Development of assays for HTS to identify inhibitors of a new PPI involved in cancer metastasis

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Abstract Breast cancer is a global problem, with 1.3 million new cases diagnosed and 430,000 deaths each year worldwide. Recently, researchers have begun developing exciting drugs to target the protein-protein interactions (PPIs) that regulate metastasis-driving signaling pathways. PPIs are critical for cellular signal transductions that play key roles in both normal and abnormal functions in cells. Changes in specificity and affinity of these interactions can lead to cellular malfunctions, such as uncontrolled cell growth and/or cell migration. A critical key in dissemination process is the ability of cells to coordinate signaling pathways with cytoskeleton dynamics. As critical coordinators of the cytoskeleton machinery, GTPases and their regulator GEFs are key players for cell movement. Many tumors show increased expression and/or activation of GTPases and GEFs. These proteins play fundamental roles in several aspects of cancer progression, metastasis and poor prognosis. Our long-term goals are to better understand the molecular mechanisms of GTPases in metastasis process and to device more effective, less toxic breast cancer therapies. Our strong preliminary data produced in our laboratory uncover a new protein-protein interaction critical in cell invasion and metastasis. The objective in this application is to implement a pilot high-throughput screen (HTS) to identify small molecule inhibitors of these novel binding partners that will represent potentially life-saving therapeutic leads for inhibiting cancer metastasis. Three specific aims will be developed in this proposal: 1) Development of fluorescence assay to detect this PPI; 2) Pilot screen: Complete pilot screen against a compound collection of 20,000 compounds and follow up assays; 3) Biological effects: perform initial characterization of active compounds on cell migration and invasion. Our HTS will involve an innovative approach that analyzes PPIs in cells and improve scientific knowledge about key signaling proteins in cell migration. This investigation will have substantial significance in understanding fundamental and defined features of cell motility, with implications for many areas of biology and pathophysiology.