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Review Non-coding RNAs as antibiotic targets Savannah Colameco, Marie A. Elliot Department of Biology and Institute for Infectious Disease Research, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4K1, Canada article info Article history: Received 26 August 2016 Accepted 12 December 2016 Available online xxxx Keywords: Antibiotic Non-coding RNA Ribosome tRNA Riboswitch abstract Antibiotics inhibit a wide range of essential processes in the bacterial cell, including replication, tran- scription, translation and cell wall synthesis. In many instances, these antibiotics exert their effects through association with non-coding RNAs. This review highlights many classical antibiotic targets (e.g. rRNAs and the ribosome), explores a number of emerging targets (e.g. tRNAs, RNase P, riboswitches and small RNAs), and discusses the future directions and challenges associated with non-coding RNAs as antibiotic targets. Ó 2016 Elsevier Inc. All rights reserved. Contents 1. Introduction .......................................................................................................... 00 2. rRNA – the most-targeted system in a bacterial cell? ......................................................................... 00 2.1. Antibiotics targeting the 16S rRNA .................................................................................. 00 2.2. Antibiotics targeting the 23S rRNA .................................................................................. 00 2.3. Towards developing new rRNA-targeting antibiotics .................................................................... 00 3. Targeting tRNAs and tRNA function ....................................................................................... 00 3.1. Inhibition of both rRNA and tRNA function by promiscuous antibiotics ..................................................... 00 3.2. Starting at the beginning: inhibiting tRNA maturation by blocking processing ............................................... 00 3.3. Short circuiting tRNA charging ...................................................................................... 00 4. Inhibiting trans-translation by targeting tmRNA activity ...................................................................... 00 5. Small RNAs – a future target? ............................................................................................ 00 6. Riboswitches – bacterial-specific drug targets ............................................................................... 00 6.1. Antibiotics targeting bacterial riboswitches ........................................................................... 00 6.2. Strategies for future drug discovery .................................................................................. 00 7. Challenges and future directions .......................................................................................... 00 Acknowledgements .................................................................................................... 00 References ........................................................................................................... 00 1. Introduction Historically, infectious disease has been a major cause of human morbidity and mortality. Antibiotics were first discovered in the early 1900’s [1], and their subsequent wide-spread application has revolutionized health-care, and with it, improved our quality of life. Our ability to readily treat infections is, however, being stea- dily eroded with the rise in antibiotic resistance [2]. Consequently it is critical that we continue to search for new antibiotics, and devise innovative strategies to combat infectious disease. Antibiotics – or more specifically for the purposes of this review, antibacterials – target a range of essential cellular processes, including DNA replication, transcription, translation and cell wall synthesis, among many others [3]. Despite a tendency to take a protein-centric view of the cell, non-coding RNAs are central to many of the processes currently targeted by antibiotics. As we http://dx.doi.org/10.1016/j.bcp.2016.12.015 0006-2952/Ó 2016 Elsevier Inc. All rights reserved. Corresponding author at: Department of Biology, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4K1, Canada. E-mail address: [email protected] (M.A. Elliot). Biochemical Pharmacology xxx (2016) xxx–xxx Contents lists available at ScienceDirect Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm Please cite this article in press as: S. Colameco, M.A. Elliot, Non-coding RNAs as antibiotic targets, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j. bcp.2016.12.015
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Page 1: Non-coding RNAs as antibiotic targets › fcl › elliot › web › pdf... · Here we explore the mechanisms by which antibiotics target non-coding RNAs in bacteria, from the classical

Biochemical Pharmacology xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Biochemical Pharmacology

journal homepage: www.elsevier .com/locate /b iochempharm

Review

Non-coding RNAs as antibiotic targets

http://dx.doi.org/10.1016/j.bcp.2016.12.0150006-2952/� 2016 Elsevier Inc. All rights reserved.

⇑ Corresponding author at: Department of Biology, McMaster University, 1280Main Street West, Hamilton, ON L8S 4K1, Canada.

E-mail address: [email protected] (M.A. Elliot).

Please cite this article in press as: S. Colameco, M.A. Elliot, Non-coding RNAs as antibiotic targets, Biochem. Pharmacol. (2016), http://dx.doi.org/10bcp.2016.12.015

Savannah Colameco, Marie A. Elliot ⇑Department of Biology and Institute for Infectious Disease Research, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4K1, Canada

a r t i c l e i n f o a b s t r a c t

Article history:Received 26 August 2016Accepted 12 December 2016Available online xxxx

Keywords:AntibioticNon-coding RNARibosometRNARiboswitch

Antibiotics inhibit a wide range of essential processes in the bacterial cell, including replication, tran-scription, translation and cell wall synthesis. In many instances, these antibiotics exert their effectsthrough association with non-coding RNAs. This review highlights many classical antibiotic targets(e.g. rRNAs and the ribosome), explores a number of emerging targets (e.g. tRNAs, RNase P, riboswitchesand small RNAs), and discusses the future directions and challenges associated with non-coding RNAs asantibiotic targets.

� 2016 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002. rRNA – the most-targeted system in a bacterial cell? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

2.1. Antibiotics targeting the 16S rRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.2. Antibiotics targeting the 23S rRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.3. Towards developing new rRNA-targeting antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

3. Targeting tRNAs and tRNA function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

3.1. Inhibition of both rRNA and tRNA function by promiscuous antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.2. Starting at the beginning: inhibiting tRNA maturation by blocking processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.3. Short circuiting tRNA charging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

4. Inhibiting trans-translation by targeting tmRNA activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 005. Small RNAs – a future target? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 006. Riboswitches – bacterial-specific drug targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

6.1. Antibiotics targeting bacterial riboswitches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 006.2. Strategies for future drug discovery. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

7. Challenges and future directions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction

Historically, infectious disease has been a major cause of humanmorbidity and mortality. Antibiotics were first discovered in theearly 1900’s [1], and their subsequent wide-spread applicationhas revolutionized health-care, and with it, improved our quality

of life. Our ability to readily treat infections is, however, being stea-dily eroded with the rise in antibiotic resistance [2]. Consequentlyit is critical that we continue to search for new antibiotics, anddevise innovative strategies to combat infectious disease.

Antibiotics – or more specifically for the purposes of this review,antibacterials – target a range of essential cellular processes,including DNA replication, transcription, translation and cell wallsynthesis, among many others [3]. Despite a tendency to take aprotein-centric view of the cell, non-coding RNAs are central tomany of the processes currently targeted by antibiotics. As we

.1016/j.

