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

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.