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RNA-binding protein Musashi-1 (MSI1) is a key regulator of several stem cell populations. It is involved in tumor proliferation and maintenance, and it regulates target mRNAs at the translational level. The known mRNA targets of MSI1 include Numb, APC and P21WAF-1, key regulators of Notch, Wnt signaling and cell cycle progression, respectively. In this study, we aim to identify small molecule inhibitors of MSI1-mRNA interaction thus blocking the growth of cancer cells with high levels of MSI1. Using a fluorescence polarization (FP) assay, we screened small molecules from several chemical libraries for those that disrupt the binding of MSI1 to its consensus RNA binding site. Two clusters of hit compounds have been identified, one of which is composed of derivatives of secondary metabolites from Aspergillus nidulans. The top hit, Aza-9, from this cluster was further validated by surface plasmon resonance and nuclear magnetic resonance which revealed that Aza-9 binds to MSI1 directly, in the RNA binding pocket. Next, we tested whether Aza-9 has anti-cancer potential in cells and whether such activities work through MSI1. Due to poor cellular uptake, we used PEGylated liposomes to facilitate Aza-9 entry into the cells. Aza-9-lipo inhibits colon cancer cell proliferation, induces apoptosis and autophagy, and down-regulates Notch/Wnt signaling in colon cancer cell lines. In conclusion, we identified a series of potential lead compounds for inhibiting MSI1 function while establishing a framework for identifying small molecule inhibitors of RNA binding proteins using FP-based screening methodology. Citation Format: Lan Lan, Jiajun Liu, Amber Amith, Carl Appelman, Xiaoqing Wu, Ke Li, Anuradha Roy, Ragul Gowthaman, John Karanicolas, Amber Somoza, Clay Wang, Berl Oakley, Roberto De Guzman, Kristi Neufeld, Liang Xu. Identification and validation of an Aspergillus nidulanssecondary metabolite derivative as an inhibitor of the Musashi1-RNA interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3059.