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Fig. 1. Ribosome-targeting antibiotics. Schematic of ribosome activity, highlighting the points at which different antibiotics act. Throughout, antibiotics interacting with the23S rRNA are shown above the ribosome, while those binding the 16S are indicated below the ribosome. (A) Antibiotics targeting translation initiation. During translationinitiation, the 50S (containing the 23S rRNA) and 30S (containing the 16S rRNA) ribosomal subunits come together on the mRNA template, forming a 70S ribosome. Thisprocess requires the binding of initiation factors, and the binding of the f-Met-tRNA to the P site of the ribosome. The orthosomycins and thiopeptides (binding to the 23SrRNA) prevent binding of initiation factors to the ribosome, while kasugamycin and edeine (binding to the 16S rRNA) prevent the binding of the f-Met-tRNA. (B) Antibioticstargeting translation elongation. There are multiple stages of translation elongation that are targeted by antibiotics. The tetracyclines bind the 16S rRNA and block theaddition of new aminoacyl-tRNAs into the A-site. Linezolid, clindamycin, and chloramphenicol (binding to the 23S rRNA) block peptide bond formation. Finally, theaminoglycosides and tuberactinomycins (binding to the 16S) and erythromycin (binding to the exit channel) block translocation. (C) Antibiotics that affect translation fidelity.The decoding centre of the 16S rRNA allows only cognate tRNAs to enter the A site, ensuring accurate translation. Paromomycin and streptomycin bind to the decoding centresuch that either cognate or non-cognate tRNAs are accepted into the A site, resulting in mistranslation (illustrated by the incorporation of red amino acids).

2 S. Colameco, M.A. Elliot / Biochemical Pharmacology xxx (2016) xxx–xxx

continue to learn more about the function of non-coding RNAs inthe bacterial cell, we are presented with opportunities to exploitthis understanding, and develop new and/or improvedways of con-trolling bacterial growth and pathogenesis.

Here we explore the mechanisms by which antibiotics targetnon-coding RNAs in bacteria, from the classical rRNAs and tRNAs,through to the more recently discovered regulatory RNAs. For eachRNA class, we highlight past and present drug discovery and devel-opment efforts, and discuss future perspectives and challengesassociated with non-coding RNA activity modulation.

2. rRNA – the most-targeted system in a bacterial cell?

Protein translation is an RNA-driven process involving threemajor RNA classes: messenger RNAs (mRNAs), transfer RNAs

Please cite this article in press as: S. Colameco, M.A. Elliot, Non-coding RNAs asbcp.2016.12.015

(tRNAs) and ribosomal RNAs (rRNAs) (Fig. 1). Translation takesplace within the ribosome, which is a massive ribonucleoproteincomplex comprising three rRNAs and over 50 proteins [4,5]. Thesecomponents are divided between two subunits: the 50S subunitcontaining the 23S and 5S rRNAs, and the smaller 30S subunit con-taining the 16S rRNA. These two subunits come together whentranslation initiates, forming the mature 70S ribosome.

Translation encompasses three stages: initiation, elongation,and termination [6,7]. The first stage involves assembling the 70Sribosome on the mRNA transcript, and positioning it in frame sotranslation can initiate (Fig. 1A). Elongation next requires theamino acid-bearing tRNAs to cycle through three positions withinthe ribosome: the A-, P-, and E-sites (Fig. 1A,B). Incomingaminoacyl-tRNAs (aa-tRNAs) first localize to the A-site, or aminoa-cyl site, where they pair with their cognate codon on the mRNA

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transcript. The tRNA then shifts to the P-site, where the growingpeptide chain is added to this translocated tRNA, and peptide bondformation occurs (the initiation f-Met-tRNA enters at the P-site).This cycle is repeated when a new tRNA enters the A-site, and oncethe peptide chain is transferred from one tRNA to another, thenewly unloaded tRNA is ejected from the ribosome via the E- (orexit) site. This continues until the ribosome reaches a stopcodon/termination signal, at which point the nascent polypeptidechain is released from the ribosome and the ribosomal subunitsare recycled.

Within the ribosome, the rRNAs provide the scaffold onto whichthe ribosomal proteins are assembled, and generally direct ribo-some function [8]. When the 30S and 50S subunits come togetherto give the functional 70S ribosome, the 16S and 23S rRNAs formthe A-, P- and E-sites for tRNAs (Fig. 1A). The eukaryotic ribosomeshares a similar overall architecture, only it has a greater proteincomplement, and its rRNAs are much larger (due to the presenceof multiple insertions not found in their bacterial counterparts).When considering the inherent differences between bacterial andeukaryotic ribosomal systems, the structural and functional com-plexity of the ribosome, and the essential nature of protein trans-lation, the bacterial ribosome represents an outstandingantibiotic target.

There already exists an impressive repertoire of ribosome-targeting antibiotics that interfere with distinct steps in the trans-lation process. The majority of these antibiotics exert their effectsthrough interactions with the rRNAs [9]. These molecules affecttranslation initiation or elongation, and less frequently, translationtermination. Here, we present an overview of the antibiotics tar-geting rRNA and the processes they disrupt (Fig. 1). Details onthe binding sites and precise mechanisms of action for specificantibiotics have been recently reviewed elsewhere [9–13].

2.1. Antibiotics targeting the 16S rRNA

The 16S rRNA within the 30S ribosome has a critical structuralrole: it forms the base of the A-, P- and E-sites. It is further essentialfor mRNA decoding, ensuring translational fidelity by only accept-ing amino-acylated tRNAs having the correct anticodon. The twobest-characterized classes of antibiotics associating with the 16SrRNA are the aminoglycosides [14–16] and the tetracyclines[14,17]. Clinically, the aminoglycosides are notable for their usein treating Gram-negative bacterial infections [18] (e.g. tobramycinis a front-line antibiotic used in treating Pseudomonas aeruginosainfections [19]), as well as mycobacterial infections (e.g. strepto-mycin was the first antibiotic used to treat tuberculosis [20]). Incontrast, the tetracyclines are broad spectrum bacteriostaticantibiotics that are currently used in the treatment of Lyme disease[21] and other spirochete infections.

