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Nov 1, 2013 (Vol. 33, No. 19)

Interrupting Cell Signaling

  • Protein kinases, enzymes that phosphorylate proteins and other organic molecules, are increasingly important targets for drug discovery.

    Inhibiting these enzymes can disrupt signal-transduction pathways that play a critical role in a variety of disorders, particularly cancer and inflammatory diseases.

    At CHI’s recent “Next-Gen Kinase Inhibitors” conference, presenters discussed recent advances and ongoing challenges in targeting kinases for drug discovery. Examples of the innovative work described include novel screening methods to quantify protein-protein interactions in live cell-based screening systems, approaches for selective targeting of protein kinases, and design strategies for generating potent kinase inhibitors.

    As a target class, kinases have “proven to be highly druggable and a rich source of clinical agents and approved therapies, some of which have been identified using relatively new approaches such as fragment-based design,” said James E. Dowling, Ph.D., principal scientist at AstraZeneca’s R&D Boston facility. “These accomplishments are quite impressive given the early views of many scientists who felt that the conserved ATP-binding pocket would present a challenge to the identification of selective inhibitors.”

    As with all drug candidates, balancing factors such as potency, selectivity for the target, off-target effects, bioavailability, pharmacokinetics, and other drug-like properties is a challenge in the development of kinase inhibitors as therapeutic agents.

    In the early stages of drug discovery, potency and chemical diversity are perhaps most important. “We also monitor parameters such as solubility, in vitro metabolic stability, and lipophilicity, and we use these parameters to help rank emerging chemical series,” said Dr. Dowling. “There tends to be a willingness to embrace emerging series with potential selectivity issues as long as there are strategies available for increasing primary target potency without simultaneously enhancing off-target effects. Another early objective is to identify early probe compounds with sufficient selectivity to enable successful validation of the target of interest.”

  • Screening on µ-Patterned Surfaces

    Click Image To Enlarge +
    Scientists at the University of Applied Sciences, Upper Austria, Wels use cell signaling to determine the selectivity of protein kinase inhibitors and their use in drug targeting. In recent years protein kinases have become the most studied class of drug target. Many protein kinase inhibitors have now been approved as anticancer drugs and many more are undergoing clinical trials. [Gustoimages/Science Source]

    Julian Weghuber, Ph.D., principal investigator at the University of Applied Sciences, Upper Austria, Wels, described a strategy for evaluating protein-protein interactions in live cells for drug discovery. Dr. Weghuber and colleagues are using microstructured surfaces, called µ-patterned surfaces, to analyze the interactions of various signaling proteins, such as tyrosine kinases, and membrane-bound receptors. Detection and quantification of the interactions in the context of a living cell is achieved with total internal reflection fluorescence (TIRF) microscopy. This µ-patterning technique has the advantage of being applicable for high-throughput screening.

    To illustrate the mechanism of the µ-patterning assay used to detect protein-protein interactions, Dr. Weghuber refers to the membrane protein of interest as the “bait” and the fluorophore-labeled protein as the “prey.” Grids of µ-patterned antibody specific to the membrane protein bait are printed on glass surfaces. In cells grown on the µ-patterned biochips, the bait in the cell membranes will arrange on the surface according to the antibody µ-pattern. Introduction of the fluorophore-labeled protein prey will result in a homogeneous distribution of prey if it does not interact with the bait, or in co-patterning with the antibody in the presence of bait-prey interactions.

    The researchers are applying this µ-pat-terning technique as a characterization tool to analyze medically relevant drug targets, and specifically to target the interaction between epidermal growth factor receptor (EGFR) and growth factor receptor-bound protein 2 (Grb2) to identify new drugs that affect EGRF-based signaling. According to Dr. Weghuber, the researchers “performed an in-depth analysis of the interaction properties of these molecules and then used different pharmaceuticals to modify this interaction,” including monoclonal antibodies and kinase inhibitors.

    Although the current methodology is not suited to large-scale drug screening, Dr. Weghuber cited several refinements his team is exploring to automate the technique and increase throughput rates. “These attempts include µ-patterned surfaces generated in 96- or 384-well plates, customized software, and the development of a TIRF-capable well-plate reader,” said Dr. Webhuber.

  • Selective Kinase Inhibition

    Piqur Therapeutics is a Swiss pharmaceutical company focusing on the discovery and development of cancer therapeutics targeting the PI3K-Akt-mTOR signal pathway. (In this pathway, PI3K refers to phosphatidylinositide 3-kinases; Atk, to protein kinase B; and mTOR, to the mammalian target of rapamycin inhibition.) In particular, the company is working on overcoming the problems of dual PI3K-mTOR inhibition.

    Mutations and amplifications in the components of the P13K-Akt-mTOR pathway contribute to cancer cell growth, survival, and proliferation. The main challenge in targeting PI3K kinase, as in the kinase field, “is to obtain highly selective inhibitors, which allow us to understand the consequences of pharmacological inhibition of a particular P13K isoform,” said Doriano Fabbro, Ph.D., CSO.

    In his presentation, Dr. Fabbro discussed the current understanding of kinase inhibition based on the knowledge base gained from studies of small molecule binding to the ATP pocket of kinases. In addition, he described an example of how selective kinase inhibitors can have an effect on their overall structure.

    Despite the central role kinases play in human biology, “and their sizeable potential as therapeutic targets, only a small fraction of the human kinome has been functionally annotated with ‘selective’ small molecule inhibitors,” said Dr. Fabbro. “Thus, the kinase field appears to be frozen in targeting the obvious. This is true with respect to target, site of inhibition (that is, ATP binding site), as well as indication (mainly oncology).”

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