Riboswitches are structured domains within the non-coding portions of some mRNAs, where they serve as metabolite-sensing genetic switches. Metabolite binding causes allosteric changes in the mRNA that bring about changes in gene-expression processes such as transcription termination and translation initiation.
Riboswitches comprise two domains: an aptamer and an expression platform. The aptamer is highly conserved even in distantly related organisms, and serves as a precise sensor for its target metabolite. The expression platform is far more variable in sequence and in structure as it can function by assuming one of many structural forms to control gene expression.
Experimental data that are now known to correspond to riboswitch function date back at least 30 years. Recent studies have confirmed that a variety of gene-control 'mysteries' described in the literature over the past decades can be explained by the presence of seven distinct classes of riboswitches.
The aptamer domains of riboswitches exhibit surprising selectivity and specificity that compares favourably with protein receptors. These findings, along with the possibility that modern riboswitches might be evolutionary hold outs of an ancient form of gene-control system, indicate that the performance characteristics of riboswitches are competitive with those that are exhibited by proteins.
The mechanisms of gene control by bacterial riboswitches are largely based on transcription termination and translation initiation. However, the discovery of a riboswitch that has ribozyme function, and evidence which indicates that eukaryotes might use riboswitches for splicing control, hint at the potential for far greater diversity for riboswitch function in ancient and modern organisms.
New studies indicate that bacteria express numerous new RNA motifs and small non-coding RNAs. These findings suggest that more riboswitches will be identified, and so riboswitches seem to be a significant form of genetic control in bacteria.
Riboswitches are complex folded RNA domains that serve as receptors for specific metabolites. These domains are found in the non-coding portions of various mRNAs, where they control gene expression by harnessing allosteric structural changes that are brought about by metabolite binding. New findings indicate that riboswitches are robust genetic elements that are involved in regulating fundamental metabolic processes in many organisms.
Subscribe to Journal
Get full journal access for 1 year
only $22.08 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Ptashne, M. & Gann, A. Genes and Signals (Cold Spring Harbor Laboratory Press, New York, 2002).
Hannon, G. J. RNA interference. Nature 418, 244–251 (2002).
Dykxhoorn, D. M., Novina, C. D. & Sharp, P. A. Killing the messenger: short RNAs that silence gene expression. Nature Rev. Mol. Cell Biol. 4, 457–467 (2003).
McManus, M. T. & Sharp, P. A. Gene silencing in mammals by small interfering RNAs. Nature Rev. Genetics 3, 737–747 (2002).
Carrington, J. C. & Ambros, V. Role of microRNAs in plant and animal development. Science 301, 336–338 (2003).
Nahvi, A. et al. Genetic control by a metabolite binding mRNA. Chem. Biol. 9, 1043–1049 (2002). The first demonstrations that mRNAs bind metabolites directly in the absence of proteins are described in this paper and in reference 7.
Winkler, W., Nahvi, A. & Breaker, R. R. Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature 419, 952–956 (2002). This paper and references 32, 57 and 58 report evidence for the existence of two main forms of riboswitch gene control: transcription termination and translation initiation.
Lai, E. C. RNA sensors and riboswitches: self-regulating messages. Curr. Biol. 13, R285–R291 (2003).
Müller, S. Another face of RNA: metabolite-induced 'riboswitching' for regulation of gene expression. Chembiochem 4, 817–819 (2003).
Winkler, W. C. & Breaker, R. R. Genetic control by metabolite-binding riboswitches. Chembiochem 4, 1024–1032 (2003).
Nudler, E. & Mironov, A. S. The riboswitch control of bacterial metabolism. Trends Biochem. Sci. 29, 11–17 (2004).
Vitreschak, A. G., Rodionov, D. A., Mironov, A. A. & Gelfand, M. S. Riboswitches: the oldest mechanism for the regulation of gene expression? Trends Genet. 20, 44–50 (2004).
Monod, J. & Jacob, F. General conclusions: teleonic mechanisms in cellular metabolism, growth, and differentiation. Cold Spring Harbor Symp. Quant. Biol. 26, 389–401 (1961).
Monod, J., Changeux, J. -P. & Jacob, F. Allosteric proteins and cellular control systems. J. Mol. Biol. 6, 306–329 (1963).
