Structural Characterization and Chemical Inhibition of the ARNT/TACC3 Complex

My research project focuses on mechanistic studies of a new group of transcriptional coactivators (coiled-coil coactivators: TACC3, TRIP230, CoCoA), involved in cancer development and progression. Normally, these coactivators play an essential role in the HIF hypoxia response, directly interacting with the ARNT subunit of HIF in a novel and promoter-specific way. However, misregulation by overexpression or activating fusions (for example, FGFR-TACC3) is sufficient for transformation and associated with the development of glioblastoma, renal cell carcinoma and other cancers. In light of this connection between coiled-coil coactivators (CCCs) and HIF signaling, tools that inhibit HIF/CCC complex formation might present opportunities to interrogate the linkage between different CCC-containing pathways and may offer a novel route to blocking cancer formation and progression. As a member of a new group of transcription coactivators, knowing how TACC3 interacts with ARNT is critical in understanding the general role of CCCs in HIF-dependent transcription machinery. In the first half of my study, I characterized the ARNT/TACC3 complex with various biophysical and biochemical methods including solution NMR, X-ray crystallography, circular dichroism, luminescence proximity and numerous cell-based assays. A 3.15 Å ARNT/TACC3 crystal structure was solved by molecular replacement, revealing details of this protein complex and providing a structural funcation for coactivator recruitment in HIF signaling pathway. The second half of this study focuses on the search for ARNT/TACC3 inhibitors with in vitro screens to regulate ARNT/CCCs activity in a rapid and flexible way. From a fragment-based NMR screen, I identified small molecules that specifically bound within the second PER-ARNT-SIM (PAS) domain of ARNT and perturb its interaction with TACC3. However, these small molecules have drawbacks, such as low potency or unclear modes of action. To identify higher potency small molecules targeting ARNT/TACC3 complexes, I developed an AlphaScreen-based high throughput screen. Hopefully the discovery of artificial ligands with known mode-of-action that inhibit this typically "undruggable" protein complex will provide new perspectives in small molecule inhibitor development, and also serve as a very useful tool in cell biology for studying pathways utilizing ARNT/TACC3.