A Modular Approach to the Discovery and Affinity Maturation of Sequence-Selective RNA-Binding Compounds

  • Benjamin L. MillerEmail author
Part of the Topics in Medicinal Chemistry book series (TMC, volume 27)


This chapter describes a strategy developed at the University of Rochester that relies on a two-step process for the generation of high-affinity, sequence-selective RNA-binding compounds with target-relevant biological activity. First, a natural product-inspired dynamic combinatorial library (DCL) is employed to rapidly produce “hit” compounds able to bind the target RNA. Second, a process of analog synthesis is employed to enhance affinity, bioavailability, and sequence selectivity. This strategy has been used to successfully produce compounds able to bind target RNAs with high (nanomolar) affinity, and target-relevant activity in cellular assays and in vivo. In particular, approaches to RNA targets of critical importance in Myotonic Dystrophy (a triplet repeat RNA-mediated disease) and in the life cycle of HIV will be discussed.


Bioisosteres Dynamic combinatorial chemistry Frameshifting HIV Myotonic dystrophy Natural products 


  1. 1.
    Dervan PB, Burli RW (1999) Sequence-specific DNA recognition by polyamides. Curr Opin Chem Biol 3:688–693CrossRefGoogle Scholar
  2. 2.
    Thomas JR, Hergenrother PJ (2008) Targeting RNA with small molecules. Chem Rev 108:1171–1224CrossRefGoogle Scholar
  3. 3.
    Georgianna WE, Young DD (2011) Development and utilization of non-coding RNA-small molecule interactions. Org Biomol Chem 9:7969–7978CrossRefGoogle Scholar
  4. 4.
    Guan L, Disney MD (2012) Recent advances in developing small molecules targeting RNA. ACS Chem Biol 7:73–86CrossRefGoogle Scholar
  5. 5.
    Corbett PT, Leclaire J, Vial L, West KR, Wietor JL, Sanders JKM, Otto S (2006) Dynamic combinatorial chemistry. Chem Rev 106:3652–3711CrossRefGoogle Scholar
  6. 6.
    Ramström O, Bunyapaiboonsri T, Lohmann S, Lehn JM (2002) Chemical biology of dynamic combinatorial libraries. Biochim Biophys Acta 1572:178–186CrossRefGoogle Scholar
  7. 7.
    Miller BL (ed) (2009) Dynamic combinatorial chemistry. Wiley, New York, NYGoogle Scholar
  8. 8.
    Rideout D (1986) Self-assembling cytotoxins. Science 233:561–563CrossRefGoogle Scholar
  9. 9.
    Feldman KS, Bobo JS, Ensel SM, Lee YB, Weinreb PH (1990) Template-controlled oligomerization support studies. Template synthesis and functionalization. J Org Chem 55:474–481CrossRefGoogle Scholar
  10. 10.
    Miller BL, Bonner WA (1995) Enantioselective autocatalysis. III. Configurational and conformational studies on a 1,4-benzodiazepinooxazole derivative. Orig Life Evol Biosph 25:539–547CrossRefGoogle Scholar
  11. 11.
    Krämer R, Lehn JM, Marquis-Rigault A (1993) Self-recognition in helicate self-assembly: spontaneous formation of helical metal complexes from mixtures of ligands and metal ions. Proc Natl Acad Sci U S A 90:5394–5398CrossRefGoogle Scholar
  12. 12.
    Brady PA, Sanders JKM (1997) Thermodynamically-controlled cyclisation and interconversion of oligocholates: metal ion templated “living” macrolactonisation. J Chem Soc Perkin Trans 1 1997:3237–3253CrossRefGoogle Scholar
  13. 13.
    Hioki H, Still WC (1998) Chemical evolution: a model system that selects and amplifies a receptor for the tripeptide (D)Pro(L)Val(D)Val. J Org Chem 63:904–905CrossRefGoogle Scholar
  14. 14.
    Huc I, Lehn JM (1997) Virtual combinatorial libraries: dynamic generation of molecular and supramolecular diversity by self-assembly. Proc Natl Acad Sci U S A 94:2106–2110CrossRefGoogle Scholar