Kuganov, B. I. Allosteric enzymes. (John Wiley & Sons Ltd., New York, 1978).
Jacob, F. & Monod, J. Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol. 3, 318–356 (1961).
Gilbert, W. The RNA world. Nature 319, 618 (1986).
Joyce, G. F. The antiquity of RNA-based evolution. Nature 418, 214–221 (2002).
Kruger, K., Grabowski, P. J., Zaug, A. J., Sands, J., Gottschling, D. E. & Cech, T. R. Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena. Cell 31, 147–157 (1982).
Guerrier-Takada, C., Gardiner, K., Marsh, T., Pace, N. & Altman, S. The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell 35, 849–857 (1983).
Gold, L., Polisky, B., Uhlenbeck, O. & Yarus, M. Diversity of oligonucleotide functions. Annu. Rev. Biochem. 64, 763–797 (1995).
Osborne, S. E. & Ellington, A. D. Nucleic acid selection and the challenge of combinatorial chemistry. Chem. Rev. 97, 349–370 (1997).
Hermann, T. & Patel, D. J. Adaptive recognition by nucleic acid aptamers. Science 287, 820–825 (2000).
Tang, J. & Breaker, R. R. Rational design of allosteric ribozymes. Chem. Biol. 4, 453–459 (1997). First demonstration that engineered RNAs can function as allosteric molecular switches and respond to small metabolites.
Soukup, G. A. & Breaker, R. R. Engineering precision RNA molecular switches. Proc. Natl Acad. Sci. USA 96, 3584–3589 (1999).
Seetharaman, S., Zivarts, M., Sudarsan, N. & Breaker, R. R. Immobilized RNA switches for the analysis of complex chemical and biological mixtures. Nature Biotechnol. 19, 336–341 (2001).
Breaker, R. R. Engineered allosteric ribozymes as biosensor components. Curr. Opin. Biotechnol. 13, 31–39 (2002).
Silverman, S. K. Rube Goldberg goes (ribo)nuclear? Molecular switches and sensors made from RNA. RNA 9, 377–383 (2003).
Gold, L., Brown, D., He, Y. -Y., Shtatland, T., Singer, B. S. & Wu, Y. From oligonucleotide shapes to genomic SELEX: novel biological regulatory loops. Proc. Natl Acad. Sci. USA 94, 59–64 (1997). Although unpublished speculation that riboswitches might exist had been ongoing for several years, this is an early publication that briefly mentions the possibility.
Gold, L., Singer, B., He, Y. -Y. & Brody, E. SELEX and the evolution of genomes. Curr. Opin. Genet. Dev. 7, 848–851 (1997).
Gelfand, M. S., Mironov, A. A., Jomantas, J., Kozlov, Y. I. & Perumov, D. A. A conserved RNA structure element involved in the regulation of bacterial riboflavin synthesis genes. Trends Genet. 15, 439–442 (1999). This publication, as well as reference 49, used sequence comparisons to make the first secondary-structure models for genetic-control elements that have since proven to be riboswitches.
Nou, X. & Kadner, R. J. Adenosylcobalamin inhibits ribosome binding to btuB RNA. Proc. Natl Acad. Sci. USA 97, 7190–7195 (2000).
Stormo, G. D. & Ji, Y. Do mRNAs act as direct sensors of small molecules to control their expression? Proc. Natl Acad. Sci. USA 98, 9465–9467 (2001).
Miranda-Rios, J., Navarro, M. & Soberón, M. A conserved RNA structure (thi box) is involved in regulation of thiamin biosynthetic gene expression in bacteria. Proc. Natl Acad. Sci. USA 98, 9736–9741 (2001).
Lundrigan, M. D., Köster, W. & Kadner, R. J. Transcribed sequences of the Escherichia coli btuB gene control its expression and regulation by vitamin B12 . Proc. Natl Acad. Sci. USA 88, 1479–1483 (1991).
Ravnum, S. & Andersson, D. I. Vitamin B12 repression of the btuB gene in Salmonella typhimurium is mediated via a translational control which requires leader and coding sequences. Mol. Microbiol. 23, 35–42 (1997).
