(Dis)similar Analogues of Riboswitch Metabolites as Antibacterial

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(Dis)similar Analogues of Riboswitch Metabolites as Antibacterial Lead Compounds Miniperspective Daniel Matzner and Günter Mayer* Life and Medical Sciences Institute, University of Bonn, Gerhard-Domagk-Strasse 1, 53121 Bonn, Germany ABSTRACT: The rise of antimicrobial resistance in human pathogenic bacteria has increased the necessity for the discovery of novel, yet unexplored antibacterial drug targets. Riboswitches, which are embedded in untranslated regions of bacterial messenger RNA (mRNA), represent such an interesting target structure. These RNA elements regulate gene expression upon binding to natural metabolites, second messengers, and inorganic ions, such as fluoride with high affinity and in a highly discriminative manner. Recently, efforts have been directed toward the identification of artificial riboswitch activators by establishing highthroughput screening assays, fragment-based screening, and structureguided ligand design approaches. Emphasis in this review is placed on the special requirements and synthesis of new potential antibiotic drugs that target riboswitches in which dissimilarity is an important aspect in the design of potential lead compounds.

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THREAT OF MULTIDRUG RESISTANT BACTERIA Antimicrobial resistance (AMR) is a major global health concern. In particular, multidrug resistant bacteria or methicillin-resistant Staphylococcus aureus (MRSA) is responsible for mounting healthcare costs and complications in treatment and deaths. MRSA is one of the most widespread causes of antibiotic-resistant healthcare-associated infections (HAIs) worldwide. In the U.S. 23 000 people die each year because of drug-resistant infections, adding enormous costs to the healthcare system.1 These expenses are hard to estimate but the additional costs in all U.S. hospitals for treating resistant infections could reach U.S. $25−35 billion. In the EU extra healthcare costs and productivity losses are estimated at up to €1.5 billion per year.1−3 According to the European annual epidemiological report of 2012, the occurrence of MRSA has stabilized or decreased, but in general, these data show large variations and the percentage of MRSA among all S. aureus cases isolated was still above 25%.4 The development of widespread resistance among different human pathogens is a consequence of antibiotic (mis)use. Antibiotics are among the most commonly prescribed drugs, and large amounts of antibiotics are preventatively used on healthy animals in food production.5 This incurs a high selective pressure on bacteria to develop resistances, which occur quickly, in comparison to the date of introduction of an antibiotic regimen and the observation of antibiotic resistant strains. For example, methicillin was introduced in 1960 and MRSA was identified only 2 years later.1 The use of antibiotics in food production or to treat infections caused by inadequately identified bacteria goes hand in hand with the application of broad-spectrum antibiotics. These drugs kill pathogenic bacteria © XXXX American Chemical Society

but also normally colonized microbes that are part of the inevitable microbiome in humans. With this in mind, novel approaches to identify new antibiotics with specific mode of action are urgently needed. Besides traditional systemic approaches, in which new compounds are investigated for their general potential to inhibit bacterial growth and/or virulence, new antibacterial drug targets have to be identified. Well-established antibiotics target a narrow spectrum of cellular processes, as they affect the function of only four bacterial life processes.6 In this regard, novel drug targets and new chemical entities targeting them need to be identified and developed, respectively. Riboswitches represent such a novel class of potential target structures, and in recent years we have seen the offspring of endeavors for developing compounds with antibacterial activity that actually act on these RNA elements. This is reflected by several review papers dealing with the discovery of small molecules that specifically bind to bacterial riboswitches and therefore could function as antibacterial drug candidates.7,8 Riboswitches are typically found in the 5′-untranslated region of bacterial mRNA and are composed of an aptamer domain and an expression platform. The aptamer domain senses the presence of a cognate metabolite at a defined concentration. Upon binding, the metabolite usually triggers a negative feedback-loop mechanism, whereby conformational changes are induced and relayed to the expression platform that modulates gene expression (Figure 1). The most common mechanisms to control gene expression are either the induction Received: June 9, 2014

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DOI: 10.1021/jm500868e J. Med. Chem. XXXX, XXX, XXX−XXX