  15. 15.
    Klug A (2010) The discovery of zinc fingers and their applications in gene regulation and genome manipulation. Annu Rev Biochem 79:213–231CrossRefGoogle Scholar
  16. 16.
    Schepartz A, McDevitt JP (1989) Self-assembling ionophores. J Am Chem Soc 111:5976–5977CrossRefGoogle Scholar
  17. 17.
    Klekota B, Hammond MH, Miller BL (1997) Generation of novel DNA-binding compounds by selection and amplification from self-assembled combinatorial libraries. Tetrahedron Lett 38:8639–8643CrossRefGoogle Scholar
  18. 18.
    Klekota B, Miller BL (1999) Selection of DNA_binding compounds via multistage molecular evolution. Tetrahedron 55:11687–11697CrossRefGoogle Scholar
  19. 19.
    Karan C, Miller BL (2001) RNA-selective coordination complexes identified via dynamic combinatorial chemistry. J Am Chem Soc 123:7455–7456CrossRefGoogle Scholar
  20. 20.
    Ludlow RF, Otto S (2008) Two-vial, LC-MS identification of ephedrine receptors from a solution-phase dynamic combinatorial library of over 9000 components. J Am Chem Soc 130:12218–12219CrossRefGoogle Scholar
  21. 21.
    Bugaut A, Jantos K, Wietor JL, Rodriguez R, Sanders JKM, Balasubramanian S (2008) Exploring the differential recognition of DNA G-quadruplex targets by small molecules using dynamic combinatorial chemistry. Angew Chem Int Ed Engl 47:2677–2680CrossRefGoogle Scholar
  22. 22.
    Buhler E, Sreenivasachary N, Candau SJ, Lehn JM (2007) Modulation of the supramolecular structure of G-quartet assemblies by dynamic covalent decoration. J Am Chem Soc 129:10058–10059CrossRefGoogle Scholar
  23. 23.
    Harrington CA, Rosenow C, Retief J (2000) Monitoring gene expression using DNA microarrays. Curr Opin Microbiol 3:285–291CrossRefGoogle Scholar
  24. 24.
    Tan DS, Burbaum JJ (2000) Ligand discovery using encoded combinatorial libraries. Curr Opin Drug Discov Devel 3:439–453Google Scholar
  25. 25.
    Hattori K, Koike K, Okuda K, Hirayama T, Ebihara M, Takenaka M, Nagasawa H (2016) Solution-phase synthesis and biological evaluation of triostin A and its analogues. Org Biomol Chem 14:2090–2111CrossRefGoogle Scholar
  26. 26.
    Szajewski RP, Whitesides GM (1980) Rate constants and equilibrium-constants for thiol-disulfide interchange reactions involving oxidized glutathione. J Am Chem Soc 102:2011–2026CrossRefGoogle Scholar
  27. 27.
    McNaughton BR, Miller BL (2006) Resin-bound dynamic combinatorial chemistry. Org Lett 8:1803–1806CrossRefGoogle Scholar
  28. 28.
    Addess KJ, Sinsheimer JS, Feigon J (1993) Solution structure of a complex between [N-MeCys3, N-MeCys7]TANDEM and [d(GATATC)]2. Biochemistry 32:2498–2508CrossRefGoogle Scholar
  29. 29.
    Addess KJ, Feigon J (1994) Sequence specificity of quinoxaline antibiotics. 2. NMR studies of the binding f [N-MeCys3, N-MCys7]TANDEM and triostin A to DNA containing a CpI step. Biochemistry 33:12397–12404CrossRefGoogle Scholar
  30. 30.
    Mankodi A, Teng-Umnuay P, Krym M, Henderson D, Swanson M, Thornton CA (2003) Ribonuclear inclusions in skeletal muscle in myotonic dystrophy types 1 and 2. Ann Neurol 54:760–768CrossRefGoogle Scholar
  31. 31.
    Ashizawa T, Dubel JR, Harati Y (1993) Somatic instability of CTG repeat in myotonic dystrophy. Neurology 43:2674–2678CrossRefGoogle Scholar
  32. 32.
    Thornton CA, Johnson K, Moxley RT (1994) Myotonic dystrophy patients have larger CTG expansions in skeletal muscle than in leukocytes. Ann Neurol 35:104–107CrossRefGoogle Scholar
  33. 33.
