RESUMEN
RNA helicases impact RNA structure and metabolism from transcription through translation, in part through protein interactions with transcription factors. However, there is limited knowledge on the role of transcription factor influence upon helicase activity. RNA helicase A (RHA) is a DExH-box RNA helicase that plays multiple roles in cellular biology, some functions requiring its activity as a helicase while others as a protein scaffold. The oncogenic transcription factor EWS-FLI1 requires RHA to enable Ewing sarcoma (ES) oncogenesis and growth; a small molecule, YK-4-279 disrupts this complex in cells. Our current study investigates the effect of EWS-FLI1 upon RHA helicase activity. We found that EWS-FLI1 reduces RHA helicase activity in a dose-dependent manner without affecting intrinsic ATPase activity; however, the RHA kinetics indicated a complex model. Using separated enantiomers, only (S)-YK-4-279 reverses the EWS-FLI1 inhibition of RHA helicase activity. We report a novel RNA binding property of EWS-FLI1 leading us to discover that YK-4-279 inhibition of RHA binding to EWS-FLI1 altered the RNA binding profile of both proteins. We conclude that EWS-FLI1 modulates RHA helicase activity causing changes in overall transcriptome processing. These findings could lead to both enhanced understanding of oncogenesis and provide targets for therapy.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteína EWS de Unión a ARN/metabolismo , ARN Helicasas DEAD-box/antagonistas & inhibidores , ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/genética , Indoles/farmacología , Modelos Moleculares , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , ARN/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
Transcription factors have long been deemed 'undruggable' targets for therapeutics. Enhanced recognition of protein biochemistry as well as the need to have more targeted approaches to treat cancer has rendered transcription factors approachable for therapeutic development. Since transcription factors lack enzymatic domains, the specific targeting of these proteins has unique challenges. One challenge is the hydrophobic microenvironment that affects small molecules gaining access to block protein interactions. The most attractive transcription factors to target are those formed from tumor specific chromosomal translocations that are validated oncogenic driver proteins. EWS-FLI1 is a fusion protein that results from the pathognomonic translocation of Ewing sarcoma (ES). Our past work created the small molecule YK-4-279 that blocks EWS-FLI1 from interacting with RNA Helicase A (RHA). To fulfill long-standing promise in the field by creating a clinically useful drug, steps are required to allow for in vivo administration. These investigations identify the need for continuous presence of the small molecule protein-protein inhibitor for a period of days. We describe the pharmacokinetics of YK-4-279 and its individual enantiomers. In vivo studies confirm prior in vitro experiments showing (S)-YK-4-279 as the EWS-FLI1 specific enantiomer demonstrating both induction of apoptosis and reduction of EWS-FLI1 regulated caveolin-1 protein. We have created the first rat xenograft model of ES, treated with (S)-YK-4-279 dosing based upon PK modeling leading to a sustained complete response in 2 of 6 ES tumors. Combining laboratory studies, pharmacokinetic measurements, and modeling has allowed us to create a paradigm that can be optimized for in vivo systems using both in vitro data and pharmacokinetic simulations. Thus, (S)-YK-4-279 as a small molecule drug is ready for continued development towards a first-in-human, first-in-class, clinical trial.