Identification and Validation of an Aspergillus nidulans Secondary Metabolite Derivative as an Inhibitor of the Musashi-RNA Interaction

Abstract

RNA-binding protein Musashi-1 (MSI1) is a key regulator of several stem cell populations. MSI1 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 interactions, which could block 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. One cluster of hit compounds is the derivatives of secondary metabolites from Aspergillus nidulans. One of the top hits, Aza-9, from this cluster was further validated by surface plasmon resonance and nuclear magnetic resonance spectroscopy, which demonstrated that Aza-9 binds directly to MSI1, and the binding is at the RNA binding pocket. We also show that Aza-9 binds to Musashi-2 (MSI2) as well. To test whether Aza-9 has anti-cancer potential, we used liposomes to facilitate Aza-9 cellular uptake. Aza-9-liposome inhibits proliferation, induces apoptosis and autophagy, and down-regulates Notch and Wnt signaling in colon cancer cell lines. In conclusion, we identified a series of potential lead compounds for inhibiting MSI1/2 function, while establishing a framework for identifying small molecule inhibitors of RNA binding proteins using FP-based screening methodology.

Musashi1 Contribution to Glioblastoma Development via Regulation of a Network of DNA Replication, Cell Cycle and Division Genes

“…A single agent is unlikely to be effective in treating high aggressive tumors like glioblastoma. We and others have developed inhibitors against Musashi proteins [ 7 , 35 , 36 , 37 , 38 , 116 , 117 , 118 ]. We have identified a Msi1 inhibitor, luteolin, which showed synergistic interactions with radiation, Olaparib and Camptothecin.…”

Musashi–1—A Stemness RBP for Cancer Therapy?

“…Recently, several synthetic small molecules and naturally occurring substances were identified to modulate the RNA–binding capacity of the Musashi protein family. While some of the compounds, such as Ro 08–2750 [ 105 ] or Aza–9 [ 106 ], were shown to inhibit both MSI1 and MSI2, others were more specific to MSI1.…”

Mechanism of RNA recognition by a Musashi RNA-binding protein

“…These structures have greatly facilitated structure-based modeling and drug design targeting the MSI-RNA interactions ( Lan et al., 2015 ; Clingman et al., 2014 ; Lan et al., 2018 ; Lan et al., 2020 ). For example, we have recently identified one potent compound Aza-9 by combining fluorescence polarization (FP) assay, surface plasmon resonance (SPR), nuclear magnetic resonance (NMR) spectroscopy and molecular docking ( Lan et al., 2020a , Lan et al., 2020b ). However, experimental structures are rather static images and the dynamic mechanism of MSI-RNA interactions remains unknown, which has largely hindered the development of potent inhibitors of MSI proteins.…”