Abstract 2681: EWS-FLI1 as a molecular target: Small molecule inhibitors for a disordered protein
Authors: Barber-Rotenberg Julie S. , Kong Yali , Schnure Nilan , Dakshanamurthy Sivanesan , Frazier Peter , Erkizan Hayriye V. , Brown Milton L. , Uren Aykut , Toretsky Jeffrey A. 
Published: February 2011

Ewing's sarcoma family of tumors are characterized by the EWS-FLI1 oncogenic fusion protein. Our previous studies show that RNA Helicase A (RHA) enhances EWS-FLI1 driven oncogenesis, and interruption of this protein-protein complex by small molecule inhibitors validate this interaction as a unique therapeutic target (NatMed 2009 Jul;15(7):750-6). EWS-FLI1 is a significantly hydrophobic disordered protein with unknown three-dimensional structure, which therefore precludes standard structure-based small molecule design. Given the challenges of drug design targeted to EWS-FLI1, we hypothesize that the optimization of a peptide displacement assay using small molecules can predict and direct the design of more potent analogs. Fluorescence polarization (FP) was originally used to measure RHA peptide displacement from EWS-FLI1, but is time consuming and reagent limiting. To identify new strategies for higher-throughput screening, a TECAN ULTRA 384 was used for assay detection in a 96-well plate format. However, this resulted in high background and low signal. A second approach, AlphaScreen (amplified luminescent proximity homogenous assay, PerkinElmer), uses a bead-based interaction system based on energy conversion and generation of chemiluminescence when acceptor and donor beads are brought together during binding. Results show a 6-fold dynamic range between bound and quenched beads, in comparison to just 2.5-fold for FP and less than two-fold for TECAN. Our small molecule, YK-4-279, is able to reduce the signal down to baseline, indicating that YK-4-279 dissociates the RHA peptide and EWS-FLI1. Key negative controls support specific binding between EWS-FLI1 and the RHA peptide. In addition, YK-4-279 was unable to quench the signal from a positive control using immunoglobulin, supporting the conclusion that YK-4-279 disrupts the specific protein-peptide binding rather than directly extinguishing the chemiluminescence. Data from the AlphaScreen will be used as input to a quantitative-structure activity relationship (QSAR) method. This QSAR method will predict biological activity from chemical structure and the knowledge gained from AlphaScreen will aid in the optimization and design of future analogs. In our initial work, we used the Free-Wilson QSAR method, converting the structure of the small molecules into a numerical format allowing for a mathematical comparison between structures, to populate a table with 32 small molecule analogs of YK-4-279. The AlphaScreen data may also be used with other QSAR methods, to which we may transition in later work. AlphaScreen technology provides a method to quantitatively compare binding of small molecules in a high-throughput screening system, yields a greater dynamic range with more specificity, and provides data that will help to predict the binding of other analogs with similar chemical structures.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2681.