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May 15, 2005 (Vol. 25, No. 10)

Signal Pathways Making Inroads in Discovery

New Methods and Technologies Expand Approaches for Understanding Complex Systems

  • Signal pathways control a vast range of cellular events and keep cells operating cohesively. Extracellular changes are communicated inside cells via cell-surface receptors, like tyrosine kinase, G-protein coupled receptors, and ion-channel receptors. A signal-receptor complex triggers a cascade of protein interactions.

    Several companies will be presenting information on their novel approaches and technologies for characterizing cell signal pathways at the Cambridge Healthtech Institute's "Systems Biology Conference" in San Francisco this June.

  • Understanding Receptors

    Merrimack Pharmaceuticals (www.merrimackpharma.com) has developed a computational model of the ErbB receptor network. "This presentation will show how network biology can help you to understand these complex systems and perhaps help you to either develop better drugs or target a specific patient population," says Birgit Schoeberl, Ph.D., associate director, computational biology.

    The ErbB (epidermal growth factor receptor family) signal transduction pathway comprises four receptors with 12 different ligands known to bind to those receptors. Because of its complexity, it is difficult to develop drugs high in specificity.

    "The idea is, if we better understand how these four different receptors interact and how these downstream signal transduction pathways influence each other, it will help us develop better drugs," Dr. Schoeberl explains. She also plans to present the response patterns for ten different tumor cell lines to anti-EGFR drugs comparing simulation results with wet-lab experimental data.

    Utilizing high-density antibody microarrays with proprietary surface chemistry, the company is able to quantify receptors on different tumor cell lines via a high throughput method that also makes data generation more efficient.

    The computational model, which is based on up to 500 differential equations, is trained with experimental data (from A431 cells) using protein expression levels and time course data of protein phosphorylation, specifically ERK and AKT phosphorylation. These are two kinases downstream of the ErbB receptor and essential to inducing transcription factors, which leads to cell proliferation.

    "We use ERK and AKT phosphorylation as a read out for the inhibitor efficacy. Then we validate the model, making predictions of how an inhibitor would inhibit, for example, ErbB1 phosphorylation in the cell lines. Then we compare the experiments with the model prediction.

    "We tested this on eight different types of breast and ovarian cancer cell lines, and the model predicted those responses quite well," summarizes Dr. Schoeberl. The company says its approach is unique because of the tight interaction between experiments, modeling, and antibody engineering.

    GeneGo's (www.genego.com) MetaCore is a software platform that can link high throughput data to signaling pathways. "The idea is to present several case studies to show how we can use our system to cross-validate different types of data," states Tatiana Nikolskaya, Ph.D., CSO of GeneGo.

    Each high throughput method provides researchers with an idea of the real cellular processes. Different methods provide different data. MetaCore can align that information and compare it. "Such cross-validation of different HT data allows us to reconstruct cellular processes more accurately and provides better drug targets," explains Dr. Nikolskaya.

    She says the firm's biggest challenge now is to gain access to different types of data created on the same data set, especially in proteomics and metabolomics where there is not a lot of data available in the public domain. However, she adds that the company does have metabolomics data available in a separate network, which will soon be merged with their signaling network.

    Many signaling cascades have an effect on metabolic levels when they turn off and on certain metabolic subsystems. Dr. Nikolskaya says they are hoping to be able to show cascades from the receptor through kinases to transcriptional factors and then to particular metabolic pathways.

    Currently, the software's main application is mining of microarray data. "It's nice to have the same system across different departments because it's not enough to have integrated data and store and access them; it's very important to be able to mine them at the same time."

  • Studying Signal Networks Proving Valuable

    Although AstraZeneca (www. astrazeneca.com) only recently established a systems biology program, it has had some early successes in utilizing this approach for drug discovery. Adriano Henney, Ph.D., director, pathways capability, will be presenting how the company has set up its systems biology capabilities and some of its current projects (i.e., EGFR pathway).

    "I'll be trying to convey our approach in working with real projects and seeing whether a systems biology dimension can offer opportunities in a way that classical approaches historically may not," says Dr. Henney.

    A main component of the company's approach is to have focused partnerships with key academic centers. "That's a way to try and get successful answers on the true benefits of these approaches to the pharmaceutical industry."

    Since systems biology includes many disciplines, Dr. Henney's group includes molecular and cellular biologists, protein chemists, mass spectroscopy experts, engineers, and statisticians.

    He says their work spans classical pathway mapping approaches in terms of protein-protein interactions, analysis of protein complexes, and the use of mammalian and yeast cells to analyze and map protein interactions.

    Computational biologists develop and create new mathematical models of simulations, which are verified and validated by an iterative cycle of computation and wet biology to refine what's in the computer and test its application.

    "What's important is how these approaches are going to impact the most important issues that we face in the industry (e.g., toxicity, unpredicted side effects). I think the question is whether what we can offer in systems biology can help address some of those issues and provide insight into how we might modify the way we go about doing things," summarizes Dr. Henney.

  • Focusing On Kinase Pathways

    Cellular Genomics' (www.cellulargenomics.com) drug discovery platform incorporates several proprietary tools to characterize specific cell signaling pathways, and recently presented this at the IBC meeting in San Diego.

    "We're first and foremost a kinase-centric company. We have focused on understanding what happens if you inhibit a kinase using our ASKA (Analog Sensitive Kinase Alleles) technology, and now we've moved forward into drug discovery in cancer and inflammation," states Scott Mitchell, Ph.D., principal scientist.

    ASKA is a tool for mapping kinase pathways and determining function of individual kinases and their roles in disease. These functional, genetically engineered kinases can be modulated by proprietary reference compounds. Analyses using AKSA knock-in mouse models are highly predictive of wild-type kinase function.

    "Once a kinase target has been chosen, our drug discovery approach is to make arrays of focused libraries to facilitate quick advancement of our internal programs," explains Dr. Mitchell. High Speed Analog Chemistry (HSAC) is used to rapidly synthesize hundreds of analogs to explore the chemical space related to a particular lead compound.

    High throughput screening, HALO (high throughput accelerated lead optimization), generates biological data quickly for initial assessment of target activity.

    The company is currently targeting several kinases involved in angiogenesis, such as the EphB4 receptor. "We have identified kinases involved in the vascularization of tumors and discovered potent inhibitors against those targets," adds Dr. Mitchell.

    At a recent drug discovery conference, Axxam (www. axxam.com) provided a presentation of its luminescent cell-based assay, Photina.

  • Measuring Calcium Release in a Flash

    This measures calcium release in high throughput compound screening to identify novel modulators of important targets like G-protein coupled receptors and ion channels.

    This photoprotein has been optimized for mammalian cells and created with a more general calcium binding site. It generates a "flash" luminescence signal, allowing the detection of light signals from as few as 100 cells.

    "There are several advantages with Photina if you compare it to fluorescent dyes," explains Germano Carganico, Ph.D., CBO. "Fluorescent dyes can be calcium-sensitive and can create some toxic problems in you have a long incubation time. You can also avoid problems if you are running high throughput screening because in some compounds you can have a high number of artifacts."

    Dr. Carganico adds that Photina can be applied to a large number of different screening platforms, and works very well with CCD-camera detection because it is very sensitive to the luminescence.

    The company says this novel photoprotein is being used in very early drug discovery, mostly for primary screening on new chemical libraries. A European patent was granted in March and the company recently signed a collaboration agreement with CyBio to develop new technologies for drug discovery using automated luminescence screening platforms.



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