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Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes
Published on: January 30, 2019
Identification of small-molecule inhibitors against the interaction of RNA-binding protein PSF and its target RNA for
Ken-Ichi Takayama1, Seiji Matsuoka2, Shungo Adachi3
1Department of Systems Aging Science and Medicine, Tokyo Metropolitan Institute for Geriatrics and Gerontology, itabashi-ku, Tokyo 173-0015, Japan.
Abstract:
Diverse cellular activities are modulated through a variety of RNAs, including long noncoding RNAs (lncRNAs), by binding to certain proteins. The inhibition of oncogenic proteins or RNAs is expected to suppress cancer cell proliferation. We have previously demonstrated that PSF interaction with its target RNAs, such as androgen-induced lncRNA CTBP1-AS, is critical for hormone therapy resistance in prostate and breast cancers. However, the action of protein-RNA interactions remains almost undruggable to date. High-throughput screening (HTS) has facilitated the discovery of drugs for protein-protein interactions. In the present study, we developed an in vitro alpha assay using Flag peptide-conjugated lncRNA, CTBP1-AS, and PSF. We then constructed an effective HTS screening system to explore small compounds that inhibit PSF-RNA interactions. Thirty-six compounds were identified and dose-dependently inhibited PSF-RNA interaction in vitro. Moreover, chemical optimization of these lead compounds and evaluation of cancer cell proliferation revealed two promising compounds, N-3 and C-65. These compounds induced apoptosis and inhibited cell growth in prostate and breast cancer cells. By inhibiting PSF-RNA interaction, N-3 and C-65 up-regulated signals that are repressed by PSF, such as the cell cycle signals by p53 and p27. Furthermore, using a mouse xenograft model for hormone therapy-resistant prostate cancer, we revealed that N-3 and C-65 can significantly suppress tumor growth and downstream target gene expression, such as the androgen receptor (AR). Thus, our findings highlight a therapeutic strategy through the development of inhibitors for RNA-binding events in advanced cancers.
Insights
Researchers developed a high-throughput screening method to find compounds that inhibit protein-RNA interactions, crucial for cancer resistance. Two compounds, N-3 and C-65, effectively suppressed tumor growth in preclinical models by targeting PSF-RNA interactions.
Area of Science:
- Molecular Biology
- Cancer Research
- Drug Discovery
Background:
- Long noncoding RNAs (lncRNAs) and proteins modulate cellular activities, with their interactions implicated in cancer progression.
- PSF protein interaction with CTBP1-AS lncRNA is critical for hormone therapy resistance in prostate and breast cancers.
- Targeting protein-RNA interactions for therapeutic purposes remains a significant challenge due to their undruggable nature.
Purpose of the Study:
- To develop a high-throughput screening (HTS) system to identify small compounds inhibiting PSF-RNA interactions.
- To evaluate the efficacy of identified compounds in suppressing cancer cell proliferation and tumor growth.
- To explore a novel therapeutic strategy targeting RNA-binding events in advanced cancers.
Main Methods:
- Development of an in vitro alpha assay using Flag peptide-conjugated CTBP1-AS lncRNA and PSF.
- Construction of an HTS system to screen for small compounds inhibiting PSF-RNA interactions.
- Chemical optimization of lead compounds and evaluation in cancer cell lines and a mouse xenograft model.
Main Results:
- Thirty-six compounds were identified that dose-dependently inhibited PSF-RNA interaction in vitro.
- Two optimized compounds, N-3 and C-65, induced apoptosis and inhibited cell growth in prostate and breast cancer cells.
- N-3 and C-65 suppressed tumor growth in a hormone therapy-resistant prostate cancer mouse model, downregulating AR expression.
Conclusions:
- Inhibitors of PSF-RNA interaction, such as N-3 and C-65, represent a promising therapeutic strategy for advanced cancers.
- Targeting protein-RNA interactions offers a novel approach to overcome hormone therapy resistance.
- The developed HTS system provides a platform for discovering new drugs targeting RNA-binding events.
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