Structure, Function, and Inhibition of the RhoGEF Trio - a Target in Uveal Melanoma Uveal melanoma (UM) is a cancer of the pigment-containing cells of the eye which has an extremely high metastasis and fatality rate. The standard of care for metastatic UM is poor, and therapies in current clinical trials do not significantly improve overall survival. Recent efforts to elucidate the biochemical mechanism of uveal melanoma have revealed an aberrant signal transduction cascade mediated primarily by the C-terminal guanine nucleotide exchange factor (GEF) module of Trio (TrioC). This module is regulated via an autoinhibitory constraint that is released upon binding of the heterotrimeric G protein G?q. I have solved the 2.7 crystal structure of TrioC in an inactive state, yielding molecular insight into the autoinhibition mechanism. Using site directed mutagenesis and biochemical assays, I have obtained data which supports that individual residues in the ?6-?N linker helix and the?3-?4 loop of TrioC are responsible for stabilizing this novel conformation. Several point mutations are observed in cancer patients in these regulatory regions of TrioC (cBioPortal database) and they may represent activating mutations important for the development of cancer. I plan to verify this by generating thesesite- specific variants and testing their activity in biochemical assays and in model cell lines. In parallel, I seek to determine the structure of a selective inhibitory peptide with low micromolar potency in complex with TrioC, and conduct an activity-based FP high-throughput screen to discover novel scaffolds which inhibit TrioC. Such molecules could serve as the first step towards significantly improving the standard of care for metastatic uveal melanoma and other Trio-linked cancers.
The RhoGEF TrioC has recently been implicated in the pathogenesis of uveal melanoma. The proposed aims focus on biochemical and cell-based characterization of a small library of TrioC mutants observed in cancer patients, the use of a chemical probe to pursue rational inhibitor discovery, and the use of a FP screen to discover new small molecule inhibitors of TrioC. Successful completion of this proposal will yield insight into the pathological signaling, transcription, and growth phenotypes influenced by hyperactive TrioC, and produce valuable small molecule probes for TrioC and possible leads for the uveal melanoma indication.
