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High-throughput fluorescence anisotropy screen for inhibitors of the oncogenic mRNA binding protein, IMP-1
Lily Mahapatra1, Chengjian Mao, Neal Andruska
11Department of Molecular and Integrative Physiology, University of Illinois, Urbana, IL, USA.
Abstract:
Cancer cell proliferation is regulated by oncogenes, such as c-Myc. An alternative approach to directly targeting individual oncogenes is to target IMP-1, an oncofetal protein that binds to and stabilizes messenger RNAs (mRNAs), leading to elevated expression of c-Myc and other oncogenes. Expression of IMP-1 is tightly correlated with a poor prognosis and reduced survival in ovarian, lung, and colon cancer. Small-molecule inhibitors of IMP-1 have not been reported. We established a fluorescence anisotropy/polarization microplate assay (FAMA) for analyzing binding of IMP-1 to a fluorescein-labeled 93 nucleotide c-Myc mRNA target (flMyc), developed the assay as a highly robust (Z' factor = 0.60) FAMA-based high-throughput screen for inhibitors of binding of IMP-1 to flMyc, and carried out a successful pilot screen of 17,600 small molecules. Our studies support rapidly filtering out toxic nonspecific inhibitors using an early cell-based assay in control cells lacking the target protein. The physiologic importance of verified hits from the in vitro high-throughput screen was demonstrated by identification of the first small-molecule IMP-1 inhibitor, a lead compound that selectively inhibits proliferation of IMP-1-positive cancer cells with very little or no effect on proliferation of IMP-1-negative cells.
Insights
Researchers identified the first small-molecule inhibitor for IMP-1, a protein linked to poor cancer prognosis. This compound selectively targets IMP-1-positive cancer cells, offering a potential new therapeutic strategy for ovarian, lung, and colon cancers.
Area of Science:
- Oncology
- Molecular Biology
- Drug Discovery
Background:
- Oncogenes like c-Myc regulate cancer cell proliferation.
- The oncofetal protein IMP-1 stabilizes mRNAs, increasing oncogene expression and correlating with poor cancer prognosis.
- No small-molecule inhibitors for IMP-1 have been previously reported.
Purpose of the Study:
- To develop a high-throughput screening method for identifying IMP-1 inhibitors.
- To discover the first small-molecule inhibitor of IMP-1.
- To evaluate the selective efficacy of IMP-1 inhibitors in cancer cells.
Main Methods:
- Established a fluorescence anisotropy/polarization microplate assay (FAMA) to analyze IMP-1 binding to c-Myc mRNA.
- Developed a robust FAMA-based high-throughput screen (Z' factor = 0.60).
- Screened 17,600 small molecules and utilized cell-based assays to filter out nonspecific inhibitors.
Main Results:
- Successfully developed and validated a FAMA-based high-throughput screen for IMP-1 inhibitors.
- Identified the first small-molecule inhibitor of IMP-1.
- The lead compound selectively inhibited proliferation of IMP-1-positive cancer cells.
Conclusions:
- IMP-1 is a viable therapeutic target for cancer treatment.
- The developed FAMA assay is effective for high-throughput screening of IMP-1 inhibitors.
- The identified lead compound demonstrates selective anti-cancer activity, warranting further investigation.
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