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Sequence-based design of bioactive small molecules that target precursor microRNAs

Nature Chemical Biology volume 10, pages 291–297 (2014)Cite this article

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Abstract

Oligonucleotides are designed to target RNA using base pairing rules, but they can be hampered by poor cellular delivery and nonspecific stimulation of the immune system. Small molecules are preferred as lead drugs or probes but cannot be designed from sequence. Herein, we describe an approach termed Inforna that designs lead small molecules for RNA from solely sequence. Inforna was applied to all human microRNA hairpin precursors, and it identified bioactive small molecules that inhibit biogenesis by binding nuclease-processing sites (44% hit rate). Among 27 lead interactions, the most avid interaction is between a benzimidazole (1) and precursor microRNA-96. Compound 1 selectively inhibits biogenesis of microRNA-96, upregulating a protein target (FOXO1) and inducing apoptosis in cancer cells. Apoptosis is ablated when FOXO1 mRNA expression is knocked down by an siRNA, validating compound selectivity. Markedly, microRNA profiling shows that 1 only affects microRNA-96 biogenesis and is at least as selective as an oligonucleotide.

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Figure 1: The Inforna approach to design small molecules that target RNA from sequence applied to human miRNA precursors.
Figure 2: Designer small molecules targeting miRNA precursors identified via Inforna are bioactive.
Figure 3: Effects of small molecules on the downstream targets of miR-96.
Figure 4: Profiling of disease-associated miRNAs that are affected upon addition of 40 μM of 1 or a miR-96 antagomir.

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References

  1. Arambula, J.F., Ramisetty, S.R., Baranger, A.M. & Zimmerman, S.C. A simple ligand that selectively targets CUG trinucleotide repeats and inhibits MBNL protein binding. Proc. Natl. Acad. Sci. USA 106, 16068–16073 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  2. Childs-Disney, J.L., Hoskins, J., Rzuczek, S.G., Thornton, C.A. & Disney, M.D. Rationally designed small molecules targeting the RNA that causes myotonic dystrophy type 1 are potently bioactive. ACS Chem. Biol. 7, 856–862 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Ofori, L.O., Hoskins, J., Nakamori, M., Thornton, C.A. & Miller, B.L. From dynamic combinatorial 'hit' to lead: in vitro and in vivo activity of compounds targeting the pathogenic RNAs that cause myotonic dystrophy. Nucleic Acids Res. 40, 6380–6390 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Kumar, A. et al. Chemical correction of pre-mRNA splicing defects associated with sequestration of muscleblind-like 1 protein by expanded r(CAG)-containing transcripts. ACS Chem. Biol. 7, 496–505 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Bose, D. et al. The tuberculosis drug streptomycin as a potential cancer therapeutic: inhibition of miR-21 function by directly targeting its precursor. Angew. Chem. Int. Edn Engl. 51, 1019–1023 (2012).

    Article  CAS  Google Scholar 

  6. Stelzer, A.C. et al. Discovery of selective bioactive small molecules by targeting an RNA dynamic ensemble. Nat. Chem. Biol. 7, 553–559 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Parsons, J. et al. Conformational inhibition of the hepatitis C virus internal ribosome entry site RNA. Nat. Chem. Biol. 5, 823–825 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Davidson, A. et al. Simultaneous recognition of HIV-1 TAR RNA bulge and loop sequences by cyclic peptide mimics of Tat protein. Proc. Natl. Acad. Sci. USA 106, 11931–11936 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Guan, L. & Disney, M.D. Recent advances in developing small molecules targeting RNA. ACS Chem. Biol. 7, 73–86 (2012).

    Article  CAS  PubMed  Google Scholar 

  10. Thomas, J.R. & Hergenrother, P.J. Targeting RNA with small molecules. Chem. Rev. 108, 1171–1224 (2008).

    Article  CAS  PubMed  Google Scholar 

  11. Yonath, A. & Bashan, A. Ribosomal crystallography: initiation, peptide bond formation, and amino acid polymerization are hampered by antibiotics. Annu. Rev. Microbiol. 58, 233–251 (2004).

