells.”

    The challenges here are both conceptual and technical, Dr. Ren explains. What happens to a complex receptor is not a single event but a cascade of activities. “We need to figure out what is the best step to monitor receptor activation and what is the best assay technology available.

    “We ultimately decided to use AlphaScreen, a luminscent homogeneous immunoassay. This is a simple but effective method to monitor molecular interactions. When two biomolecules form a complex, they also bring a pair of donor and acceptor beads to close proximity. Which, in turn, generates luminescent signals when excited with a laser. That’s how you measure activities in this type of assay.”

    Using PerkinElmer’s AlphaScreen technology, Dr. Ren developed several robust cellular RTK assays measuring receptor autophosphorylation. The high assay sensitivity enables miniaturization, and the homogenous nature of these assays renders efficient automation with liquid handlers and robotics.

    “We need a lot of cells to do a large screening—one billion cells a day. We discovered another technology on the market, HyperFlask™ by Corning that enabled us to grow multiple layers of cells in the same volume of flask. This is a much more manageable way to keep up with the huge cell culture need,” says Dr. Ren.


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