    Pushechnikov A, Lee MM, Childs-Disney JL, Sobczak K, French JM, Thornton CA, Disney MD (2009) Rational design of ligands targeting triplet repeating transcripts that cause RNA dominant disease: application to myotonic muscular dystrophy type 1 and spinocerebellar ataxia type 3. J Am Chem Soc 131:9767–9779CrossRefGoogle Scholar
  34. 34.
    Arambula JF, Ramisetty SR, Baranger AM, Zimmerman SC (2009) A simple ligand that selectively targets CUG trinucleotide repeats and inhibits MBNL protein binding. Proc Natl Acad Sci U S A 106:16068–16073CrossRefGoogle Scholar
  35. 35.
    Warf MB, Nakamori M, Matthys CM, Thornton CA, Berglund JA (2009) Pentamidine reverses the splicing defects associated with myotonic dystrophy. Proc Natl Acad Sci U S A 106:18551–118556CrossRefGoogle Scholar
  36. 36.
    Liquori CL, Ricker K, Moseley ML, Jacobsen JF, Kress W, Naylor SL, Day JW, Ranum LPW (2001) Myotonic dystrophy type 2 caused by a CCTG expansion in intron 1 of ZNF9. Science 293:864–867CrossRefGoogle Scholar
  37. 37.
    Tian B, White RJ, Xia T, Welle S, Turner DH, Mathews MB, Thornton CA (2000) Expanded CUG repeat RNAs form hairpins that activate the double-stranded RNA-dependent protein kinase PKR. RNA 6:79–87CrossRefGoogle Scholar
  38. 38.
    Belew AT, Dinman JD (2015) Cell cycle control (and more) by programmed -1 ribosomal frameshifting: implications for disease and therapeutics. Cell Cycle 14:172–178CrossRefGoogle Scholar
  39. 39.
    Atkins JF, Loughran G, Bhatt PR, Firth AE, Baranov PV (2016) Ribosomal frameshifting and transcriptional slippage: from genetic steganography and cryptography to adventitious use. Nucleic Acids Res 44:7007–7078Google Scholar
  40. 40.
    Biswas P, Jiang X, Pacchia AL, Dougherty JP, Peltz SW (2004) The human immunodeficiency virus type 1 ribosomal frameshifting site is an invariant sequence determinant and an important target for antiviral therapy. J Virol 78:2082–2087CrossRefGoogle Scholar
  41. 41.
    Staple DW, Butcher SE (2005) Solution structure and thermodynamic investigation of the HIV-1 frameshift inducing element. J Mol Biol 349:1011–1023CrossRefGoogle Scholar
  42. 42.
    Low JT, Garcia-Miranda P, Mouzakis KD, Gorelick RJ, Butcher SE, Weeks KM (2014) Structure and dynamics of the HIV-1 frameshift element RNA. Biochemistry 53:4282–4291CrossRefGoogle Scholar
  43. 43.
    Shehu-Xhilaga M, Crowe SM, Mak J (2001) Maintenance of the Gag/Gag-Pol ratio is important for human immunodeficiency virus type 1 RNA dimerization and viral infectivity. J Virol 75:1834–1841CrossRefGoogle Scholar
  44. 44.
    Dulude D, Berchiche YA, Gendron K, Brakier-Gingras L, Heveker N (2006) Decreasing the frameshift efficiency translates into an equivalent reduction of the replication of the human immunodeficiency virus type 1. Vaccine 345:127–136Google Scholar
  45. 45.
    Hung M, Patel P, Davis S, Green SR (1998) Importance of ribosomal frameshifting for human immunodeficiency virus type 1 particle assembly and replication. J Virol 72:4819–4824Google Scholar
  46. 46.
    Marcheschi RJ, Tonelli M, Kumar A, Butcher SE (2011) Structure of the HIV-1 frameshift site RNA bound to a small molecule inhibitor of viral replication. ACS Chem Biol 6:857–864CrossRefGoogle Scholar
  47. 47.
    Tan DS, Foley MA, Stockwell BR, Shair MD, Schreiber SL (1999) Synthesis and preliminary evaluation of a library of polycyclic small molecules for use in chemical genetic assays. J Am Chem Soc 121:9073–9087CrossRefGoogle Scholar
  48. 48.
