Riboswitches: discovery of drugs that target bacterial gene-regulatory RNAs.

Riboswitches are messenger RNA (mRNA) domains that regulate gene function in response to the intracellular concentration of a variety of metabolites and second messengers. They control essential genes in many pathogenic bacteria, thus representing an inviting new class of biomolecular target for the development of antibiotics and chemical-biological tools. In this Account, we briefly review the discovery of riboswitches in the first years of the 21st century and their ensuing characterization over the past decade. We then discuss the progress achieved so far in using riboswitches as a focus for drug discovery, considering both the value of past serendipity and the particular challenges that confront current researchers. Five mechanisms of gene regulation have been determined for riboswitches. Most bacterial riboswitches modulate either transcription termination or translation initiation in response to ligand binding. All known examples of eukaryotic riboswitches, and some bacterial riboswitches, control gene expression by alternative splicing. The glmS riboswitch, which is widespread in Gram-positive bacteria, is a catalytic RNA activated by ligand binding: its self-cleavage destabilizes the mRNA of which it is part. Finally, one example of a trans-acting riboswitch is known. Three-dimensional structures have been determined for representatives of 13 structurally distinct riboswitch classes, providing atomic-level insight into their mechanisms of ligand recognition. While cellular and viral RNAs have attracted widespread interest as potential drug targets, riboswitches show special promise due to the diversity of small-molecule recognition strategies that are on display in their ligand-binding pockets. Moreover, riboswitches have evolved to recognize small-molecule ligands, which is unique among known structured RNA domains. Structural and biochemical advances in the study of riboswitches provide an impetus for the development of methods for the discovery of novel riboswitch activators and inhibitors. Recent rational drug design efforts focused on select riboswitch classes have yielded a small number of candidate antibiotic compounds, including one active in a mouse model of Staphylococcus aureus infection. The development of high-throughput methods suitable for riboswitch-specific drug discovery is ongoing. A fragment-based screening approach employing equilibrium dialysis that may be generically useful has demonstrated early success. Riboswitch-mediated gene regulation is widely employed by bacteria; however, only the thiamine pyrophosphate-responsive riboswitch has thus far been found in eukaryotes. Thus, riboswitches are particularly attractive as targets for antibacterials. Indeed, antimicrobials with previously unknown mechanisms have been found to function by binding riboswitches and causing aberrant gene expression.

[1]  A. Ferré-D’Amaré,et al.  Structural Basis of glmS Ribozyme Activation by Glucosamine-6-Phosphate , 2006, Science.

[2]  H. Naganawa,et al.  THE ABSOLUTE STRUCTURE OF ORYZOXYMYCIN , 1974 .

[3]  E. Westhof,et al.  Binding of tobramycin leads to conformational changes in yeast tRNAAsp and inhibition of aminoacylation , 2002, The EMBO journal.

[4]  R. Batey,et al.  Crystal Structure of the Lysine Riboswitch Regulatory mRNA Element* , 2008, Journal of Biological Chemistry.

[5]  J. Sutcliffe Improving on nature: antibiotics that target the ribosome. , 2005, Current opinion in microbiology.

[6]  R. Batey,et al.  Structure of the SAM-II riboswitch bound to S-adenosylmethionine , 2008, Nature Structural &Molecular Biology.

[7]  Mijeong Kang,et al.  Structural Insights into riboswitch control of the biosynthesis of queuosine, a modified nucleotide found in the anticodon of tRNA. , 2009, Molecular cell.

[8]  Y. Lu,et al.  Identification of aecA mutations in Bacillus subtilis as nucleotide substitutions in the untranslated leader region of the aspartokinase II operon. , 1991, Journal of general microbiology.

[9]  T. Henkin,et al.  Mechanisms of resistance to an amino acid antibiotic that targets translation. , 2007, ACS chemical biology.

[10]  A. Ferré-D’Amaré,et al.  Recognition of the bacterial second messenger cyclic diguanylate by its cognate riboswitch , 2009, Nature Structural &Molecular Biology.

[11]  Harald Schwalbe,et al.  Metal-ion binding and metal-ion induced folding of the adenine-sensing riboswitch aptamer domain , 2007, Nucleic acids research.