    Article  CAS  PubMed  Google Scholar 

  12. Mathews, D.H. et al. Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure. Proc. Natl. Acad. Sci. USA 101, 7287–7292 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Woese, C. & Pace, N. The RNA World 2nd edn. (eds. Gesteland, R.F., Cech, T.R. & Atkins, J.F.) 91–117 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1993).

  14. Batey, R.T., Rambo, R.P. & Doudna, J.A. Tertiary motifs in RNA structure and folding. Angew. Chem. Int. Edn Engl. 38, 2326–2343 (1999).

    Article  CAS  Google Scholar 

  15. Spahn, C.M. et al. Hepatitis C virus IRES RNA-induced changes in the conformation of the 40s ribosomal subunit. Science 291, 1959–1962 (2001).

    Article  CAS  PubMed  Google Scholar 

  16. Disney, M.D. et al. Two-dimensional combinatorial screening identifies specific aminoglycoside-RNA internal loop partners. J. Am. Chem. Soc. 130, 11185–11194 (2008).

    Article  CAS  PubMed  Google Scholar 

  17. Velagapudi, S.P., Seedhouse, S.J. & Disney, M.D. Structure-activity relationships through sequencing (StARTS) defines optimal and suboptimal RNA motif targets for small molecules. Angew. Chem. Int. Edn Engl. 49, 3816–3818 (2010).

    Article  CAS  Google Scholar 

  18. Velagapudi, S.P., Seedhouse, S.J., French, J. & Disney, M.D. Defining the RNA internal loops preferred by benzimidazole derivatives via 2D combinatorial screening and computational analysis. J. Am. Chem. Soc. 133, 10111–10118 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Jiang, Q. et al. miR2Disease: a manually curated database for microRNA deregulation in human disease. Nucleic Acids Res. 37, D98–D104 (2009).

    Article  CAS  PubMed  Google Scholar 

  20. Griffiths-Jones, S., Saini, H.K., van Dongen, S. & Enright, A.J. miRBase: tools for microRNA genomics. Nucleic Acids Res. 36, D154–D158 (2008).

    Article  CAS  PubMed  Google Scholar 

  21. Bartel, D.P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297 (2004).

    Article  CAS  PubMed  Google Scholar 

  22. Ambros, V. et al. A uniform system for microRNA annotation. RNA 9, 277–279 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Wu, M. & Turner, D.H. Solution structure of (rGCGGACGC)2 by two-dimensional NMR and the iterative relaxation matrix approach. Biochemistry 35, 9677–9689 (1996).

    Article  CAS  PubMed  Google Scholar 

  24. SantaLucia, J. Jr. & Turner, D.H. Structure of (rGGCGAGCC)2 in solution from NMR and restrained molecular dynamics. Biochemistry 32, 12612–12623 (1993).

    Article  CAS  PubMed  Google Scholar 

  25. Kozomara, A. & Griffiths-Jones, S. miRBase: integrating microRNA annotation and deep-sequencing data. Nucleic Acids Res. 39, D152–D157 (2011).

    Article  CAS  PubMed  Google Scholar 

  26. Krützfeldt, J. et al. Silencing of microRNAs in vivo with 'antagomirs'. Nature 438, 685–689 (2005).

    Article  CAS  PubMed  Google Scholar 

  27. Ebert, M.S. & Sharp, P.A. MicroRNA sponges: progress and possibilities. RNA 16, 2043–2050 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Obad, S. et al. Silencing of microRNA families by seed-targeting tiny LNAs. Nat. Genet. 43, 371–378 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Mathews, D.H. Using an RNA secondary structure partition function to determine confidence in base pairs predicted by free energy minimization. RNA 10, 1178–1190 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Childs-Disney, J.L., Wu, M., Pushechnikov, A., Aminova, O. & Disney, M.D. A small molecule microarray platform to select RNA internal loop-ligand interactions. ACS Chem. Biol. 2, 745–754 (2007).

