Lan 2013

Screening for inhibitors of RNA-binding protein Musashi-1

Lan Lan1, Carl Appelman1, Amber Smith1, Jia Yu1, Rebecca Marquez1, Philip Gao2, Na Zhang2, Anuradha Roy3, Asokan Anbanandam4, Ragul Gowthaman1,5, John Karanicolas1,5, Jeffrey Aubé6, Kristi Neufeld1, and Liang Xu1  

1Department of Molecular Biosciences, 2Protein Production Group, NIH COBRE in Protein Structure and Function, 3High Throughput Screening Laboratory, 4Bio-NMR Core Facility,  5Center for Bioinformatics, 6Department of Medicinal Chemistry, The University of Kansas.

The Notch signaling pathway is involved in several important cellular processes, such as cell proliferation, differentiation, and apoptosis. Dysregulation of Notch signaling can enhance tumorigenesis, by stimulating proliferation and maintenance of a small population of cancer cells, called cancer stem cells. Another major signaling pathway, Wnt signaling pathway, has been shown to have cross-talk between Notch pathway in cancer development. An important regulator of Notch signaling is Musashi-1 (Msi1). Msi1 negatively regulates expression of Numb, a protein that inhibits Notch signaling. Msi1 binds to the 3’ untranslated region of Numb mRNA, preventing translation of Numb protein, which results in de-repression of Notch signaling. Our hypothesis is that small molecules that disrupt the Msi1-Numb binding would result in down regulation of Notch signaling, and therefore inhibit cancer stem cell maintenance and self-renewal. Using in vitro fluorescence polarization (FP) assay, we have screened chemical libraries and identified a series of Msi1 inhibitors with sub-micromolar inhibition constants (Ki). The hits are confirmed through orthogonal SPR and NMR assays. These Msi1 inhibitors may potentially inhibit cancer cell growth by inhibiting Wnt- and Notch-pathways. By combining the NMR data of our lead compounds with computational efforts, we have developed structural models of Msi1 bound to these inhibitors and identified a new series of Msi1 inhibitors. These studies will provide a stepping stone for future validation assays in vitro and in vivo. The long term goal is to discover Msi1 inhibitors with hopes of inhibiting Notch/Wnt signaling pathways and thereby inhibiting cancer stem cell maintenance and self-renewal.