Targeting the EWS-FLI1 pre-mRNA in Ewing Sarcoma through small molecule microarray screening

Authors

• RE Boer
• C Neckles
• S Kim
• DR Calabrese
• JS Schneekloth
• N Caplen

Abstract

A recently published study showed that the processing of the EWS-FLI1 fusion pre-mRNA expressed in approximately a third of Ewing sarcomas (ES) requires the RNA-binding protein HNRNPH1, which binds to two G-rich sequences present in EWSR1 exon 8. To determine if the interaction of HNRNPH1 with EWSR1 exon 8 is potentially targetable using small molecules, we used our Small Molecule Microarray (SMM) platform to identify compounds that bind selectively to the first G-rich sequence (seq 1) of EWSR1 exon 8 in the hopes of uncovering novel therapeutic options for a subset of ES patients. We have demonstrated that RNA oligomers corresponding to the G-rich sequences in EWSR1 exon 8 form G-quadruplex (G4) structures in vitro. Through our SMM screen, we identified an azoloquinazoline compound that bound with moderate selectivity over other RNA and DNA G4s. Next, Surface Plasmon Resonance (SPR) experiments showed that this compound has KD of 3 uM. We developed an ALPHAScreen assay and demonstrated that this compound can inhibit the binding of recombinant HNRNPH1 to EWSR1 RNA with an IC50 of 14 uM. We synthesized a library of derivatives to understand the structure activity relationship of the azoloquinoline scaffold and identified an improved analog with a KD of 1.2 uM. Future work will involve conjugating this lead compound to a pan-G4 binding compound that can target the second G-rich region of EWSR1 exon 8 (seq 2) to improve potency and specificity, allowing for evaluation of these compounds in inhibiting pre-mRNA splicing and protein function in ES cells.

Scientific Focus Area: Chemical Biology

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