    Article  CAS  PubMed  Google Scholar 

  31. Pilch, D.S. et al. Binding of a hairpin polyamide in the minor groove of DNA: sequence-specific enthalpic discrimination. Proc. Natl. Acad. Sci. USA 93, 8306–8311 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Pinto, I.G., Guilbert, C., Ulyanov, N.B., Stearns, J. & James, T.L. Discovery of ligands for a novel target, the human telomerase RNA, based on flexible-target virtual screening and NMR. J. Med. Chem. 51, 7205–7215 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Disney, M.D. et al. A small molecule that targets r(CGG)exp and improves defects in fragile X-associated tremor ataxia syndrome. ACS Chem. Biol. 7, 1711–1718 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Luzhkov, V.B. et al. Virtual screening and bioassay study of novel inhibitors for dengue virus mRNA cap (nucleoside-2′O)-methyltransferase. Bioorg. Med. Chem. 15, 7795–7802 (2007).

    Article  CAS  PubMed  Google Scholar 

  35. Xu, S., Witmer, P.D., Lumayag, S., Kovacs, B. & Valle, D. MicroRNA (miRNA) transcriptome of mouse retina and identification of a sensory organ–specific miRNA cluster. J. Biol. Chem. 282, 25053–25066 (2007).

    Article  CAS  PubMed  Google Scholar 

  36. Stenvang, J., Petri, A., Lindow, M., Obad, S. & Kauppinen, S. Inhibition of microRNA function by antimiR oligonucleotides. Silence. 3, 1 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Xie, L. et al. FOXO1 is a tumor suppressor in classical Hodgkin lymphoma. Blood 119, 3503–3511 (2012).

    Article  CAS  PubMed  Google Scholar 

  38. Guttilla, I.K. & White, B.A. Coordinate regulation of FOXO1 by miR-27a, miR-96, and miR-182 in breast cancer cells. J. Biol. Chem. 284, 23204–23216 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Dansen, T.B. & Burgering, B.M. Unravelling the tumor-suppressive functions of FOXO proteins. Trends Cell Biol. 18, 421–429 (2008).

    Article  CAS  PubMed  Google Scholar 

  40. Huang, H. & Tindall, D.J. FOXO factors: a matter of life and death. Future Oncol. 2, 83–89 (2006).

    Article  CAS  PubMed  Google Scholar 

  41. Lewis, B.P., Burge, C.B. & Bartel, D.P. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120, 15–20 (2005).

    Article  CAS  PubMed  Google Scholar 

  42. Tran, T. & Disney, M.D. Identifying the preferred RNA motifs and chemotypes that interact by probing millions of combinations. Nat. Commun. 3, 1125 (2012).

    Article  CAS  PubMed  Google Scholar 

  43. Hawkins, P.C., Skillman, A.G. & Nicholls, A. Comparison of shape-matching and docking as virtual screening tools. J. Med. Chem. 50, 74–82 (2007).

    Article  CAS  PubMed  Google Scholar 

  44. Han, J. et al. Molecular basis for the recognition of primary microRNAs by the Drosha–DGCR8 complex. Cell 125, 887–901 (2006).

    Article  CAS  PubMed  Google Scholar 

  45. Berezikov, E. et al. Phylogenetic shadowing and computational identification of human microRNA genes. Cell 120, 21–24 (2005).

    Article  CAS  PubMed  Google Scholar 

  46. Michlewski, G., Guil, S., Semple, C.A. & Caceres, J.F. Posttranscriptional regulation of miRNAs harboring conserved terminal loops. Mol. Cell 32, 383–393 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Lünse, C.E. et al. An aptamer targeting the apical-loop domain modulates pri-miRNA processing. Angew. Chem. Int. Edn Engl. 49, 4674–4677 (2010).

    Article  CAS  Google Scholar 

  48. Zeng, Y. & Cullen, B.R. Sequence requirements for micro RNA processing and function in human cells. RNA 9, 112–123 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Wolkenberg, S.E. & Boger, D.L. Mechanisms of in situ activation for DNA-targeting antitumor agents. Chem. Rev. 102, 2477–2495 (2002).

