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

Oligonucleotides are designed to target RNA using base pairing rules, however, they are hampered by poor cellular delivery and non-specific 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 precursors and identified bioactive small molecules that inhibit biogenesis by binding to nuclease processing sites (41% hit rate). Amongst 29 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. Importantly, microRNA profiling shows that 1 only significantly effects microRNA-96 biogenesis and is more selective than an oligonucleotide.

[1]  T. Tuschl,et al.  The Human DiGeorge Syndrome Critical Region Gene 8 and Its D. melanogaster Homolog Are Required for miRNA Biogenesis , 2004, Current Biology.

[2]  M. Nakamori,et al.  From dynamic combinatorial ‘hit’ to lead: in vitro and in vivo activity of compounds targeting the pathogenic RNAs that cause myotonic dystrophy , 2012, Nucleic acids research.

[3]  C. Burge,et al.  Conserved Seed Pairing, Often Flanked by Adenosines, Indicates that Thousands of Human Genes are MicroRNA Targets , 2005, Cell.

[4]  W D Wilson,et al.  Targeting RNA with small molecules. , 2000, Current medicinal chemistry.

[5]  B. Burgering,et al.  Unravelling the tumor-suppressive functions of FOXO proteins. , 2008, Trends in cell biology.

[6]  P. Bevilacqua,et al.  Thermodynamic analysis of an RNA combinatorial library contained in a short hairpin. , 1998, Biochemistry.

[7]  Phillip A Sharp,et al.  MicroRNA sponges: progress and possibilities. , 2010, RNA.

[8]  I. Tinoco,et al.  Absorbance melting curves of RNA. , 1989, Methods in enzymology.

[9]  Matthew D. Disney,et al.  Identifying the Preferred RNA Motifs and Chemotypes that Interact by Probing Millions of Combinations , 2012, Nature Communications.

[10]  J. SantaLucia,et al.  Nearest-neighbor thermodynamics and NMR of DNA sequences with internal A.A, C.C, G.G, and T.T mismatches. , 1999, Biochemistry.

[11]  Thomas Hermann,et al.  Conformational inhibition of the hepatitis C virus internal ribosome entry site RNA. , 2009, Nature chemical biology.

[12]  B. White,et al.  Coordinate Regulation of FOXO1 by miR-27a, miR-96, and miR-182 in Breast Cancer Cells , 2009, The Journal of Biological Chemistry.

[13]  J. SantaLucia,et al.  A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[14]  D. Mathews.  Using an RNA secondary structure partition function to determine confidence in base pairs predicted by free energy minimization. , 2004, RNA.

[15]  Stijn van Dongen,et al.  miRBase: microRNA sequences, targets and gene nomenclature , 2005, Nucleic Acids Res..

[16]  D. Tindall,et al.  FOXO factors: a matter of life and death. , 2006, Future oncology.

[17]  A. Bashan,et al.  Ribosomal crystallography: initiation, peptide bond formation, and amino acid polymerization are hampered by antibiotics. , 2004, Annual review of microbiology.

[18]  M. Disney,et al.  Recent advances in developing small molecules targeting RNA. , 2012, ACS chemical biology.

[19]  Steven J. Seedhouse,et al.  Defining the RNA internal loops preferred by benzimidazole derivatives via 2D combinatorial screening and computational analysis. , 2011, Journal of the American Chemical Society.

[20]  P. Dervan,et al.  Binding of a hairpin polyamide in the minor groove of DNA: sequence-specific enthalpic discrimination. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[21]  Anders Karlén,et al.  Corrigendum to “Virtual screening and bioassay study of novel inhibitors for dengue virus mRNA cap (nucleoside-2′O)-methyltransferase” [Bioorg. Med. Chem. 15 (2007) 7795–7802] , 2008 .

[22]  Stijn van Dongen,et al.  miRBase: tools for microRNA genomics , 2007, Nucleic Acids Res..

[23]  Batey,et al.  Tertiary Motifs in RNA Structure and Folding. , 1999, Angewandte Chemie.

[24]  J. Stenvang,et al.  Inhibition of microRNA function by antimiR oligonucleotides , 2012, Silence.

[25]  Matthew D Disney,et al.  Chemical correction of pre-mRNA splicing defects associated with sequestration of muscleblind-like 1 protein by expanded r(CAG)-containing transcripts. , 2012, ACS chemical biology.

[26]  Steven J. Seedhouse,et al.  Structure-activity relationships through sequencing (StARTS) defines optimal and suboptimal RNA motif targets for small molecules. , 2010, Angewandte Chemie.

[27]  D. Valle,et al.  MicroRNA (miRNA) Transcriptome of Mouse Retina and Identification of a Sensory Organ-specific miRNA Cluster* , 2007, Journal of Biological Chemistry.

[28]  B. Cullen,et al.  Sequence requirements for micro RNA processing and function in human cells. , 2003, RNA.

[29]  D. Turner,et al.  Solution structure of (rGCGGACGC)2 by two-dimensional NMR and the iterative relaxation matrix approach. , 1996, Biochemistry.

[30]  M. Famulok,et al.  An aptamer targeting the apical-loop domain modulates pri-miRNA processing. , 2010, Angewandte Chemie.

