RNA-binding proteins (RBPs) can act as stem cell modulators and oncogenic drivers, but have been largely
ignored by the pharmaceutical industry as potential therapeutic targets for cancer. The MUSASHI (MSI) family has
recently been demonstrated to be an attractive clinical target in the most aggressive cancers. Therefore, the discovery and
development of small molecule inhibitors could provide a novel therapeutic strategy. In order to find novel compounds
with MSI RNA binding inhibitory activity, we have developed a fluorescence polarization (FP) assay and optimized it for
high throughput screening (HTS) in a 1536-well microtiter plate format. Using a chemical library of 6,208 compounds,
we performed pilot screens, against both MSI1 and MSI2, leading to the identification of 7 molecules for MSI1, 15 for
MSI2 and 5 that inhibited both. A secondary FP dose-response screen validated 3 MSI inhibitors with IC50 below 10 μM.
Out of the 25 compounds retested in the secondary screen only 8 demonstrated optical interference due to high
fluorescence. Utilizing a SYBR-based RNA electrophoresis mobility shift assay (EMSA), we further verified MSI
inhibition of the top 3 compounds. Surprisingly, even though several aminoglycosides were present in the library, they
failed to demonstrate MSI inhibitor activity challenging the concept that these compounds are pan-active against RBPs. In
summary, we have developed an in vitro strategy to identify MSI specific inhibitors using an FP HTS platform, which will
facilitate novel drug discovery for this class of RBPs.