Richter-Dahlfors, A. A., Ravnum, S. & Andersson, D. I. Vitamin B12 repression of the cob operon in Salmonella typhimurium: translational control of the cbiA gene. Mol. Microbiol. 13, 541–553 (1994).
Ravnum, S. & Andersson, D. I. An adenosyl-cobalamin (coenzyme-B12)-repressed translational enhancer in the cob mRNA of Salmonella typhimurium. Mol. Microbiol. 39, 1585–1594 (2001).
Soukup, G. A. & Breaker, R. R. Relationship between internucleotide linkage geometry and the stability of RNA. RNA 5, 1308–1325 (1999).
Li, Y. & Breaker, R. R. Kinetics of RNA degradation by specific base catalysis of transesterification involving the 2′-hydroxyl group. J. Am. Chem. Soc. 121, 5364–5372 (1999).
Soukup, G. A., DeRose, E. C., Koizumi, M. & Breaker, R. R. Generating new ligand-binding RNAs by affinity maturation and disintegration of allosteric ribozymes. RNA 7, 524–536 (2001).
Nahvi, A., Barrick, J. E. & Breaker, R. R. Coenzyme B12 riboswitches are widespread genetic control elements in prokaryotes. Nucleic Acids Res. 32, 143–150 (2004).
Bond, C. M., Lees, K. A. & Enever, R. P. Photolytic decomposition of three cobalamins. A quantitative study. J. Pharm. Pharmacol. 24 (Suppl.), 143P (1972).
Vitreschak, A. G., Rodionov, D. A., Mironov, A. A. & Gelfand, M. S. Regulation of the vitamin B12 metabolism and transport in bacteria by a conserved RNA structural element. RNA 9, 1084–1097 (2003).
Webb, E., Claas, K. & Downs, D. thiBPQ encodes an ABC transporter required for transport of thiamine and thiamine pyrophosphate in Salmonella typhimurium. J. Biol. Chem. 273, 8946–8950 (1996).
Mironov, V. N., Perumov, D. A., Kraev, A. S., Stepanov, A. I. & Skryabin, K. G. Unusual structure in the regulation region of the Bacillus subtilis riboflavin biosynthesis operon. Mol. Biol. 24, 256–261 (1990) (in Russian).
Kreneva, R. A. & Perumov, D. A. Genetic mapping of regulatory mutations of Bacillus subtilis riboflavin operon. Mol. Gen. Genet. 222, 467–469 (1990).
Kil, Y. V., Mironov, V. N., Gorishin, I. Y., Kreneva, R. A. & Perumov, D. A. Riboflavin operon of Bacillus subtilis: unusual symmetric arrangement of the regulatory region. Mol. Gen. Genet. 233, 483–486 (1992).
Grundy, F. J. & Henkin, T. M. The S box regulon: a new global transcription termination control system for methionine and cysteine biosynthesis genes in Gram-positive bacteria. Mol. Microbiol. 30, 737–749 (1998).
Ebbole, D. J. & Zalkin, H. Cloning and characterization of a 12-gene cluster from Bacillus subtilis encoding nine enzymes for de novo purine nucleotide synthesis. J. Biol. Chem. 262, 8274–8287 (1987).
Christiansen, L. C., Schou, S., Nygaard, P. & Saxild, H. H. Xanthine metabolism in Bacillus subtilis: characterization of the xpt-pbuX operon and evidence for purine- and nitrogen-controlled expression of genes involved in xanthine salvage and catabolism. J. Bacteriol. 179, 2540–2550 (1997).
Vold, B., Szulmajster, J. & Carbone, A. Regulation of dihydrodipicolinate synthase and aspartate kinase in Bacillus subtilis. J. Bacteriol. 121, 970–974 (1975).
Lu, Y., Chen, N. Y. & Paulus, H. Identification of aecA mutations in Bacillus subtilis as nucleotide substitutions in the untranslated leader region of the aspartokinase II operon. J. Gen. Microbiol. 137, 1135–1141 (1991). This paper defines the precise locations of mutations that confer resistance to the toxic effects of a lysine analogue. The resistant phenotype, first described nearly 20 years earlier, is now known to be caused by mutations in a lysine-specific riboswitch.
Kochhar, S. & Paulus, H. Lysine-induced premature transcription termination in the lysC operon of Bacillus subtilis. Microbiol. 142, 1635–1639 (1996).