[31]  P. Hawkins,et al.  Comparison of shape-matching and docking as virtual screening tools. , 2007, Journal of medicinal chemistry.

[32]  D. Turner,et al.  Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[33]  Matthew D Disney,et al.  A small molecule microarray platform to select RNA internal loop-ligand interactions. , 2007, ACS chemical biology.

[34]  Dalia Cohen,et al.  Functional genomics to new drug targets , 2004, Nature Reviews Drug Discovery.

[35]  J. Stenvang,et al.  Silencing of microRNA families by seed-targeting tiny LNAs , 2011, Nature Genetics.

[36]  Harald J. Maier,et al.  FOXO1 is a tumor suppressor in classical Hodgkin lymphoma. , 2012, Blood.

[37]  G. Ruvkun,et al.  A uniform system for microRNA annotation. , 2003, RNA.

[38]  Gabriele Varani,et al.  Simultaneous recognition of HIV-1 TAR RNA bulge and loop sequences by cyclic peptide mimics of Tat protein , 2009, Proceedings of the National Academy of Sciences.

[39]  C. Semple,et al.  Posttranscriptional Regulation of miRNAs Harboring Conserved Terminal Loops , 2008, Molecular cell.

[40]  Byoung-Tak Zhang,et al.  Molecular Basis for the Recognition of Primary microRNAs by the Drosha-DGCR8 Complex , 2006, Cell.

[41]  N. Rajewsky,et al.  Silencing of microRNAs in vivo with ‘antagomirs’ , 2005, Nature.

[42]  Matthew D Disney,et al.  Two-dimensional combinatorial screening identifies specific aminoglycoside-RNA internal loop partners. , 2008, Journal of the American Chemical Society.

[43]  D. Bartel.  MicroRNAs Genomics, Biogenesis, Mechanism, and Function , 2004, Cell.

[44]  Yadong Wang,et al.  miR2Disease: a manually curated database for microRNA deregulation in human disease , 2008, Nucleic Acids Res..

[45]  A. Baranger,et al.  A simple ligand that selectively targets CUG trinucleotide repeats and inhibits MBNL protein binding , 2009, Proceedings of the National Academy of Sciences.

[46]  Christophe Guilbert,et al.  Discovery of ligands for a novel target, the human telomerase RNA, based on flexible-target virtual screening and NMR. , 2008, Journal of medicinal chemistry.

[47]  Carl R. Woese,et al.  4 Probing RNA Structure, Function, and History by Comparative Analysis , 1993 .

[48]  Colin Echeverría Aitken,et al.  Purification and characterization of transcribed RNAs using gel filtration chromatography , 2007, Nature Protocols.

[49]  I. Andricioaei,et al.  Discovery of selective bioactive small molecules by targeting an RNA dynamic ensemble. , 2011, Nature chemical biology.

[50]  Jessica L. Childs-Disney,et al.  Rationally designed small molecules targeting the RNA that causes myotonic dystrophy type 1 are potently bioactive. , 2012, ACS chemical biology.

[51]  D. Turner,et al.  Structure of (rGGCGAGCC)2 in solution from NMR and restrained molecular dynamics. , 1993, Biochemistry.

[52]  Ana Kozomara,et al.  miRBase: integrating microRNA annotation and deep-sequencing data , 2010, Nucleic Acids Res..

[53]  V. Luzhkov,et al.  Virtual screening and bioassay study of novel inhibitors for dengue virus mRNA cap (nucleoside-2'O)-methyltransferase. , 2007, Bioorganic & medicinal chemistry.

[54]  D. Boger,et al.  Mechanisms of in situ activation for DNA-targeting antitumor agents. , 2002, Chemical reviews.

[55]  B. White,et al.  Coordinate Regulation of FOXO 1 by miR-27 a , miR-96 , and miR-182 in Breast Cancer Cells , 2009 .

[56]  D. Turner,et al.  Solution structure of (rGGCAGGCC)2 by two-dimensional NMR and the iterative relaxation matrix approach. , 1996, Biochemistry.

[57]  Hakluyt's Voyages,et al.  Annotation , 1936, Glasgow Medical Journal.

[58]  Harald J. Maier,et al.  FOXO 1 is a tumor suppressor in classical Hodgkin lymphoma * , 2012 .

[59]  Souvik Maiti,et al.  The tuberculosis drug streptomycin as a potential cancer therapeutic: inhibition of miR-21 function by directly targeting its precursor. , 2012, Angewandte Chemie.

[60]  Matthew D Disney,et al.  A small molecule that targets r(CGG)(exp) and improves defects in fragile X-associated tremor ataxia syndrome. , 2012, ACS chemical biology.

[61]  R. Rando,et al.  Specific binding of aminoglycoside antibiotics to RNA. , 1995, Chemistry & biology.

[62]  D. Turner,et al.  Contributions of individual nucleotides to tertiary binding of substrate by a Pneumocystis carinii group I intron. , 2000, Biochemistry.

[63]  J Frank,et al.  Hepatitis C Virus IRES RNA-Induced Changes in the Conformation of the 40S Ribosomal Subunit , 2001, Science.