Advertisement

Identification of Inhibitors of MicroRNA Function from Small Molecule Screens

  • Colleen M. Connelly
  • Alexander Deiters
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1095)

Abstract

Aberrant expression of microRNAs (miRNAs) has been linked to many human diseases including cancer, immune disorders, heart disease, and viral infections. Thus, small molecule inhibitors of miRNAs have potential as new therapeutic agents, as probes for the elucidation of detailed mechanisms of miRNA function, and as tools for the discovery of new targets for the treatment of human diseases. In order to identify small molecule inhibitors of specific miRNAs, functional assays have been developed and applied to the screening of small molecule libraries. Here, we report the application of a luciferase-based reporter assay of miRNA miR-122 function to the discovery of small molecule miR-122 inhibitors.

Key words

MicroRNA Cell-based assay Luciferase High-throughput screen Small molecule inhibitor 

References

  1. 1.
    Tong AW, Nemunaitis J (2008) Modulation of miRNA activity in human cancer: a new paradigm for cancer gene therapy? Cancer Gene Ther 15:341–355PubMedCrossRefGoogle Scholar
  2. 2.
    Sevignani C, Calin G, Siracusa L, Croce C (2006) Mammalian microRNAs: a small world for fine-tuning gene expression. Mamm Genome 17:189–202PubMedCrossRefGoogle Scholar
  3. 3.
    Port JD, Sucharov C (2010) Role of microRNAs in cardiovascular disease: therapeutic challenges and potentials. J Cardiovasc Pharmacol 56:444–453PubMedCrossRefGoogle Scholar
  4. 4.
    Lindsay MA (2008) microRNAs and the immune response. Trends Immunol 29:343–351PubMedCrossRefGoogle Scholar
  5. 5.
    Cullen BR (2011) Viruses and microRNAs: RISCy interactions with serious consequences. Genes Dev 25:1881–1894PubMedCrossRefGoogle Scholar
  6. 6.
    Deiters A (2010) Small molecule modifiers of the microRNA and RNA interference pathway. AAPS J 12:51–60PubMedCrossRefGoogle Scholar
  7. 7.
    Meister G, Landthaler M, Dorsett Y, Tuschl T (2004) Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing. RNA 10:544–550PubMedCrossRefGoogle Scholar
  8. 8.
    Krützfeldt J, Rajewsky N, Braich R, Rajeev K, Tuschl T, Manoharan M et al (2005) Silencing of microRNAs in vivo with “antagomirs”. Nature 438:685–689PubMedCrossRefGoogle Scholar
  9. 9.
    Ebert MS, Neilson JR, Sharp PA (2007) MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells. Nat Methods 4:721–726PubMedCrossRefGoogle Scholar
  10. 10.
    Zhang S, Chen L, Jung E, Calin G (2010) Targeting microRNAs with small molecules: from dream to reality. Clin Pharmacol Ther 87:754–758PubMedCrossRefGoogle Scholar
  11. 11.
    Garzon R, Marcucci G, Croce C (2010) Targeting microRNAs in cancer: rationale, strategies and challenges. Nat Rev Drug Discov 9:775–789PubMedCrossRefGoogle Scholar
  12. 12.
    Shi XB, Tepper CG, deVere White RW (2008) Cancerous miRNAs and their regulation. Cell Cycle 7:1529–1538PubMedCrossRefGoogle Scholar
  13. 13.
    Gumireddy K, Young D, Xiong X, Hogenesch J, Huang Q, Deiters A (2008) Small-molecule inhibitors of microrna miR-21 function. Angew Chem Int Ed Engl 47:7482–7484PubMedCrossRefGoogle Scholar
  14. 14.
    Young D, Connelly C, Grohmann C, Deiters A (2010) Small molecule modifiers of microRNA miR-122 function for the treatment of hepatitis C virus infection and hepatocellular carcinoma. J Am Chem Soc 132:7976–7981PubMedCrossRefGoogle Scholar
  15. 15.
    Connelly CM, Thomas M, Deiters A (2012) High-throughput luciferase reporter assay for small-molecule inhibitors of microRNA function. J Biomol Screen 17:822–828PubMedCrossRefGoogle Scholar
  16. 16.
    Shan G, Li Y, Zhang J, Li W, Szulwach K, Duan R et al (2008) A small molecule enhances RNA interference and promotes microRNA processing. Nat Biotechnol 26:933–940PubMedCrossRefGoogle Scholar
  17. 17.
    Watashi K, Yeung M, Starost M, Hosmane R, Jeang K (2010) Identification of small molecules that suppress microRNA function and reverse tumorigenesis. J Biol Chem 285:24707–24716PubMedCrossRefGoogle Scholar
  18. 18.
    Bose D, Jayaraj G, Suryawanshi H, Agarwala P, Pore SK, Banerjee R et al (2012) The tuberculosis drug streptomycin as a potential cancer therapeutic: inhibition of miR-21 function by directly targeting its precursor. Angew Chem Int Ed Engl 51:1019–1023PubMedCrossRefGoogle Scholar
  19. 19.
    Chen X, Huang C, Zhang W, Wu Y, Chen X, Zhang C et al (2012) A universal activator of microRNAs identified from photoreaction products. Chem Commun 48:6432–6434CrossRefGoogle Scholar
  20. 20.
    Jopling C, Yi M, Lancaster A, Lemon S, Sarnow P (2005) Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA. Science 309:1577–1581PubMedCrossRefGoogle Scholar
  21. 21.
    Lanford R, Hildebrandt-Eriksen E, Petri A, Persson R, Lindow M, Munk M et al (2010) Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection. Science 327:198–201PubMedCrossRefGoogle Scholar
  22. 22.
    Assay Guidance Manual Version 5.0, Eli Lilly and Company and NIH Chemical Genomics Center. Available online at: http://www.ncgc.nih.gov/guidance/manual_toc.html. Accessed Jan 2011Google Scholar
  23. 23.
    Sebaugh JL (2011) Guidelines for accurate EC50/IC50 estimation. Pharm Stat 10:128–134PubMedCrossRefGoogle Scholar
  24. 24.
    Thorne N, Inglese J, Auld DS (2010) Illuminating insights into firefly luciferase and other bioluminescent reporters used in chemical biology. Chem Biol 17:646–657PubMedCrossRefGoogle Scholar
  25. 25.
    Auld DS, Thorne N, Nguyen DT, Inglese J (2008) A specific mechanism for nonspecific activation in reporter-gene assays. ACS Chem Biol 3:463–470PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Colleen M. Connelly
    • 1
  • Alexander Deiters
    • 1
  1. 1.Department of ChemistryNorth Carolina State UniversityRaleighUSA

Personalised recommendations