Patte, J. C. in Escherichia coli and Salmonella: Cellular and Molecular Biology Vol. 1 (eds Neidhardt, F. C. et al.) 528–541 (American Society for Microbiology Press, Washington DC, 1996).
Patte, J. -C., Akrim, M. & Méjean, V. The leader sequence of the Escherichia coli lysC gene is involved in the regulation of LysC synthesis. FEMS Microbiol. Lett. 169, 165–170 (1998).
Winkler, W. C., Cohen-Chalamish, S. & Breaker, R. R. An mRNA structure that controls gene expression by binding FMN. Proc. Natl Acad. USA 99, 15908–15913 (2002).
Mironov, A. S. et al. Sensing small molecules by nascent RNA: a mechanism to control transcription in bacteria. Cell 111, 747–756 (2002).
Mandal, M., Boese, B., Barrick, J. E., Winkler, W. C. & Breaker, R. R. Riboswitches control fundamental biochemical pathways in Bacillus subtilis and other bacteria. Cell 113, 577–586 (2003).
McDaniel, B. A. M., Grundy, F. J., Artsimovitch, I. & Henkin, T. M. Transcription termination control of the S box system: direct measurement of S-adenosylmethionine by the leader RNA. Proc. Natl Acad. Sci. USA 100, 3083–3088 (2003).
Epshtein, V., Mironov, A. S. & Nudler, E. The riboswitch-mediated control of sulfur metabolism in bacteria. Proc. Natl Acad. Sci. USA 100, 5052–5056 (2003).
Winkler, W. C., Nahvi, A., Sudarsan, N., Barrick, J. E. & Breaker, R. R. An mRNA structure that controls gene expression by binding S-adenosylmethionine. Nature Struct. Biol. 10, 701–707 (2003).
Grundy, F. J., Lehman, S. C. & Henkin, T. M. The L box regulon: lysine sensing by leader RNAs of bacterial lysine biosynthesis genes. Proc. Natl Acad. Sci. USA 100, 12057–12062 (2003).
Sudarsan, N., Wickiser, J. K., Nakamura, S., Ebert, M. S. & Breaker, R. R. An mRNA structure in bacteria that controls gene expression by binding lysine. Genes Dev. 17, 2688–2697 (2003).
Sudarsan, N., Barrick, J. E. & Breaker, R. R. Metabolite-binding RNA domains are present in the genes of eukaryotes. RNA 9, 644–647 (2003). Demonstration that some eukaryotic mRNAs carry metabolite-binding domains that are probably components of riboswitches.
Kubodera, T. et al. Thiamine-regulated gene expression of Aspergillus oryzae thiA requires splicing of the intron containing a riboswitch-like domain in the 5′-UTR. FEBS Lett. 555, 516–520 (2003).
Gusarov, I. & Nudler, E. The mechanism of intrinsic transcription termination. Mol. Cell 3, 495–504 (1999).
Yarnell, W. S. & Roberts, J. W. (1999) Mechanism of intrinsic transcription termination and antitermination. Science 284, 611–615 (1999).
White, H. B. III Coenzymes as fossils of an earlier metabolic state. J. Mol. Evol. 7, 101–104 (1976).
White, H. B. III in The Pyridine Nucleotide Coenzymes. 1–17 (Academic Press, New York, 1982).
Benner, S. A., Ellington, A. D. & Tauer, A. Modern metabolism as a palimpsest of the RNA world. Proc. Natl Acad. Sci. USA 86, 7054–7058 (1989).
Jeffares, D. C., Poole, A. M. & Penny, D. Relics from the RNA world. J. Mol. Evol. 46, 18–36 (1998).
Rodionov, D. A., Vitreschak, A. G., Mironov, A. A. & Gelfand, M. S. Comparative genomics of thiamin biosynthesis in prokaryotes. New genes and regulatory mechanisms. J. Biol. Chem. 276, 5093–5100 (2002).
Mandal, M. & Breaker, R. R. Adenine riboswitches and gene activation by disruption of a transcription terminator. Nature Struct. Mol. Biol. 11, 29–35 (2004). Demonstration of a riboswitch that activates gene expression.
