Small molecule inhibition of multiple RNA binding proteins underlies Musashi-2 independent phenotypes

RNA binding proteins (RBPs) are key regulators of normal and pathological gene expression. Small molecules targeting RBP-RNA interactions are a rapidly emerging class of therapeutics. Ro-08-2750 (Ro) was previously identified as a competitive inhibitor of Musashi-RNA interactions. Here we show Ro potently inhibits steroidogenesis and viability independent of Musashi-2 in multiple cell lines. We identified Ro-interacting proteins using an unbiased proteome-wide approach. Other RBPs were a prominent class of Ro-interacting proteins and leveraging large-scale ENCODE data we found a subset of these phenocopy Ro inhibition. We provide a general framework for validating the specificity and identifying targets of RBP inhibitors in a cellular context. H295R cells were stimulated with 10nM AngII and treated with DMSO control or 5 uM Ro. Cells were harvested at 0, 4, 8, 12, 16, 24 hours post-stimulation. Additionally MUSASHI2 RIP-Seq was performed in unstimulated, untreated H295R cells to found RNAs bound by this RBP.