| Summary: | The ultimate goal of this project is to control the invasive behavior of cancer cells. Despite its critical role in cancer progression and outcome, metastasis is largely unaddressed in cancer therapies. Most available drugs are directed to inhibit cell proliferation and do not specifically block processes such as cell migration and adhesion that are essential for tumor spread and invasion. To target these processes underlying tumor metastasis, we aim to develop a novel inhibitors against focal adhesion kinase (FAK), a protein-tyrosine kinase that is often found overexpressed/activated on highly invasive tumors, and therefore, it is consider a potential target for treating metastasis.
Although there are a few small molecule inhibitors available against FAK, these have not been effective in clinical applications. Instead of employing the classical approach of generating small-molecules to inhibit the kinase activity, a strategy that presents major well-documented disadvantages, I propose to block the interaction of FAK with its interaction partners as an orthogonal and complementary way to inhibit the enzyme's activity and its role in promoting malignant cell phenotypes.
To address this challenge, I utilized monobodies, synthetic antibody-mimics based on the human fibronectin scaffold. These custom-designed proteins constitute a powerful platform to inhibit protein-protein interactions, not only because monobodies can bind with remarkably high affinity and specificity, but also because they can be expressed functionally in the cell cytosol of mammalian cells, thus, acting as minimally invasive, genetically encoded tools to control signal-transduction pathways. Using directed-evolution methods, I generated several monobody binders against different domains of FAK and SRC. To test their potential applications on cell lines, I focused on the characterization of a single monobody directed against the FAT localization-domain of FAK. This monobody, named L07, bound tightly to FAT, with nanomolar affinity, and also bound selectively, since it did not bind the closest FAT domain homolog form PTK2B. In vitro, this monobody acted as a competitive inhibitor the interaction of the FAT domain and paxillin, an adaptor protein that anchors FAK to focal adhesions. When expressed in cancer cells, it prevented the localization of FAK at focal adhesions, and its co-localization with paxillin. The expression of this monobody also inhibited the cell migration, attachment and invasion.
The L07 monobody generated and characterized in this dissertation constitutes the first inhibitor of FAK that targets its localization at focal adhesions. Together, this work identified the interaction between FAK and paxillin as a potentially druggable target for controlling metastasis, and demonstrates the utility of synthetic protein design in advancing mechanistic studies of signaling networks and in discovering novel strategies to control human malignancies.
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