    Article  CAS  PubMed  Google Scholar 

  50. Kramer, R. & Cohen, D. Functional genomics to new drug targets. Nat. Rev. Drug Discov. 3, 965–972 (2004).

    Article  CAS  PubMed  Google Scholar 

  51. Bevilacqua, J.M. & Bevilacqua, P.C. Thermodynamic analysis of an RNA combinatorial library contained in a short hairpin. Biochemistry 37, 15877–15884 (1998).

    Article  CAS  PubMed  Google Scholar 

  52. McKenna, S.A. et al. Purification and characterization of transcribed RNAs using gel filtration chromatography. Nat. Protoc. 2, 3270–3277 (2007).

    Article  CAS  PubMed  Google Scholar 

  53. Peyret, N., Seneviratne, P.A., Allawi, H.T. & SantaLucia, J. Jr. Nearest-neighbor thermodynamics and NMR of DNA sequences with internal A.A, C.C, G.G, and T.T mismatches. Biochemistry 38, 3468–3477 (1999).

    Article  CAS  PubMed  Google Scholar 

  54. SantaLucia, J. Jr. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc. Natl. Acad. Sci. USA 95, 1460–1465 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Puglisi, J.D. & Tinoco, I. Jr. Absorbance melting curves of RNA. Methods Enzymol. 180, 304–325 (1989).

    Article  CAS  PubMed  Google Scholar 

  56. Wang, Y. & Rando, R.R. Specific binding of aminoglycoside antibiotics to RNA. Chem. Biol. 2, 281–290 (1995).

    Article  CAS  PubMed  Google Scholar 

  57. Disney, M.D., Gryaznov, S.M. & Turner, D.H. Contributions of individual nucleotides to tertiary binding of substrate by a Pneumocystis carinii group I intron. Biochemistry 39, 14269–14278 (2000).

    Article  CAS  PubMed  Google Scholar 

  58. Landthaler, M., Yalcin, A. & Tuschl, T. The human DiGeorge syndrome critical region gene 8 and its D. melanogaster homolog are required for miRNA biogenesis. Curr. Biol. 14, 2162–2167 (2004).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank B. White (Department of Cell Biology, University of Connecticut Health Center) for the luciferase plasmids containing FOXO1 3′ UTRs; C. Haga for assistance designing qRT-PCR primers; K. Lowe for assistance with flow cytometry; S. Seedhouse for preliminary studies with Inforna; B. Liu, J. Sokolow and T. Tran for compiling the secondary structures of the miRNA precursors and the RNA motif–small molecule database; and J. Childs-Disney, T. Kodadek, J. Cleveland, B. Roush, J. Joyce, M. Burkard and M. Guo for critical review of the manuscript. This work was funded by the US National Institutes of Health (R01GM097455). M.D.D. is a Camille and Henry Dreyfus Teacher-Scholar.

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Authors and Affiliations

  1. Department of Chemistry, The Scripps Research Institute, Scripps Florida, Jupiter, Florida, USA.,

    Sai Pradeep Velagapudi & Matthew D Disney

  2. The Department of Chemistry, The University at Buffalo, Buffalo, New York, USA

    Sai Pradeep Velagapudi

  3. The New York State Center of Excellence in Bioinformatics and Life Sciences, The University at Buffalo, Buffalo, New York, USA

    Steven M Gallo

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  1. Sai Pradeep Velagapudi
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Contributions

S.P.V. designed and completed all of the experiments and contributed to the writing of the manuscript; S.M.G. programmed Inforna; and M.D.D. conceived Inforna, designed experiments and wrote the manuscript.

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Correspondence to Matthew D Disney.

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Supplementary Results, Supplementary Tables 1 and 2, Supplementary Figures 1–16, and Supplementary Note. (PDF 9192 kb)

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Velagapudi, S., Gallo, S. & Disney, M. Sequence-based design of bioactive small molecules that target precursor microRNAs. Nat Chem Biol 10, 291–297 (2014). https://doi.org/10.1038/nchembio.1452

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