Johansen, L. E., Nygaard, P., Lassen, C., Agersø, Y. & Saxild, H. H. Definition of a second Bacillus subtilis pur regulon comprising the pur and xpt-pbuX operons plus pbuG, nupG (yxjA), and pbuE (ydhL). J. Bacteriol. 185, 5200–5209 (2003).
Grundy, F. J. & Henkin, T. M. The T box and S box transcription termination control systems. Frontiers Biosci. 8, D20–D31 (2003).
Rodionov, D. A., Vitreschak, A. G., Mironov, A. A. & Gelfand, M. S. Regulation of lysine biosynthesis and transport genes in bacteria: yet another RNA riboswitch? Nucleic Acids Res. 31, 6748–6757 (2003).
Winkler, W. C., Nahvi, A., Roth, A., Collins, J. A. & Breaker, R. R. Control of gene expression by a natural metabolite-responsive ribozyme. Nature 428, 281–286 (2004). A riboswitch that uses the action of a ribozyme to control gene expression.
Barrick, J. E. et al. New motifs suggest an expanded scope for riboswitches in bacterial genetic control. Proc. Natl Acad. Sci. USA 101, 6421–6426 (2004).
Gottesman, S. et al. Small RNA regulators of translation: mechanisms of action and approaches for identifying new small RNAs. Cold Spring Harbor Symp. Quant. Biol. 66, 353–362 (2001).
Massé, E., Majdalani, N. & Gottesman, S. Regulatory roles for small RNAs in bacteria. Curr. Opin. Microbiol. 6, 120–124 (2003).
Ben-Asouli, Y., Pel-Or, Y., Shir, A. & Kaempfer, R. Human interferon-γ mRNA autoregulates its translation through a pseudoknot that activates the interferon-inducible protein kinase PKR. Cell 108, 221–232 (2002).
Gold, L., Brodey, E., Heilig, J. & Singer, B. One, two, infinity: genomes filled with aptamers. Chem. Biol. 9, 1259–1264 (2002).
Johansson, J. et al. An RNA thermosensor controls expression of virulence genes in Listeria monocytogenes. Cell 110, 551–561 (2002). This paper and references 85 and 96 show that domains within certain mRNAs serve as thermosensing genetic switches.
Chowdhury, S., Ragaz, C., Kreuger, E. & Narberhaus, F. Temperature-controlled structural alterations of an RNA thermometer. J. Biol. Chem. 278, 47915–47921 (2003).
Henkin, T. M. & Yanofsky, C. Regulation by transcription attenuation in bacteria: how RNA provides instructions for transcription termination/antitermination decisions. Bioessays 24, 700–707 (2002).
Yanofsky, C. Using studies on tryptophan metabolism to answer basic biological questions. J. Biol. Chem. 278, 10859–10878 (2003).
Wassarman, K. M., Repoila, F., Rosenow, C., Storz, G. & Gottesman, S. Identification of novel small RNAs using comparative genomics and microarrays. Genes Dev. 15, 1637–1651 (2001).
Lyubetskaya, E. V., Leont'ev, L. A., Gelfand, M. S. & Lyubetsky, V. A. Search for alternative RNA secondary structures regulating expression of bacterial genes. Mol. Biol. 37, 707–715 (2003) (translated from Russian).
Klein, R. J. & Eddy, S. R. RSEARCH: Finding homologs of single structured RNA sequences. BMC Bioinformatics 4, 44 (2003).
Vogel, J. et al. RNomics in Escherichia coli detects new sRNA species and indicates parallel transcriptional output in bacteria. Nucleic Acids Res. 31, 6435–6443 (2003). The isolation of numerous small non-coding RNAs in bacteria offers the possibility that a rich diversity of RNAs might participate in various cellular functions.
Lee, J. F., Hesselberth, J. R., Meyers, L. A. & Ellington, A. D. Aptamer database. Nucleic Acids Res. 32, D95–D100 (2004).
Jenison, R. D., Gill, S. C., Pardi, A. & Polisky, B. High-resolution molecular discrimination by RNA. Science 263, 1425–1429 (1994). An early demonstration of the molecular recognition power of RNA aptamers.
