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

Lily Mahapatra, Chengjian Mao, Neal Andruska, Chen Zhang, David J. Shapiro

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)427-436
Number of pages10
JournalJournal of Biomolecular Screening
Volume19
Issue number3
DOIs
StatePublished - Mar 2014

Keywords

  • IMP-1
  • RNA-binding protein
  • cancer
  • fluorescence anisotropy/polarization
  • inhibitor

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biotechnology
  • Biochemistry
  • Molecular Medicine
  • Pharmacology
  • Drug Discovery

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