A 1536-Well Fluorescence Polarization Assay to Screen for Modulators of the MUSASHI Family of RNA-Binding Proteins

Abstract

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 10μM. 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.

RNA-binding Protein Musashi Homologue 1 Regulates Kidney Fibrosis by Translational Inhibition of p21 and Numb mRNA

“…Msi1 is known to be up-regulated in several cancers and is currently being explored as a potential therapeutic target (30). We found Msi1 to be expressed in tubular epithelial cells in the cytoplasm.…”

Small-molecule targeting of MUSASHI RNA-binding activity in acute myeloid leukemia

“…In order to identify a putative MSI inhibitor, we previously performed a fluorescence polarization (FP)-based screen using recombinant MSI1 and MSI2 and a consensus target RNA with a library of 6,208 compounds 38 . We selected Ro 08–2750 (Ro) based on its RNA-binding inhibition of both MSI1 and MSI2 38 . MSI2 RNA-binding inhibition was confirmed by FP ( IC 50 of 2.7 ± 0.4 μM) (Fig.…”

Functional screen of MSI2 interactors identifies an essential role for SYNCRIP in myeloid leukemia stem cells

“…Small molecule inhibitors that specifically block RBPs binding to RNAs or antisense oligionucleotides (ASOs) and can knockdown these proteins could provide a strategy for targeting these RBP complexes 37–40 . Overall, we propose that targeting the riboproteomic network in leukemia could be a novel therapeutic strategy in cancer.…”