Generally, antibiotics that bind the 16S rRNA impact translationinitiation [22] (Fig. 1A), impair translation elongation (either bypreventing tRNA binding or preventing ribosome translocationalong the mRNA) [23] (Fig. 1B), or affect translational proofreading[24,25] (Fig. 1C). Interestingly, within a given class of antibiotic(e.g. the aminoglycosides), individual compounds can bind differ-ent sites on the 16S rRNA, and have distinct inhibitory effects.The most prevalent means by which translation initiation isblocked is by preventing binding of the f-Met-tRNA to the P-site.Antibiotics like the aminoglycoside kasugamycin [22,26,27] (cur-rently being tested as a treatment for plant pathogens) [28] havebinding sites that overlap with the P-site and thus prevent tRNAaccess at this position (Fig. 1A). Antibiotics affecting translationelongation can act through a similar steric hindrance mechanism.For example, the tetracyclines bind within the A-site and inhibittRNA delivery [29] (Fig. 1B). Elongation-specific antibiotics can alsodisrupt tRNA translocation through the ribosome, as is the case for

Please cite this article in press as: S. Colameco, M.A. Elliot, Non-coding RNAs asbcp.2016.12.015

the tuberactinomycin class of non-ribosomal peptide antibiotics[30] and some aminoglycosides (e.g. hygromycin B [31,32])(Fig. 1B). There are also antibiotics that interfere with the decodingcentre of the ribosome. This is a region of the 16S rRNA that servesas a checkpoint, only permitting tRNAs with the matching anti-codon to enter the A site and contribute to peptide chain elonga-tion. The aminoglycoside antibiotic streptomycin [33] influencesthis ‘proofreading’ step by inducing a conformational change inthe decoding centre of the 16S rRNA such that it less effectivelydistinguishes between cognate and non-cognate tRNAs (Fig. 1C).

2.2. Antibiotics targeting the 23S rRNA

While the 16S rRNA provides the foundational framework forthe A-, P- and E-sites in the ribosome, the 23S rRNA houses the cat-alytic core. This peptidyl transferase centre (or PTC) directs peptidebond formation, and most antibiotics associating with the 23SrRNA bind within the PTC (which encompasses part of the A- andP-sites) and inhibit its catalytic function. The 23S rRNA is targetedby a number of well-known drug classes, including the oxazolidi-nones (e.g. linezolid), macrolides (e.g. erythromycin), lincosamides(e.g. clindamycin), and the phenicols (e.g. chloramphenicol).

The oxazolidinones bind within the A-site of the 23S and stabi-lize the A- and P-sites in an atypical configuration that is incompat-ible with peptide bond formation [34]. The lincosamides andphenicols also bind within the A-site, but act by preventing tRNAbinding to this position and thus inhibit peptide bond formation[35,36]. In contrast, the macrolides bind to the 23S within the exittunnel of the ribosome and allosterically alter the activity of thePTC, leading to ribosome stalling [36,37] (Fig. 1B).

In addition to its role in elongation and peptide bond formation,the 23S rRNA also directs aspects of translation initiation, by coor-dinating the assembly of the 70S ribosome. Antibiotics affectingthis process do so by preventing the binding of initiation factors,either directly by interfering with binding (e.g. the oligosaccharideorthosomycin antibiotics [38]), or indirectly by stimulating confor-mational changes in the RNA that inhibit binding (e.g. thiopeptideantibiotics [39]) (Fig. 1A).

2.3. Towards developing new rRNA-targeting antibiotics

With the structural biology revolution that is currently under-way, it is becoming possible to pursue rational drug design, andleverage in silico modelling techniques [40,41]. Most ribosome-targeting antibiotics bind to one of a few specific regions in the ribo-some. However, given the complexity of the ribosome, there maywell be unexplored targets within this macromolecule that couldbe exploited for future drug development. Top priorities willinvolve identifying molecules that target novel (and bacterial-specific) structural features important for ribosome structure and/or assembly, and developing modified variants of existing antibi-otics having superior activity (higher specificity/affinity) and/oran ability to circumvent resistance.

To identify new inhibitors of ribosome function, a number ofunconventional approaches are being explored. Phage display hasrecently been employed to identify small peptides capable of bind-ing specific regions of the 16S rRNA [42–44]. A subset of these pep-tides inhibited translation in in vitro assays [42–44]. While theseexperiments effectively demonstrated that there are additionalregions of the ribosome that can be targeted for inhibition, noneof these molecules have yet been shown to have in vivo activity.Many naturally occurring antibiotics have a peptide component(e.g. the glycopeptide antibiotic vancomycin); however, few syn-thetic peptide antibiotics have made it to the clinic, largely dueto issues with solubility, stability and general bioavailability(reviewed in [45]). There are significant efforts being made to

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develop strategies that will help to circumvent these issues(reviewed in [46]), and it may be that with time, the synthetic pep-tides become viable antibiotic candidates. However, the utility ofribosome-targeting peptides at this stage lies more in their identi-fication of novel folds to target, than in their clinical antibioticpotential.

In parallel with the pursuit of peptide-based antibiotics, anti-sense RNA-based inhibitors have also attracted considerable inter-est. In the case of ribosomes, this involves the application ofoligonucleotides complementary to specific rRNA sequences thatultimately block ribosome function [47]. Antisense inhibition wasfirst attempted in the late 1970’s [48], and while there are draw-backs to this technology analogous to those of peptide antibiotics– notably, challenges in delivery, oligonucleotide stability, targetsite accessibility, and dosage - these obstacles are gradually beingovercome [49–55]. In particular, novel oligonucleotide configura-tions are being developed as alternatives to RNA or DNA oligonu-cleotides, as these are less prone to nuclease degradation, and insome cases pair more stably and specifically with their targetsequences than natural nucleic acids. These include locked nucleicacids (LNAs) [56], peptide nucleic acids (PNAs) [57] and phospho-rodiamidate morpholino oligomers (PMOs) [58], which have mod-ified backbones and exhibit increased half-lives relative tostandard oligonucleotides [59,60]. The addition of different uptakesignals to the ends of these molecules is currently being investi-gated as a means of circumventing the uptake challenge (reviewedin [61]). In vivo efficacy has been demonstrated for some nucleicacid therapeutics (e.g. [47]), supporting their potential for futuredevelopment as antibiotic candidates. At this point, however, theirgreatest applicability is as biological probes for exploring theimportance of different rRNA regions [62].

In addition to identifying new types of ribosome inhibitors andnew regions of the ribosome that are worth exploring with thepeptide- and nucleic acid therapeutics, the repurposing and/orrescreening of existing antibiotics is proving to be a fruitful strat-egy [13]. Historically, many promising antibiotic candidates wereabandoned for a variety of reasons, including host cell toxicity

Fig. 2. Antibiotics affecting tRNA maturation and charging. A. tRNAs are transcribed as ‘prremoved through the action of RNase P, while the 30 end is processed by a myriad of nucleacids (blue circle) are added to the 30 end of tRNAs by dedicated aminoacyl tRNA synthetawith the aaRSs, by antibiotics binding to the 30 tail of the tRNA, or by antibiotics binding t(A) and (B), compounds marked with an asterisk (*) are clinically relevant antibiotics, whunmarked have demonstrated in vitro activity, but their in vivo activities are unknown.