    McNaughton BR, Gareiss PC, Miller BL (2007) Identification of a selective small-molecule frameshift-inducing stem-loop RNA from an 11,325 member resin bound dynamic combinatorial library. J Am Chem Soc 129:11306–11307CrossRefGoogle Scholar
  49. 49.
    Gareiss PC, Sobczak K, McNaughton BR, Palde PB, Thornton CA, Miller BL (2008) Dynamic combinatorial selection of molecules capable of inhibiting the (CUG) repeat RNA-MBNL1 interaction in vitro: discovery of lead compounds targeting myotonic dystrophy (DM1). J Am Chem Soc 130:16254–16261CrossRefGoogle Scholar
  50. 50.
    Olson KR, Eglen RM (2007) Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol 5:137–144CrossRefGoogle Scholar
  51. 51.
    McGovern SL, Helfand BT, Feng B, Shoichet BK (2003) A specific mechanism of nonspecific inhibition. J Med Chem 46:4265–4272CrossRefGoogle Scholar
  52. 52.
    Feng BY, Toyama BH, Wille H, Colby DW, Collins SR, May BC, Prusiner SB, Weissman J, Shoichet BK (2008) Small-molecule aggregates inhibit amyloid polymerization. Nat Chem Biol 4:197–199CrossRefGoogle Scholar
  53. 53.
    Schmidt MW, Baldridge KK, Boatz JA, Elbert ST, Gordon MS, Jensen JH, Koseki S, Matsunaga N, Nguyen KA, Su SJ, Windus TL, Dupuis M, Montgomery JA (1993) General atomic and molecular electronic structure system. J Comput Chem 14:1347–1363CrossRefGoogle Scholar
  54. 54.
    Fotouhi N, Joshi P, Tilley JW, Rowan K, Schwinge V, Wolitzky B (2000) Cyclic thioether peptide mimetics as VCAM-VLA-4 antagonists. Bioorg Med Chem Lett 10:1167–1169CrossRefGoogle Scholar
  55. 55.
    Stymiest JL, Mitchell BF, Wong S, Vederas JC (2003) Synthesis of biologically active dicarba analogues of the peptide hormone oxytocin using ring-closing metathesis. Org Lett 5:47–49CrossRefGoogle Scholar
  56. 56.
    Berezowska I, Chung NN, Lemieux C, Wilkes BC, Schiller PW (2007) Dicarba analogues of the cyclic enkephalin peptides H-Tyr-c[D-Cys-Gly-Phe-D(or L)-Cys]NH(2) retain high opioid activity. J Med Chem 50:1414–1417CrossRefGoogle Scholar
  57. 57.
    Mollica A, Guardiani G, Davis P, Ma S, Porreca F, Lai J, Mannina L, Sobolev AP, Hruby VJ (2007) Synthesis of stable and potent delta/mu opioid peptides: analogues of H-Tyr-c[D-Cys-Gly-Phe-D-Cys]-OH by ring-closing metathesis. J Med Chem 50:3138–3142CrossRefGoogle Scholar
  58. 58.
    Nicolaou KC, Hughes R, Cho SY, Winssinger N, Smethurst C, Labischinski H, Endermann R (2000) Target-accelerated combinatorial synthesis and discovery of highly potent antibiotics effective against vancomycin-resistant bacteria. Angew Chem Int Ed Engl 39:3823–3828CrossRefGoogle Scholar
  59. 59.
    Neto BAD, Lapis AAM (2009) Recent developments in the chemistry of deoxyribonucleic acid (DNA) intercalators: principles, design, synthesis, applications and trends. Molecules 14:1725–1746CrossRefGoogle Scholar
  60. 60.
    Chatterjee J, Gilon C, Hoffman A, Kessler H (2008) N-methylation of peptides: a new perspective in medicinal chemistry. Acc Chem Res 41:1331–1342CrossRefGoogle Scholar
  61. 61.
    Luedtke N, Tor Y (2000) A novel solid-phase assembly for identifying potent and selective RNA ligands. Angew Chem Int Ed Engl 39:1788–1790CrossRefGoogle Scholar
  62. 62.
    Ofori LO, Hilimire TA, Bennett RP, Brown NW, Smith HC, Miller BL (2014) High-affinity recognition of HIV-1 frameshift-stimulating RNA alters frameshifting in vitro and interferes with HIV-1 infectivity. J Med Chem 57:723–732CrossRefGoogle Scholar
  63. 63.
    Buttke TMT, McCubrey JAJ, Owen TCT (1993) Use of an aqueous soluble tetrazolium/formazan assay to measure viability and proliferation of lymphokine-dependent cell lines. J Immunol Methods 157:233–240CrossRefGoogle Scholar
