Ewing sarcoma family of tumors (ESFT) consists of highly malignant tumors of the bone and soft tissue. Ninety-five percent of cases contain a balanced t(11;22) or t(21;22) rearrangement, combining the amino-terminus of EWS to the carboxy-terminus of FLI1 or ERG, which contain the highly conserved ets DNA binding domain. As the EWS-FLI1 protein is found only in ESFT cells and its expression is required for the oncogenic phenotype, it presents a promising molecular target for anti-cancer therapies. EWS-FLI1 is a hydrophobic disordered protein with unknown three-dimensional structure, precluding standard structure-based drug design. RNA Helicase A (RHA) enhances EWS-FLI1 driven oncogenesis and interruption of this protein-protein complex validates this interaction as a unique therapeutic target. Surface plasmon resonance screening identified compounds that bind to EWS-FLI1, including a lead compound that induces apoptosis in ESFT cells and reduces the growth of xenografts. Our compound, YK-4-279, has a chiral center and can be separated into enantiomers, only one of which is able to specifically target the protein-protein interaction. This work is significant for its identification of a single enantiomer effect upon a protein-protein interaction suggesting that small molecule targeting of intrinsically disordered proteins can be highly specific. Given the challenges of drug design targeted to EWS-FLI1, we proposed that characterization of the physical interaction points between EWS-FLI1 and RHA would allow us to better alter the lead compound to block this protein-protein interaction. While full length EWS-FLI1 is able to pull down RHA, fragments of the protein are not. Although we can successfully crosslink EWS-FLI1 and RHA, we have yet to identify what region of EWS-FLI1 is involved. We are able to show specific regions of order and disorder of EWS-FLI1, which may lead to the identification of the binding site for YK-4-279. The development of higher-throughput methods for testing small molecules that bind to or inhibit EWS-FLI1 function will allow us to further investigate protein structure and function. These data are a contribution to the future development of small molecules in an era where novel approaches to cancer therapy are critical for improving patient care.
|Advisor:||Toretsky, Jeffrey A.|
|Commitee:||Brown, Milton, Crooke, Elliott, Metallo, Steven, Uren, Aykut|
|School Location:||United States -- District of Columbia|
|Source:||DAI-B 73/08(E), Dissertation Abstracts International|
|Keywords:||Disordered proteins, Ewing sarcoma, Protein-protein interactions, RNA helicase A, Small molecules|
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