Please cite this article in press as: S. Colameco, M.A. Elliot, Non-coding RNAs asbcp.2016.12.015

[13]. However, given the structural information available for bothbacterial and eukaryotic ribosomes, it is now possible to determinethe binding sites of these previously discovered antibiotics. This inturn permits a rational approach to modifying the antibiotic scaf-fold to maximize specificity for the bacterial ribosome, and con-comitantly minimize cytotoxicity to eukaryotic cells [13]. Similarapproaches are being used to help circumvent antibiotic resistance,through the development of semi-synthetic derivatives of existingantibiotics having greater affinity for their mutated or modifiedtargets [9]. This has proven to be highly effective, as can be seenfor tigecycline (derived from tetracycline) (e.g. [63]), and telithro-mycin (derived from erythromycin) (e.g. [64]), and is likely to con-tinue being a successful strategy in the future.

3. Targeting tRNAs and tRNA function

While the ribosome has proven to be an exceptional system forantibiotic-directed inhibition, other translational elements havealso shown considerable promise as antibiotic targets. tRNAs arean essential component of the translation process, where theyare required for decoding the mRNA and providing amino acidsfor peptide chain elongation. This amino acid delivery functionhas also been co-opted by other cellular processes, including pep-tidoglycan biosynthesis (providing amino acids needed for peptidecrossbridges) and phospholipid modification (adding amino acidsto phosphatidylglycerol) [65]. Both protein synthesis and cell wallbiogenesis are essential processes in bacteria, and consequently,inhibiting tRNA activity has the potential to be an effective meansof eradicating pathogens [66].

A major issue with tRNA inhibition; however, is the fact thattRNAs are essential in all organisms. Thus, the most productivetRNA-specific antimicrobials would be those targeting aspects oftRNA synthesis/function that are unique to bacteria. To date, therehave been molecules identified that inhibit everything from tRNAmaturation [67], through to tRNA charging [68,69], and tRNA-mediated decoding [70] (Fig. 2), although few of these have foundclinical application as antibiotics.

e-tRNAs’, with additional sequences at their 50 and 30 ends. The 50 end sequences areases. RNase P activity is inhibited by a range of antibiotics, outlined in red. B. Aminoses (aaRS) (shown in green). This process can be inhibited by antibiotics associatingo the anticodon stem-loop region and impeding association with the aaRSs. For bothile those marked with a number symbol (#) inhibit bacterial growth. Those that are

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3.1. Inhibition of both rRNA and tRNA function by promiscuousantibiotics

tRNAs, like rRNAs, are abundant in bacterial cells, and are highlynegatively charged. The aminoglycosides are positively chargedantibiotics, and in addition to their ribosome inhibitory capabili-ties, these compounds also have off-target activity in the form ofinteractions with tRNAs. In the case of tobramycin, it not onlybinds the A-site of the ribosome and promotes mis-translation,but also binds tRNAs, having a particular affinity for tRNA(Asp)[68]. Tobramycin binding alters the tRNA conformation and indoing so, blocks the interaction between the tRNA and its cognateamino acyl tRNA synthetase, thus inhibiting tRNA charging withthe appropriate amino acid [68] (Fig. 2). Interactions have alsobeen observed between neomycin B, another aminoglycoside,and tRNA(Phe) [69] (Fig. 2). Structural studies have revealed thatthis antibiotic displaces metal ion co-factors within the tRNA,and like tobramycin, blocks tRNA association with its amino acyltRNA synthetase [69].

tRNA-aminoglycoside interactions are not specific to bacterialtRNAs [68,69]. While aminoglycoside association with multipleRNA molecules in bacterial cells may enhance their antimicrobialeffect, it may also impact host cell function leading to undesirableside effects. Generally, aminoglycoside interactions with their RNAtargets rely on appropriate structural determinants and are largelysequence independent [71]. As there is no single structural motifrecognized by all aminoglycosides, this suggests some level ofspecificity to their RNA interaction preferences. A future goal willbe to modify aminoglycosides – and other RNA-targeting antibi-otics – to enhance their specificity for particular bacterial RNA fea-tures, and to diversify the repertoire of molecules to which theycan bind strongly.

3.2. Starting at the beginning: inhibiting tRNA maturation by blockingprocessing

tRNAs are transcribed as ‘pre-tRNAs’, and these transcripts aresubject to extensive processing and modification. This typicallyinvolves cleavage of both their 50 and 30 ends, addition of a terminalCCA sequence (for some bacterial tRNAs), and modification of mul-tiple nucleotides throughout the mature tRNA sequence [72](Fig. 2). Initial processing of the pre-tRNA 50 end is directed bythe Mg2+-dependent ribonuclease, RNase P [73], and this cleavageevent is required for tRNA function. RNase P is itself a ribonucleo-protein, comprising a catalytic RNA and a variable number of pro-tein subunits: bacteria typically have a single protein subunit,while archaea have four, and eukaryotes have upwards of nine[74]. Given this divergence in enzyme composition, it becomespossible to specifically inhibit the activity of the bacterial enzyme,relative to its eukaryotic counterparts. RNase P is an essentialenzyme complex in bacteria, and notably, is present in relativelylow abundance, making it an attractive antibiotic target (reviewedin [75]).

RNase P activity, and by extension, tRNA maturation, is inhib-ited by a range of natural and synthetic compounds (Fig. 2A). Asubset of aminoglycosides, the antibiotic class affecting both ribo-some function and (in some cases) tRNA activity, is also activeagainst RNase P [76]. Neomycin B, the compound that inhibitsmetal binding of the tRNA(Phe), has a similar association withRNase P. It serves as a metal mimic, preventing divalent cationbinding and leading to loss of enzyme catalytic capabilities. Sincethis discovery, efforts have been made to improve the RNaseP-targeting of specific aminoglycosides. Success came with the dis-covery that modifying neomycin could enhance RNase P associa-tion [77]. Indeed, the conjugation of five Arg residues toneomycin resulted in a nearly 1000-fold increase in RNase P inhi-

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bition relative to the parent neomycin B compound [78]. Theseconjugated antibiotics also had much stronger affinity for bacterialRNase P enzymes than they did for those from eukaryotes, suggest-ing effective discrimination between bacterial and eukaryoticenzymes [78]. It is not clear whether these modifications affectedribosome binding of this neomycin B derivative, and it is conceiv-able that the high positive charge conferred by the Arg conjugationmight lead to more promiscuous nucleic acid association than forneomycin B itself.

