title: 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 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.
authors.forename: Gerard
authors.initials: G
authors.lastname: Minuesa
authors.forename: Christophe
authors.initials: C
authors.lastname: Antczak
authors.forename: David
authors.initials: D
authors.lastname: Shum
authors.forename: Constantin
authors.initials: C
authors.lastname: Radu
authors.forename: Bhavneet
authors.initials: B
authors.lastname: Bhinder
authors.forename: Yueming
authors.initials: Y
authors.lastname: Li
authors.forename: Hakim
authors.initials: H
authors.lastname: Djaballah
authors.affiliation: (Michael G. Kharas) Molecular Pharmacology & Chemistry Program, MSKCC, New York, USA. kharasm@mskcc.org.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Kharas
mesh_terms: D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005454:Fluorescence Polarization / Q000379:methods; D057166:High-Throughput Screening Assays; D006801:Humans; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D054852:Small Molecule Libraries / Q000737:chemistry* / Q000494:pharmacology
chemical_list: C491297:MSI1 protein, human; C438151:MSI2 protein, human; D009419:Nerve Tissue Proteins; D016601:RNA-Binding Proteins; D054852:Small Molecule Libraries