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Journal of Medicinal Chemistry

Figure 1. Negative feedback loop mechanism utilized by several riboswitches. Upon binding of a metabolite, riboswitches regulate gene expression, thereby inhibiting the production of certain proteins that are in turn important for the biosynthesis of a particular metabolite sensed by the RNA element.

of transcription attenuation or inhibition of translation initiation (Figure 2a,b).9 Moreover, riboswitch regulation is possible through a combination of translation initiation and mRNA decay as shown for the lysC riboswitch,10 Rhodependent transcription termination in the mgtA and the ribB riboswitch11 and even trans-acting riboswitches have been described.12 Apart from these, another mechanism utilized is the regulation of gene expression by self-cleavage (Figure 2c). Until now 24 classes of riboswitches are known,9 several of them are studied in depth, e.g., riboswitches that sense thiaminepyrophosphate (TPP), 13 S-adenosyl methione (SAM),14−18 flavin mononucleotide (FMN),19 lysine,20 purines,21−23 cyclic-di-GMP,24,25 cyclic-di-AMP,26 and glucosamine 6-phosphate (GlcN6P).27 Furthermore, orphan riboswitches whose ligands remain elusive yet are known, with yybP, pfl, and ykk being the most numerous orphans and several predicted riboswitch motifs that need to be investigated.9,28,29 Riboswitches were found in several bacterial species, some of them in high priority human pathogens, like Clostridium dif f icile (14 000 deaths/year), Listeria monocytogenes, Mycobacterium tuberculosis, Acinetobacter baumanii (500 deaths/year), Pseudomonas aeruginosis (440 deaths/year), Streptococcus pneumonia (7000 deaths/year), Salmonella enterica (450 deaths/year), and Staphylococcus aureus (11285 deaths/year).1,6 Other than bacteria, riboswitches were identified in eukaryotes, namely, fungi and plants, where regulation of splicing is one mode of action identified, but none have been found in humans so far.

Figure 2. Common mechanisms employed by riboswitches to regulate gene expression: (a) regulation of translation initiation by sequestering the Shine−Dalgarno (SD) sequence upon metabolite binding; (b) transcription termination by formation of a terminator hairpin followed by a consecutive stretch of uridines; (c) unique mechanism of the glmS riboswitch. GlcN6P triggers ribozyme cleavage, and subsequently translation is prohibited by induced mRNA degradation by RNase J1.

recognition mechanism30 or by a perfectly preorganized metabolite-binding cavity.31,32 Riboswitch-metabolite interactions are realized through the following: multiple hydrogen bounds (e.g., purine riboswitches),21,33 electrostatic interactions, e.g., recognition of the ammonium group in lysine,34,35 or chelating and bridging mono- or bivalent metal-ions, like Mg2+ions mediating interactions between the phosphate groups of GlcN6P31,32 or TPP36,37 to their respective riboswitch. Hydrophobic interactions of metabolites, e.g., stacking of aromatic rings with corresponding bases are common (e.g., TPP36,37 or FMN38).39 Ligand affinity differs not only between distinct riboswitch classes but also within riboswitches of one class. For example, GlcN6P activates the glmS riboswitch of S. aureus with a determined half-maximal effective concentration (EC 50 value) of ∼4 μM, 40 whereas the homologous representative from B. subtilis is activated at 50-fold higher metabolite concentrations (EC50 ≈ 200 μM).27 This observation of varying affinities for the same ligand in the same riboswitch class is supported by the identification of a tunable aptamer region in the purine riboswitch.41 A remarkable affinity has been observed for the c-di-GMP class I riboswitch, which revealed a dissociation constant of 10 pM at most. This astonishing affinity, paired with a slow kon rate that is 4 orders of magnitude below the diffusion-controlled limit,42 led Shanahan et al. to perform binding experiments.