While these studies effectively demonstrated the ability to tuneantibiotic specificity through directed modification, they did notaddress the ability of these modified antibiotics to inhibit bacterialgrowth, and thus their antibiotic potential remains unknown. Agoal in moving forward will therefore be to build on the neomycinconjugate studies in developing aminoglycoside derivatives withgreater specificity for RNase P – and other bacterial RNAs – andat the same time, ensuring effective in vivo antibacterial activity.

In addition to repurposing existing antibiotics to inhibit RNaseP, searches for novel inhibitors have also been undertaken. The firstreported high-throughput screen for bacterial RNase P inhibitorsemployed a fluorescence-based tRNA cleavage assay [67]. Thisled to the identification of guanylhydrazone and benzothiazoliumderivatives that apparently abrogated RNase P activity in vitro,but also inhibited bacterial growth [67]; whether these compoundsalso affect eukaryotic RNase P activity is not clear. Subsequentscreens have been adding to the repertoire of RNase P-inhibitorycompounds. Fluorescent pre-tRNA substrates have been used toscreen for molecules that inhibit bacterial RNase P processingand/or pre-tRNA binding, and have led to the identification of anew natural product derivative (iriginol hexaacetate) that inhibitsbacterial RNase P activity at nanomolar levels [79]. As was the casefor the neomycin conjugates, this new compound has not beentested for in vivo activity. Indeed, it was suggested that it wouldbe unlikely to function in vivo given the labile nature of the acetylgroup, but instead may serve as a valuable scaffold for futuredevelopment [79].

The RNA-based nature of the bacterial RNase P enzyme (whereRNA comprises �90% of the enzyme), has also prompted investiga-tions into the feasibility of employing antisense technology forenzyme inhibition, similar to that described for rRNA inhibition.Experimental trials have proven the utility of this approach. Short(12 to 14-mer) oligonucleotides targeting the RNase P catalyticcore inhibited enzyme activity by simultaneously interfering withbinding of the substrate (pre-tRNA) and folding of the RNase P RNA[80–83]. Enzyme inhibition by these antisense molecules was notlimited to in vitro conditions; these nucleic acids also inhibitedthe growth of Escherichia coli at micromolar concentrations [83].

Based on the preliminary successes discussed above, RNase P,and more broadly tRNA processing, can be considered to be viableantibiotic targets. The key challenges in moving forward centre onthe identification of specific RNase P inhibitors having fewer off-target effects, greater bacterial specificity, improved uptake, andenhanced potency. These challenges are not unique to RNase Pinhibitors, and represent a major hurdle in developing any antibi-otics targeting RNAs. Clearly, improving specificity for bacterialRNase P is a feasible goal. It remains to be seen whether improvedspecificity can be achieved alongside reasonable in vivo potency,and minimal host toxicity.

3.3. Short circuiting tRNA charging

Correct processing of tRNA ends is an important pre-requisitefor amino acid charging. There is a dedicated aminoacyl tRNA syn-thetase (aaRS) for each amino acid (with a few exceptions in somebacterial species) [84], and the process of tRNA charging has longbeen recognized as one with promising antibiotic targeting poten-

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tial [85–88]. Natural product inhibitors of aminoacylation havebeen shown to bind both the tRNA substrate, and the aaRS itself.Irrespective of the target, inhibiting tRNA charging leads to anabundance of uncharged tRNAs, activation of the stringentresponse, and ultimately translational repression.

The antibiotic purpuromycin was discovered in the 1970’s [89],but its mode of action remained elusive until the late 1990’s whenit was discovered to bind tRNAs with high affinity and specificallyinhibit their aminoacylation [90] (Fig. 2B). While effectivelyinhibiting bacterial translation, purpuromycin also inhibits transla-tion in eukaryotic cells. Thus while purpuromycin, and other tRNA-binding compounds may have potential chemotherapeutic (anti-cancer) utility, they are likely of limited antibacterial use giventheir potential for host toxicity.

There has been considerably more activity devoted to develop-ing inhibitors of the enzymes responsible for aminoacylation[85–88], with these enzyme inhibitors effectively abolishing thefunction of specific tRNAs in the cell. While aaRSs are conservedin all organisms, bacterial and eukaryotic aaRSs exhibit varyinglevels of structural divergence. This opens the door to developingselective inhibitors of the bacterial enzymes. To date, a numberof natural product inhibitors of bacterial aaRSs have been identi-fied, but only one has been developed for clinical application.Mupirocin, or pseudomonic acid [91], specifically binds to the bac-terial isoleucyl tRNA synthetase, inhibiting the charging of IletRNAs [92] (Fig. 2C). It is currently used as a topical antibiotic intreating Gram-positive infections, including that of methicillin-resistant Staphylococcus aureus (MRSA) [93]. Efforts have beenmade to improve the activity of mupirocin, with derivatives havingbeen developed with altered properties (e.g. reduced serum bind-ing [94]). However, there seems to have been little progress in thisarea in the last decade, suggesting that modifications that improvebioavailability may also reduce antibiotic activity.

Other natural product inhibitors characterized to date targetdifferent aaRSs (Fig. 2B). For example, indolmycin is a tryptophananalogue that is specific for TrpRS [95]; borrelidin is a nitrile-containing macrolide that targets ThrRS [96]; microcin C is a pep-tide antibiotic that associates with AspRS [97]; and albomycin is asideromycin (siderophore-linked antibiotic) that inhibits SerRS[98]. Indolmycin has activity against MRSA [99] and Helicobacterpylori [100]. In contrast, borrelidin has broad specificity, inhibitingboth eukaryotic and bacterial ThrRS enzymes. It therefore has notbeen pursued for antibacterial purposes, although borrelidin ana-logues are being investigated for their antimalarial potential[101]. Microcin C is known as a ‘Trojan horse’ antibiotic, in thatit is taken up by cells in an inactive form and is processed in thecell to generate a non-hydrolysable aspartyl adenylate [102]. Ithas bacteriostatic activity against a range of Gram-negative bacte-ria [103], and has been modified to specifically inhibit other aaRSs[104]. Unlike many antibiotics, microcin C readily traverses theGram-negative cell wall through target peptide transporters[105]. Albomycin is another ‘Trojan horse’ antibiotic that is activelytransported into cells [106], in this case through its siderophorecoupling, and has activity against both Gram-positive and -negative bacteria [107].

