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High-throughput fluorescence anisotropy screen for inhibitors of the oncogenic mRNA binding protein, IMP-1.


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.

SUBMITTER: Mahapatra L 

PROVIDER: S-EPMC4072326 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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High-throughput fluorescence anisotropy screen for inhibitors of the oncogenic mRNA binding protein, IMP-1.

Mahapatra Lily L   Mao Chengjian C   Andruska Neal N   Zhang Chen C   Shapiro David J DJ  

Journal of biomolecular screening 20131009 3


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  ...[more]

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