[12]  R. Montange,et al.  Structure of the S-adenosylmethionine riboswitch regulatory mRNA element , 2006, Nature.

[13]  T. Begley,et al.  Thi20, a remarkable enzyme from Saccharomyces cerevisiae with dual thiamin biosynthetic and degradation activities. , 2005, Bioorganic chemistry.

[14]  R. Schroeder,et al.  Inhibition of the self-cleavage reaction of the human hepatitis delta virus ribozyme by antibiotics. , 1996, Journal of molecular biology.

[15]  Catherine A. Wakeman,et al.  Structure and Mechanism of a Metal-Sensing Regulatory RNA , 2007, Cell.

[16]  Jeffrey E. Barrick,et al.  The distributions, mechanisms, and structures of metabolite-binding riboswitches , 2007, Genome Biology.

[17]  M. Hatzoglou,et al.  A stress-responsive RNA switch regulates VEGF expression , 2008, Nature.

[18]  C. Abell,et al.  A fragment-based approach to identifying ligands for riboswitches. , 2010, ACS chemical biology.

[19]  O. Uhlenbeck,et al.  Inhibition of the hammerhead ribozyme by neomycin. , 1969, RNA.

[20]  Paul J Hergenrother,et al.  Targeting RNA with small molecules. , 2008, Chemical reviews.

[21]  T. Hermann,et al.  RNA as a target for small-molecule therapeutics , 2005 .

[22]  J. C. Koedam The mode of action of pyrithiamine as an inductor of thiamine deficiency. , 1958, Biochimica et biophysica acta.

[23]  Andrew R. Leach,et al.  Molecular Complexity and Its Impact on the Probability of Finding Leads for Drug Discovery , 2001, J. Chem. Inf. Comput. Sci..

[24]  N. Ban,et al.  Structural basis of thiamine pyrophosphate analogues binding to the eukaryotic riboswitch. , 2008, Journal of the American Chemical Society.

[25]  C. Buchrieser,et al.  A trans-Acting Riboswitch Controls Expression of the Virulence Regulator PrfA in Listeria monocytogenes , 2009, Cell.

[26]  R. Batey,et al.  A structural basis for the recognition of 2'-deoxyguanosine by the purine riboswitch. , 2009, Journal of molecular biology.

[27]  R. Breaker,et al.  An mRNA structure that controls gene expression by binding FMN , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[28]  R. Nielsen A conserved RNA structure element involved in the regulation of bacterial riboflavin synthesis genes , 2022 .

[29]  S. Strobel,et al.  Structural investigation of the GlmS ribozyme bound to Its catalytic cofactor. , 2007, Chemistry & biology.

[30]  Ian R Kleckner,et al.  Tuning riboswitch regulation through conformational selection. , 2011, Journal of molecular biology.

[31]  H. Schwalbe,et al.  Phosphate‐Group Recognition by the Aptamer Domain of the Thiamine Pyrophosphate Sensing Riboswitch , 2006, Chembiochem : a European journal of chemical biology.

[32]  A. Ferré-D’Amaré,et al.  Crystal structures of the thi-box riboswitch bound to thiamine pyrophosphate analogs reveal adaptive RNA-small molecule recognition. , 2006, Structure.

[33]  R. Breaker,et al.  Riboswitches as antibacterial drug targets , 2006, Nature Biotechnology.

[34]  R. Breaker,et al.  Design and antimicrobial action of purine analogues that bind Guanine riboswitches. , 2009, ACS chemical biology.

[35]  M. Famulok,et al.  High‐Throughput‐Compatible Assay for glmS Riboswitch Metabolite Dependence , 2006, Chembiochem : a European journal of chemical biology.

[36]  Sebastian Doniach,et al.  Structural transitions and thermodynamics of a glycine-dependent riboswitch from Vibrio cholerae. , 2007, Journal of molecular biology.

[37]  R. Coccia,et al.  Thialysine utilization for protein synthesis by CHO cells. , 1986, Physiological chemistry and physics and medical NMR.