High-throughput screens are also yielding some interestinginhibitors. These include novel non-hydrolysable adenylate ana-logues that inhibit aaRS activity at nanomolar levels in vitro, andinhibit bacterial growth at micromolar levels in vivo [85]. To date,these compounds have failed to have the required level of efficacyin animal models. It is clear, however, that despite setbacks likethis, inhibitors of aaRSs have significant potential for antibacterialdevelopment. In addition to high-throughput screening endeav-ours, it is also now possible to leverage the wealth of functionaland structural information available for aaRSs in developing com-pounds with greater biochemical and biological activity, using sim-

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ilar approaches to those described for the rRNA inhibitors. Theuptake signals associated with microcin C and albomycin couldalso be exploited to promote heterologous antibiotic uptake, asaccessing cytoplasmic targets remains a major challenge forRNA-targeting/affecting antibiotics.

4. Inhibiting trans-translation by targeting tmRNA activity

Related to the tRNAs is the remarkable tmRNA, a hybrid tRNA-mRNA molecule [108]. tmRNA plays a critical role in recyclingstalled ribosomes and promoting the degradation of improperlytranslated proteins/peptides through a process known as ‘trans-translation’ [109]. This recycling pathway is found in all bacteriawhose genomes have been sequenced to date [110], and it is essen-tial in many pathogens including Mycobacterium [111] and Neisse-ria [112], two organisms for which resistance to existing antibioticsis a major issue [113–115]. Trans-translation is also required forvirulence in a number of other pathogens, including Salmonella[116], Streptococcus [117], and Yersinia [118]. tmRNA functions inassociation with a small protein co-factor SmpB [119], and tmRNAactivity requires further interaction with the translation-associated EF-Tu protein [120,121] and the ribosomal protein S1[122]. Notably, eukaryotes do not have a trans-translation system.

Given the importance of tmRNA for both viability and virulencein bacteria, and considering the number of required interactionpartners, trans-translation has great potential as a drug target[123]. Recent work has revealed trans-translation to be targetedby pyrazinamide/pyrazinoic acid, a front-line drug used in treatingtuberculosis infections [124,125]. This molecule interferes withtmRNA binding by the ribosomal S1 protein, and its antibioticactivity comes from the loss of trans-translation [126]. Morerecently, a high-throughput screen for inhibitors of trans-translation using a cell-based luciferase reporter yielded severalpotent candidates capable of inhibiting the growth of bothGram-positive and -negative bacteria [127]. Precisely how thesecompounds affect trans-translation remains to be determined,but their broad-spectrum antibacterial activity [127] suggests thatthey may provide an excellent platform for further development.

tmRNA and trans-translation were only discovered �20 yearsago [108], making them relative newcomers to the non-codingRNA world. As their importance in bacteria becomes clearer, andtheir amenability for high-throughput screening is explored moreextensively, this process has the potential to become a major targetfor drug discovery and development in the years to come.

5. Small RNAs – a future target?

One of the newest players in the bacterial non-coding RNAworld is the small regulatory RNAs (sRNAs). As their name implies,these transcripts are typically short, ranging in size from �40 to500 nucleotides (although longer – and shorter – sRNAs have alsobeen identified). sRNAs can arise through independent transcrip-tion of dedicated genes, or through the processing of 50 (rare) or30 (more common) untranslated regions of mRNAs [128–130].Most small RNAs are not essential in bacteria, and instead they typ-ically contribute to the fine-tuning of gene expression [131]. Giventheir relatively recent discovery, they have yet to be explored aspotential antibiotic targets. Their activity in bacteria is distinctfrom RNA-based regulation in eukaryotes [132], and many sRNAsplay important roles in virulence [117,133–139]. They further con-trol biofilm formation [140–144], antibiotic resistance [145], and amultitude of bacterial stress responses [146–151], all of which canreduce the effectiveness of antibiotic therapies. While it is unlikelythat targeting any given sRNA could lead to pathogen eradication,

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modulating sRNA activity could serve as an adjuvant, enhancingthe efficacy of existing antibiotic therapies.

sRNAs characterized to date exert their regulatory effects eitherthrough base-pairing with other RNAs in the cell, or by associatingwith proteins. Protein-binding sRNAs represent a minority of thosethat have been characterized, and those sRNAs that target proteinstypically block protein association with their native ligand/sub-strate through competitive binding [152–155]. In contrast, themajority of characterized sRNAs act by base-pairing with one ormore target mRNAs, usually in the vicinity of the ribosome bindingsite. A growing number of sRNAs act as positive regulators, eitherpromoting mRNA transcript stability by protecting their bindingpartners from nucleases, or enhancing translation by relievingendogenous sequestration of the ribosome binding site [156–158].Most characterized sRNAs, however, negatively regulate their targettranscripts by recruiting nucleases and enhancing mRNA transcriptdegradation, and/or sequestering the ribosome binding site andinhibiting ribosome access [159].

Base-pairing sRNAs frequently act in concert with an RNA chap-erone known as Hfq. Hfq is found in many bacteria, and is partic-ularly important for sRNA-mediated regulation in Gram-negativebacteria [160,161]. It functions both in promoting base-pairingbetween sRNAs and their mRNA targets, and in stabilizing sRNAs[162,163]. Hfq adopts a hexameric ring configuration, with fourdistinct RNA binding sites (proximal and distal faces, hexamericrim and C-terminal tail), and consequently is remarkably flexiblein its RNA interactions (reviewed in [164]). Like the sRNAs it asso-ciates with, Hfq is essential for virulence [165–171], biofilm forma-tion [172], antibiotic resistance [173], and stress responses[165,174–179] in a wide range of bacteria. More specialized RNAchaperones are now being identified and characterized [180], butthese appear to have a small number of sRNA-interaction partnersand more defined functional roles than the promiscuous Hfqchaperone.

Currently, there are no antibiotics known to directly targetsRNA pathways. However, there are many processes that couldbe disrupted, including sRNA biosynthesis, sRNA activity, sRNAinteraction partners, and sRNA turnover. Like all RNAs, sRNAexpression is directed by RNA polymerase. Bacteria have a singleRNA polymerase, whose structure is similar to that of the eukary-otic RNA polymerase II [181]. It is, however, different enough thatbacterial-specific inhibitors have been identified. There are cur-rently two classes of RNA-polymerase targeting drugs on the mar-ket: rifamycin and its derivatives [182], which are used in treatingtuberculosis [183], and fidaxomicin/lipiarmicin, which is used totreat Clostridium difficile infections [184]. RNA polymerase, likethe ribosome, is a complex macromolecular machine that presentsa wealth of opportunities for small molecule-mediated perturba-tion. A recent review nicely highlights different strategies thatare currently being pursued for RNA polymerase (and transcriptionin general) as an antibacterial target [185].