Kiga, D., Futamura, Y., Sakamoto, K. & Yokoyama, S. An RNA aptamer to the xanthine/guanine base with a distinctive mode of purine recognition. Nucleic Acids Res. 26, 1755–1760 (1998).
Burgstaller, P. & Famulok, M. Isolation of RNA aptamers for biological cofactors by in vitro selection. Angew. Chem. Int. Ed. Engl. 33, 1084–1087 (1994).
Morita, M. T. et al. Translational induction of heat shock transcription factor σ32: evidence for a built-in RNA thermosensor. Genes Dev. 13, 655–665 (1999).
We thank members of the Breaker laboratory for helpful discussions and comments on the manuscript.
The authors declare no competing financial interests.
- ALLOSTERIC ENZYME
An enzyme that is triggered to alter its function in response to the binding of a target compound at a site that is distal from the active site of the enzyme.
- RNA WORLD
A hypothetical time in early evolution, before the emergence of DNA and proteins, when biological processes were guided entirely by RNA molecules.
A nucleic-acid molecule that folds to form an active site and catalyzes a chemical reaction.
An RNA domain, either engineered or natural, that forms a precise three-dimensional structure and selectively binds a target molecule.
- IN VITRO EVOLUTION
The use of various separation and amplification techniques that serve to mimic Darwinian evolution and create variants of proteins or nucleic acids that have new or improved functions.
(Untranslated region). Stretches of untranslated sequences located upstream and downstream of the coding region of an mRNA.
- IN-LINE PROBING
An RNA-structure probing method that can be used to examine secondary-structure models and to determine whether RNAs undergo substantive structural rearrangements under different incubation conditions.
- DISSOCIATION CONSTANT
The equilibrium constant for a ligand binding to its receptor, which, in the case of riboswitches, represents the concentration of ligand that is required to convert half of the aptamers that are present in a mixture to their ligand-bound form.
- EXPRESSION PLATFORM
The part of a riboswitch that interacts with an aptamer to transduce metabolite binding into a change in gene expression.
- INTRINSIC TERMINATOR
A hairpin structure followed by a run of U residues in a nascent RNA transcript that stalls the RNA polymerase and induces transcription termination.
A hairpin structure that, on formation, precludes the formation of an intrinsic terminator and thereby permits transcription to proceed.
- RIBOSOME-BINDING SITE
Also known as the Shine–Dalgarno sequence, it is a short stretch of conserved nucleotides that is situated several nucleotides upstream of the start codon in prokaryotic mRNAs. This sequence is recognized by the ribosome during translation initiation.
- TRANSCRIPTIONAL UNIT
An RNA transcript, such as mRNA, that is transcribed separately. In the case of operons, one transcriptional unit can encode several proteins.
A collection of separate genes, the expression of which is controlled as a unit by a specific signalling compound or factor.
- SELF-CLEAVING RIBOZYME
Five of the nine known natural ribozymes catalyze self-cleavage using an internal phosphoester transfer reaction.
A non-coding segment of mRNA that is removed by splicing processes before translation by ribosomes.
- ROSE ELEMENT
An RNA sequence in certain bacteria that responds to changes in temperature and controls expression of adjacent heat-shock genes.
- AMINOACYL-tRNA SYNTHETASE
An enzyme that recognizes a specific tRNA and selectively loads each with its cognate amino acid.
- LEADER PEPTIDE
A peptide that is encoded upstream of a larger open reading frame, the translation of which is used as a sensor for adequate levels of a particular aminoacylated tRNA.
A complex formed by the trp RNA-binding attenuation protein from B. subtilis. TRAP binds tryptophan and serves as a protein factor for regulating the trp operon.
- TRANSCRIPTIONAL PAUSING
The temporary stalling of RNA polymerase during transcription that is typically caused by hairpin structures or other sequence elements within the nascent mRNA.
- RNase P
A ribonucleoprotein-enzyme complex wherein the RNA component serves as a ribozyme that processes precursor RNAs such as pre-tRNA transcripts.
About this article
Frontiers in Bioengineering and Biotechnology (2020)
Metal ions and sugar puckering balance single-molecule kinetic heterogeneity in RNA and DNA tertiary contacts
Nature Communications (2020)
International Journal of Molecular Sciences (2020)
SLAS DISCOVERY: Advancing the Science of Drug Discovery (2020)