Single Enantiomer of YK-4-279 Demonstrates Specificity in Targeting the Oncogene EWS-FLI1

Abstract

Julie S. Barber-Rotenberg 1 , Saravana P. Selvanathan 1 , Yali Kong 1 , Hayriye V. Erkizan 1 , Tara M. Snyder 2 , S. Peter Hong 3 , Christina L. Kobs 3 , Natalie L. South 3 , Steven Summer 3 , Philip J. Monroe 3 , Maksymilian Chruszcz 4 , Veselin Dobrev 5 , Perrer N. Tosso 1 , Lauren J. Scher 1 , Wladek Minor 4 , Milton L. Brown 1 , Steven J. Metallo 5 , Aykut Üren 1 , and Jeffrey A. Toretsky 1 1 Department of Oncology, Georgetown University Lombardi Comprehensive Cancer Center, Washington, DC USA 2 AMRI, Pharmaceutical and Quality Services, Albany, NY USA 3 Battelle Memorial Institute, Health and Life Sciences, Columbus, OH USA 4 Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA USA 5 Department of Chemistry, Georgetown University, Washington, DC USA Received: February 17, 2012; Accepted: February 26, 2012; Published: February 29, 2012; Keywords: PRL-3 monoclonal antibody, PRL-3 mouse/human chimeric antibody, antibody therapy, intracellular oncoprotein Correspondence: Jeffrey A. Toretsky, // // Abstract Oncogenic fusion proteins, such as EWS-FLI1, are excellent therapeutic targets as they are only located within the tumor. However, there are currently no agents targeted toward transcription factors, which are often considered to be ‘undruggable.’  A considerable body of evidence is accruing that refutes this claim based upon the intrinsic disorder of transcription factors.  Our previous studies show that RNA Helicase A (RHA) enhances the oncogenesis of EWS-FLI1, a putative intrinsically disordered protein.  Interruption of this protein-protein complex by small molecule inhibitors validates this interaction as a unique therapeutic target.  Single enantiomer activity from a chiral compound has been recognized as strong evidence for specificity in a small molecule-protein interaction.  Our compound, YK-4-279, has a chiral center and can be separated into two enantiomers by chiral HPLC.  We show that there is a significant difference in activity between the two enantiomers.  (S)-YK-4-279 is able to disrupt binding between EWS-FLI1 and RHA in an immunoprecipitation assay and blocks the transcriptional activity of EWS-FLI1, while (R)-YK-4-279 cannot.  Enantiospecific effects are also established in cytotoxicity assays and caspase assays, where up to a log-fold difference is seen between (S)-YK-4-279 and the racemic YK-4-279.  Our findings indicate that only one enantiomer of our small molecule is able to specifically target a protein-protein interaction. This work is significant for its identification of a single enantiomer effect upon a protein interaction suggesting that small molecule targeting of intrinsically disordered proteins can be specific. Furthermore, proving YK-4-279 has only one functional enantiomer will be helpful in moving this compound towards clinical trials.