There are numerous opportunities, and multiple challenges,when considering approaches to target sRNA activities in a cell.Unlike proteins, sRNAs are not broadly conserved, and thereforeany sRNA modulator would likely have a narrow activity spectrum.As these RNAs frequently lack the structural complexity of rRNAs,there would be limited functional configurations available to targetwith small molecules. Consequently, activity modulation would bemost effectively achieved using antisense technology, much likethat which has been proposed for the rRNAs and RNase P. Giventhe fine-tuning regulatory nature of most sRNAs, it is unlikely thatblocking the function of any one sRNAwould have the desired levelof antibacterial inhibition needed to clear an infection, and thuspursuing sRNA activity modulators as a stand-alone antibiotic isunlikely to be a high priority. However, from an academic perspec-tive, it would be interesting to see whether sRNA modulators have

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synergistic effects when administered in conjunction with moreconventional antibiotics, and whether they could help to sensitizeresistant or otherwise tolerant (e.g. biofilm-associated) organismsto these drugs.

Broader spectrum inhibition of sRNA activity could be morereadily achieved through the targeting of sRNA chaperones likeHfq. A recent cell-based screen for Hfq inhibitors has been reported[186]. This screen employed a fluorescence assay, in which theexpression of a fluorescent protein-encoding reporter gene wascontrolled by an Hfq-requiring sRNA [186]. A library of cyclic pep-tides was screened, and inhibitors were identified and confirmedusing in vitro experiments. This work effectively validated Hfq asa druggable target, and opened the door for additional screeningendeavours. The relative complexity of Hfq as a hexamer with mul-tiple RNA binding faces, coupled with a growing structure-functionunderstanding, could pave the way for rational drug design andscreening using strategies analogous to those employed for othernon-coding RNAs.

Finally, our understanding of sRNA function in bacteria has thepotential to guide nucleotide-based antibiotic development: [187].As described above, antisense technology is continuing to evolve,and when coupled with a better understanding of sRNA activity,could provide us with the knowledge and the tools to more pre-cisely and effectively control gene expression or RNA activity usingnucleic acid-based antibiotics.

6. Riboswitches – bacterial-specific drug targets

Riboswitches are the most recent addition to the bacterial non-coding RNA inventory. These non-coding RNA elements are typi-cally found within the 50 untranslated regions (UTRs) of the mRNAsthat they regulate [188–193]. They affect gene expression byspecifically interacting with a ligand; ligand binding changes theRNA conformation such that there is a ‘switch’ in the expressionof the downstream gene (Fig. 3). Riboswitches have two functionaldomains: an aptamer domain and an expression platform. The for-mer is the ligand-binding domain and it can range in size from �30to 200 nt in length [194–196]. Ligand binding to the aptamerdomain results in a conformational change that alters the structureof the downstream expression platform, and ultimately dictatesthe regulatory outcome. Most riboswitches affect gene expressionat transcriptional or translation levels (Fig. 3). For those affectingtranscription, the expression platform typically consists of mutu-ally exclusive terminator and anti-terminator structures. Whichof these structures forms depends on ligand presence or absence.Most commonly, ligand binding causes a switch from an anti-terminator to a terminator structure, resulting in premature tran-scription termination. For riboswitches influencing translation,ligand binding results in a conformational change in the expressionplatform such that ribosome binding site accessibility is altered,and with it, downstream translation.

The riboswitches characterized to date aremost often associatedwith primary metabolic genes, and in many cases, their cognateligand is a product (or precursor/derivative) of the biosyntheticpathways they control. For example, the lysine biosynthetic genesare controlled by a lysine-responsive riboswitch [197], while theriboflavin biosynthetic genes are regulated by a riboswitchresponding to a phosphorylated derivative of riboflavin, the flavinmononucleotide (FMN) [198]. It is worth noting that while theseriboswitches are widespread in bacteria, they are not universallyconserved (e.g. [199,200]).

Given the primary metabolic nature of many riboswitch-associated genes, there is increasing interest in targeting theseRNA elements using antibiotic therapies [201–203]. In additionto their control of essential metabolic processes, riboswitches bind

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Fig. 3. Riboswitch activity. Riboswitches are found in the 50 UTR of mRNAs (blue sequence), and control the expression of their downstream genes/operons (shown in black).They adopt one of two mutually exclusive configurations: one in the absence of ligand/analogue (shown here as ‘gene expressed’) and one in the presence of ligand (depictedhere as ‘gene repressed’). They can influence the transcription (left) or the translation (right) of their associated genes. SD = Shine Dalgarno sequence (ribosome binding site).

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small molecules with high specificity and affinity, making themexcellent candidates for chemical perturbation. Riboswitches arealso entirely absent from mammalian systems, and consequentlyprovide the potential for bacterial-specific targeting. Notably,riboswitches are common in the genomes of many pathogenic bac-teria, including Mycobacterium tuberculosis [204,205], Staphylococ-cus aureus [206–208], Streptococcus pneumoniae [209–211], andVibrio cholera [212,213], amongst others.

6.1. Antibiotics targeting bacterial riboswitches

Recent work has revealed that nature has already validatedriboswitches as an antibiotic target. A member of the antibiotic-producing Streptomyces genus, Streptomyces davawensis, producesa riboflavin analogue known as roseoflavin. Roseoflavin was dis-covered in 1974 [214], but the mechanism underlying its antimi-crobial activity remained unknown for decades. In 2009,roseoflavin was found to directly bind the FMN riboswitch, inhibit-ing riboflavin production [198]. This discovery inspired confidencein riboswitches as drug-targets, and since then, there have been anumber of productive searches for new molecules capable ofinhibiting riboswitch-controlled pathways. A major success camein 2015, with the reporting of a synthetic mimic of FMN (‘ribocil’)that was tightly bound by the FMN riboswitch, and which effec-tively inhibited bacterial growth in mouse infection models[215]. While the FMN riboswitch is an excellent antimicrobial tar-get in many organisms, there is particular interest in developingFMN-riboswitch inhibitors for treating tuberculosis. This isbecause unlike many bacteria, Mycobacterium tuberculosis lacks ariboflavin transporter, making riboflavin biosynthesis essential inthis bacterium [216].

Other riboswitches have also shown promise as antimicrobialtargets, specifically the lysine and thiamine pyrophosphate (TPP– controlling thiamine biosynthesis) riboswitches. Following thediscovery of these riboswitches [196,197], it was determined that

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the established antibiotics S-(2-aminoethyl)-L-cysteine (AEC)[197] - a lysine analogue, and pyrithiamine – a thiamine analogue[217], effectively shut down the expression of the downstreamlysine and thiamine biosynthetic gene clusters respectively. A gen-eral trend with riboswitch inhibitors identified to date, includingthose for FMN, lysine and TPP, is that they all act by mimickingthe native ligand. Furthermore, they all act by promoting repres-sion of their associated downstream genes.