[38]  R. Miura,et al.  Letter: Roseoflavin, a new antimicrobial pigment from Streptomyces. , 1974, The Journal of antibiotics.

[39]  D. Herschlag,et al.  The ligand-free state of the TPP riboswitch: a partially folded RNA structure. , 2010, Journal of molecular biology.

[40]  J. Burke The Hairpin Ribozyme , 1994 .

[41]  A. Serganov,et al.  Structural basis for discriminative regulation of gene expression by adenine- and guanine-sensing mRNAs. , 2004, Chemistry & biology.

[42]  A. Serganov,et al.  Structural basis for gene regulation by a thiamine pyrophosphate-sensing riboswitch , 2006, Nature.

[43]  A. Iwashima,et al.  Formation of pyrithiamine pyrophosphate in brain tissue. , 1976, Journal of biochemistry.

[44]  A. Serganov,et al.  Structural insights into ligand recognition by a sensing domain of the cooperative glycine riboswitch. , 2010, Molecular cell.

[45]  D. Lafontaine,et al.  Novel Riboswitch Ligand Analogs as Selective Inhibitors of Guanine-Related Metabolic Pathways , 2010, PLoS pathogens.

[46]  C. Abell,et al.  Fragment screening against the thiamine pyrophosphate riboswitchthiM , 2011 .

[47]  Hiroshi Murakami,et al.  Structural basis of specific tRNA aminoacylation by a small in vitro selected ribozyme , 2008, Nature.

[48]  R. Montange,et al.  Structure of a natural guanine-responsive riboswitch complexed with the metabolite hypoxanthine , 2004, Nature.

[49]  A. Ferré-D’Amaré RNA-modifying enzymes. , 2003, Current opinion in structural biology.

[50]  R. Micura,et al.  Folding of a transcriptionally acting PreQ1 riboswitch , 2010, Proceedings of the National Academy of Sciences.

[51]  R. Anupam,et al.  Structure-activity studies of oxazolidinone analogs as RNA-binding agents. , 2006, Bioorganic & medicinal chemistry letters.

[52]  K. Matsui,et al.  ROSEOFLAVIN, A NEW ANTIMICROBIAL PIGMENT FROM STREPTOMYCES , 1974 .

[53]  Kathryn D. Smith,et al.  Structural basis of ligand binding by a c-di-GMP riboswitch , 2009, Nature Structural &Molecular Biology.

[54]  J. Davies,et al.  Antibiotic inhibition of group I ribozyme function , 1991, Nature.

[55]  W. J. Robbins The Pyridine Analog of Thiamin and the Growth of Fungi. , 1941, Proceedings of the National Academy of Sciences of the United States of America.

[56]  J. Micklefield,et al.  Reengineering orthogonally selective riboswitches , 2010, Proceedings of the National Academy of Sciences.

[57]  A. Ferré-D’Amaré,et al.  Essential role of an active-site guanine in glmS ribozyme catalysis. , 2007, Journal of the American Chemical Society.

[58]  Andrea L Edwards,et al.  Structural basis for recognition of S-adenosylhomocysteine by riboswitches. , 2010, RNA.

[59]  T. Henkin,et al.  4,5-Disubstituted oxazolidinones: High affinity molecular effectors of RNA function. , 2008, Bioorganic & medicinal chemistry letters.

[60]  M. Mack,et al.  The RFN riboswitch of Bacillus subtilis is a target for the antibiotic roseoflavin produced by Streptomyces davawensis , 2009, RNA biology.

[61]  A. Serganov,et al.  Coenzyme recognition and gene regulation by a flavin mononucleotide riboswitch , 2009, Nature.

[62]  A. Ferré-D’Amaré,et al.  Structural basis for specific, high-affinity tetracycline binding by an in vitro evolved aptamer and artificial riboswitch. , 2008, Chemistry & biology.

[63]  R. Breaker,et al.  Development and Application of a High-Throughput Assay for glmS Riboswitch Activators , 2006, RNA biology.

[64]  A. Ferré-D’Amaré,et al.  Cocrystal structure of a class I preQ1 riboswitch reveals a pseudoknot recognizing an essential hypermodified nucleobase , 2009, Nature Structural &Molecular Biology.

