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.

Figure 1. YK-4-279 is a chiral molecule and the enantiomers were separated to purity

(A) The racemate was analyzed using a Chiralpak AD column with 60% 2-propanol in heptane with 0.1% TFA as a mobile phase. HPLC analysis of racemate clearly identified 2 single enantiomer peaks. (B) The enantiomers were crystalized in ethyl acetate and analyzed by x-ray crystallography. This resolved crystal shows the (S)-enantiomer and has an optical rotation of −191.9°. (C) Small molecule circular dichroism show 50μM of enantiomers rotate plane-polarized light in opposite directions.

Figure 2. (S)-YK-4-279 disrupts binding between EWS-FLI1 and RHA

(A) An ELISA assay measures binding between EWS-FLI1 and RHA. Small molecule (1 – 30μM) was added to EWS-FLI1, followed by the addition of RHA and detection with antibody (**, p < 0.05 compared to vehicle control, using a two-tailed Student's t-test). (B) TC32 cells were treated with 10μM small molecule for 15 hours. Immunoprecipitation of EWS-FLI1 and RHA was detected via western blot. (C) TC32 cells were treated with 0.3, 1, 3, and 10μM of small molecule for 15 hours followed by immunoprecipitation. (D) Immunoblot of 10% input for (C). (E) Densitometry was calculated for each band and the ratio RHA to EWS-FLI1 was plotted.

Figure 3. EWS-FLI1 activity is altered by (S)- but not (R)-enantiomer of YK-4-279

(A) COS7 cells were co-transfected with EWS-FLI1 and the EWS-FLI1-responsive promoter NR0B1. Transfection assays were performed in triplicate (**, p < 0.05 compared to vehicle control, using a two-tailed Student's t-test). (B) Expression of EWS-FLI1 in transfected cells was detected by western blot. (C) A673i cells were treated with vehicle or 1μM small molecule for 8 hours. Control cells were treated with tetracycline for 72 hours to reduce EWS-FLI1. qRT-PCR was used to quantify levels of CyclinD1a and CyclinD1b mRNA levels. Data is averaged from four experiments performed in duplicate (**, p < 0.05 compared to vehicle, using a two-tailed Student's t-test). (E) Immunoblot of total cell lysates from one of the four experiments shown in (D).

Figure 4. (S)-YK-4-279 is the active enantiomer in cellular assays

(A) A panel of ESFT and non-ESFT cells were treated with a dose range of small molecule. Cell viability was measured by WST after 72 hours of treatment. One representative graph from a cytotoxicity assay is shown. Graphs show IC50 values for (B) ESFT and (C) non-ESFT cells (**, p < 0.05, using a two-tailed Student's t-test). (D) ESFT and non-ESFT cells were treated with 10μM small molecule for 18 hours. Graph shows fold caspase-3 activity of treated cell lysates to control cell lysates. (E) A4573 cells were assayed for caspase-3 activation with increasing concentrations of YK-4-279 and (S)-YK-4-279 for 18 hours. For all panels, black bars represent YK-4-279, blue bars represent (S)-YK-4-279, and red bars represent (R)-YK-4-279.