  64. 64.
    Grentzmann G, Ingram JA, Kelly PJ, Gesteland RF, Atkins JF (1998) A dual-luciferase reporter system for studying recoding signals. RNA 4:479–486CrossRefGoogle Scholar
  65. 65.
    Miller JH, Presnyak V, Smith HC (2007) The dimerization domain of HIV-1 viral infectivity factor Vif is required to block virion incorporation of APOBEC3G. Retrovirology 4:81CrossRefGoogle Scholar
  66. 66.
    Finnegan CM, Rawat SS, Puri A, Wang JM, Ruscetti FW, Blumenthal R (2004) Ceramide, a target for antiretroviral therapy. Proc Natl Acad Sci U S A 101:15452–15457CrossRefGoogle Scholar
  67. 67.
    Hilimire TA, Bennett RP, Stewart RA, Garcia-Miranda P, Blume A, Becker J, Sherer N, Helms ED, Butcher SE, Smith HC, Miller BL (2016) N-methylation as a strategy for enhancing the affinity and selectivity of RNA-binding peptides: application to the HIV-1 frameshift-stimulating RNA. ACS Chem Biol 11:88–94CrossRefGoogle Scholar
  68. 68.
    Miller SC, Scanlan TS (1997) Site-selective N-methylation of peptides on solid support. J Am Chem Soc 119:2301–2302CrossRefGoogle Scholar
  69. 69.
    Ofori LO, Hoskins J, Nakamori M, Thornton CA, Miller BL (2012) 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 40:6380–6390CrossRefGoogle Scholar
  70. 70.
    Crothers DM (1968) Calculation of binding isotherms for heterogeneous polymers. Biopolymers 6:575–584CrossRefGoogle Scholar
  71. 71.
    Mankodi A, Logigian E, Callahan L, McClain C, White R, Henderson D, Krym M, Thornton CA (2000) Myotonic dystrophy in transgenic mice expressing an expanded CUG repeat. Science 298:1769–1773CrossRefGoogle Scholar
  72. 72.
    Rodriguez-Docampo Z, Otto S (2008) Orthogonal or simultaneous use of disulfide and hydrazone exchange in dynamic covalent chemistry in aqueous solution. Chem Commun 2008:5301–5303CrossRefGoogle Scholar
  73. 73.
    Escalante AM, Orrillo AG, Furlan RLE (2010) Simultaneous and orthogonal covalent exchange processes in dynamic combinatorial libraries. J Comb Chem 12:410–413CrossRefGoogle Scholar
  74. 74.
    Gromova AV, Ciszewski JM, Miller BL (2012) Ternary resin-bound dynamic combinatorial chemistry. Chem Commun 2012:2131–2133CrossRefGoogle Scholar
  75. 75.
    Dirksen A, Dirksen S, Hackeng TM, Dawson PE (2006) Nucleophilic catalysis of hydrazone formation and transimination: implications for dynamic covalent chemistry. J Am Chem Soc 128:15602–15603CrossRefGoogle Scholar
  76. 76.
    Bhat VT, Caniard AM, Luksch T, Brenk R, Campopiano DJ, Greaney MF (2010) Nucleophilic catalysis of acylhydrazone equilibration for protein-directed dynamic covalent chemistry. Nat Chem 2:490–497CrossRefGoogle Scholar
  77. 77.
    Larsen D, Pittelkow M, Karmakar S, Kool ET (2015) New organocatalyst scaffolds with high activity in promoting hydrazone and oxime formation at neutral pH. Org Lett 17:274–277CrossRefGoogle Scholar
  78. 78.
    McAnany JD, Reichert JP, Miller BL (2016) Probing the geometric constraints of RNA binding via dynamic covalent chemistry. Bioorg Med Chem 24:3940–3946CrossRefGoogle Scholar
  79. 79.
    Corbett PT, Tong LH, Sanders JKM, Otto S (2005) Diastereoselective amplification of an induced-fit receptor from a dynamic combinatorial library. J Am Chem Soc 127:8902–8903CrossRefGoogle Scholar

Copyright information

© Springer International Publishing AG 2017

Authors and Affiliations

  1. 1.Department of DermatologyUniversity of Rochester Medical CenterRochesterUSA

Personalised recommendations