Building on this early success, there now exists a growing reper-toire of ligand analogues that are being tested for their antibioticpotential through riboswitch inhibition, including molecules thattarget: glmS riboswitches [218–220], which control the productionof glucosamine 6-phosphate, a key molecule in peptidoglycanbiosynthesis; purine riboswitches [221,222], which are essentialfor nucleotide/nucleoside synthesis; cyclic-di-GMP riboswitches[223], which are important for controlling motility/biofilm deci-sions; and T-box riboswitches [224,225], which govern the charg-ing of tyrosine-specific tRNAs.

6.2. Strategies for future drug discovery

There are �30 characterized riboswitch classes, and this num-ber continues to grow. Only a handful of these, however, have beensubjected to screening for analogues with antibiotic potential.Structure-guided ligand docking and a variety of high-throughputscreening strategies are currently being investigated as means bywhich to identify novel riboswitch inhibitors. Structure-guidedligand docking is a well-established technique in protein-smallmolecule interaction studies [226]. As more riboswitch structuresbecome available, equivalent RNA-small molecule investigationsare becoming feasible. In a proof-of-principle experiment, Daldropet al. [227] modified a protein-specific program, and screened alibrary of compounds for interaction with a purine riboswitch.They successfully identified several compounds that could interactwith this riboswitch [227], and while there is no information avail-

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able on whether these compounds affected bacterial growth, itsuggests that structure-guided ligand docking could be employedfor RNA-targets having established structures.

The small molecule-binding nature of riboswitches also meansthey are natural candidates for high-throughput screens. The chal-lenge to date has been in developing appropriate assays to use inidentifying ligands of interest. Fluorescent strategies are appearingto provide a productive approach to addressing this issue. A recentstudy focusing on the SAM-I riboswitch involved screening alibrary of non-fluorescent small molecules for the ability to dis-place a fluorescently labeled analogue from the riboswitch [228].This displacement-type screening could be readily adapted forany riboswitch for which a fluorescent ligand analogue could bedeveloped. Alternative approaches are employing fluorescence res-onance energy transfer (FRET) and fluorescence polarization tech-niques, where the riboswitch is labelled, and ligand binding (inthe context of a high-throughput screen) results in a change in flu-orescence output [218,219,229].

While riboswitches possess many characteristics that makethem desirable drug targets (largely bacterial specific, associatedwith essential primary metabolic pathways, complex structureswith demonstrated inhibition potential), not all riboswitches willbe equally effective targets. A subset of riboswitches act to pro-mote the expression of their downstream metabolic genes (e.g.[230,231]), and thus ligand analogues would be expected to acti-vate (not inhibit) these important metabolic pathways. Thesewould not be effective candidates for antibiotic targeting. Manyriboswitches also do not operate in an entirely binary fashion, inthat they tune downstream gene expression up or down, but donot confer stringent on/off control [232] This could be expectedto limit their utility as antibiotic targets; however, it surprisinglyhas not seemed to be a major factor for the lysine riboswitch,which is the target of validated antibiotics. Whether this is alsothe case for other riboswitches remains to be determined.

7. Challenges and future directions

A major challenge facing all antibiotic applications is resistance.Resistance arises most commonly through target modification (e.g.mutation or post-transcriptional modification), antibiotic inactiva-tion, or drug efflux [233]. Significant efforts in both academic andindustrial laboratories are being dedicated to establishing ways ofcircumventing resistance. One approach that has proven successfulis the development of (semi-) synthetic compounds derived fromestablished antibiotics [234]. These new compounds usually targetthe same site as existing antibiotics, but are modified in ways thatminimize the effect of target modification. This is, however, des-tined to be an endless cycle, with resistance to these second gener-ation drugs having already been observed. This is necessitating thedevelopment of successive generations of drugs derived from thesame scaffold. In an attempt to break this resistance cycle, combina-tion therapies are gaining traction [235]. This typically involves thesimultaneous administration of several antibiotics targeting dis-tinct cellular processes (e.g. ribosome assembly, and translation ini-tiation/elongation). As we develop a more detailed mechanisticunderstanding of non-coding RNA action in the bacterial cell, thereis the potential to exploit the activities of antibiotics targeting dif-ferent systems (or different aspects of the same system), in devisingthe most effective combinatorial strategies.

When considering antibiotic development, specificity is a par-ticular concern for antibiotics targeting non-coding RNAs. Manybacterial non-coding RNAs have equivalent counterparts ineukaryotic cell, and thus off-target effects, and their accompanyingtoxicity, are a major concern. Maximizing specificity and efficacywill be an important goal in future drug development. This will

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almost certainly be aided by advances in structural biology, andan unprecedented ability to understand – at a molecular level –the interactions between antibiotics and their targets, and willbenefit from the ability to compare complex structures fromorganisms inhabiting different kingdoms. It is important to notethat there may also be species-specific differences in the bindingof antibiotics to different non-coding RNAs/associated proteins.These differences may lead to altered antibiotic activities in differ-ent species. While there are obvious benefits associated withbroad-spectrum antibiotics, there is also an increasing interest inspecies-specific drugs capable of targeting pathogens, while leav-ing the host natural microbiota unperturbed. As such there maybe a place for species-specific drugs in the future.

Finally, there are obstacles to be circumvented in moving fromthe bench, where interesting molecules inhibit RNA action in atest-tube, to the clinic, where these molecules inhibit disease/pathogenesis in mammalian systems. Identifying compounds cap-able of inhibiting essential biochemical reactions represents animportant first step. Those compounds that inhibit bacterialgrowth are obvious candidates to prioritize, although as discussedabove, there are many strategies being explored to enhance antibi-otic uptake. Further modifying antibiotics of interest to have thenecessary stability, bioavailability, and lack of toxicity for clinicalapplication represents a final hurdle, and one that presents a sig-nificant challenge. Despite these challenges, it is critical that wecontinue populating the discovery/developmental pipelines withpossible antibiotic candidates, and continue exploring the biologyof essential systems - such as those directed by non-coding RNAs- to better understand their function and how to manipulate or dis-rupt their activities. Every molecule that fails to make it to clinicalapplication has potential utility as a probe of biological activity. Inthe case of non-coding RNAs, expanding the repertoire of chemicalprobes that affect RNA activity can only serve to enhance ourunderstanding of non-coding RNA function, and ultimatelyimprove our ability to rationally alter its activity.

Acknowledgements

This work was supported by an NSERC Discovery Grant (No.04681), and an NSERC Discovery Accelerator Supplement to M.A.E.

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