[65]  R. Breaker,et al.  Antibacterial lysine analogs that target lysine riboswitches. , 2007, Nature chemical biology.

[66]  Sarah R. Kirk,et al.  Neomycin−Acridine Conjugate: A Potent Inhibitor of Rev-RRE Binding , 2000 .

[67]  V. Méjean,et al.  The leader sequence of the Escherichia coli lysC gene is involved in the regulation of LysC synthesis. , 1998, FEMS Microbiology Letters.

[68]  A. Ferré-D’Amaré,et al.  The glmS ribozyme: use of a small molecule coenzyme by a gene-regulatory RNA , 2010, Quarterly Reviews of Biophysics.

[69]  H. Sticht,et al.  Structural Rearrangements of HIV-1 Tat-responsive RNA upon Binding of Neomycin B* , 2000, The Journal of Biological Chemistry.

[70]  P. Seeburg,et al.  Structural mechanism for STI-571 inhibition of abelson tyrosine kinase. , 2000, Science.

[71]  Margaret S. Ebert,et al.  An mRNA structure in bacteria that controls gene expression by binding lysine. , 2003, Genes & development.

[72]  R. Breaker,et al.  Thiamine pyrophosphate riboswitches are targets for the antimicrobial compound pyrithiamine. , 2005, Chemistry & biology.

[73]  A. Ferré-D’Amaré,et al.  Idiosyncratically tuned switching behavior of riboswitch aptamer domains revealed by comparative small-angle X-ray scattering analysis. , 2010, RNA.

[74]  A. Virtanen,et al.  Aminoglycoside binding displaces a divalent metal ion in a tRNA–neomycin B complex , 2001, Nature Structural Biology.

[75]  A. Ferré-D’Amaré,et al.  Crystal structure of pseudouridine synthase RluA: indirect sequence readout through protein-induced RNA structure. , 2006, Molecular cell.

[76]  T. Henkin,et al.  Crystal structures of the SAM-III/SMK riboswitch reveal the SAM-dependent translation inhibition mechanism , 2008, Nature Structural &Molecular Biology.

[77]  R. Montange,et al.  Free state conformational sampling of the SAM-I riboswitch aptamer domain. , 2010, Structure.

[78]  N. Ban,et al.  Structure of the Eukaryotic Thiamine Pyrophosphate Riboswitch with Its Regulatory Ligand , 2006, Science.

[79]  J. Hugenholtz,et al.  Riboflavin Production in Lactococcus lactis: Potential for In Situ Production of Vitamin-Enriched Foods , 2004, Applied and Environmental Microbiology.

[80]  G. F. Joyce,et al.  Forty years of in vitro evolution. , 2007, Angewandte Chemie.

[81]  A. Ferré-D’Amaré,et al.  Ribozymes and riboswitches: modulation of RNA function by small molecules , 2009, Biochemistry.

[82]  A. Ferré-D’Amaré,et al.  Requirement of helix P2.2 and nucleotide G1 for positioning the cleavage site and cofactor of the glmS ribozyme. , 2007, Journal of molecular biology.

[83]  M. Brännvall,et al.  Inhibition of RNase P RNA cleavage by aminoglycosides. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[84]  Vahe Bandarian,et al.  The Structural Basis for Recognition of the PreQ0 Metabolite by an Unusually Small Riboswitch Aptamer Domain*♦ , 2009, Journal of Biological Chemistry.

[85]  B. Suess,et al.  Engineered riboswitches: Overview, problems and trends , 2008, RNA biology.

[86]  A. Serganov,et al.  Structural insights into amino acid binding and gene control by a lysine riboswitch , 2008, Nature.

[87]  C. Yanofsky,et al.  Biochemical Features and Functional Implications of the RNA-Based T-Box Regulatory Mechanism , 2009, Microbiology and Molecular Biology Reviews.

[88]  Ronald R. Breaker,et al.  Roseoflavin is a natural antibacterial compound that binds to FMN riboswitches and regulates gene expression , 2009, RNA biology.