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LIGAND BINDING AND RNA SWITCHING Riboswitches distinguish a particular metabolite with high affinity from a complex repository of compounds of varying concentrations. This implies specific interactions between RNA and ligand, combined with a perfectly evolved binding pocket that covers the ligand, most likely through an adaptive B


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Journal of Medicinal Chemistry They tested a fluorescently labeled variant of the class I c-diGMP binding riboswitch from Vibrio cholerae exhibiting a lower affinity for c-di-GMP and faster achievement of binding equilibrium.43 With this modified riboswitch, they were able to measure binding affinities of c-di-GMP analogues and elucidate ligand moieties important for binding. High affinity is not the sole factor for successful gene regulation by riboswitches. Riboswitches exert ligand binding while at the same time transcription is in progress.44 This makes clear that kinetic factors are important for efficient regulation because ligand binding may not reach thermodynamic equilibrium. One important kinetic factor is the speed of the RNA polymerase relative to the rate constant for ligand association to the correctly folded riboswitch or at least the upstream aptamer domain.9 Thus, riboswitches under kinetic control might require significantly higher ligand concentration than their actual Kd suggests to productively regulate gene expression.45 A first step toward addressing riboswitches as an antibacterial drug target is setting up appropriate screening systems, which allow the identification of potent artificial activators acting on riboswitch classes present in human pathogenic bacteria. As of yet, efforts have been taken to identify and design ligands by various approaches. For example, traditional screening assays of chemical compound libraries for glmS riboswitch activators,46,47 fragment-based screening,48,49 and structure-guided compound design of, for example, lysine analogues have been developed and employed with varying success.35 All of these ideas do not necessarily implicate success in finding a better or even comparable potent riboswitch activator. An important prerequisite for those compounds is the ability to activate the riboswitch, most likely by inducing conformational changes relayed to the expression platform. This factor may sound trivial, but most screening assays would utilize affinity as a first observable parameter. Conformational changes in the expression domain or rather the whole riboswitch are hard to predict even in cases where riboswitch classes are well characterized and supported by high-resolution crystal structures. For most of the riboswitches, the isolated aptamer domain was investigated by X-ray structural analysis; however, even the crystal structures of whole riboswitches, e.g., the SAM riboswitch, allow insight only into a static situation. Dynamic methods, e.g., electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), small-angle X-ray scattering (SAXS),50,51 or fluorescence resonance energy transfer (FRET), would allow advanced prediction and insight into adaptive rearrangements upon ligand binding.52,53 Development of direct readout formats that give not only access to binding but also the activation status of a defined riboswitch upon ligand binding is desirable. This has been realized in the case of the self-cleaving glmS riboswitch, which is even adaptable to state-of-the-art high-throughput screening formats.40,46 The riboswitches’ strict discrimination between closely related molecules through multiple interactions and ligandencircling binding pockets make artificial ligand identification a challenging task. Thus, design and testing of metabolite analogues are limited by a small scope that allows incorporation of variations at different positions of the natural ligand without loss of binding. However, in-depth understanding of the underlying principles and molecular architectures that are required for ligand binding and most importantly for riboswitch activation might reveal modifiable sites of ligands able to form new contacts with the riboswitch of choice. In this regard, the

possible alterations are manifold. The acceptance of artificial ligands with a scope of modifications could be shown for the TPP riboswitch, to which binding and activation have been shown for pyrithiaminepyrophosphate (PTPP),54 and the same is true for the class of purine riboswitches.33 PTPP is a long known antibiotic whose activity is at least partially due to action on the TPP riboswitch.54 Also validated as artificial activators are the long known lysine analogue L-aminoethylcysteine (AEC),55 PC1,56 a guanine analogue, and carba-GlcN6P,40 a pseudo-sugar analogue of GlcN6P.

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HIGH-THROUGHPUT SCREENING A straightforward approach to identify small molecules that could function as antibacterial lead compounds is the use of screening methods of which high-throughput methods allow rapid investigation of diverse chemical compounds libraries that would otherwise not be accessible. Hickey et al. were able to synthesize a fluorescent analogue of S-adenosyl methione that allows high throughput screening for SAM-I riboswitch binders via a fluorescence polarization assay.57 Also Furukawa et al. were able to devise an assay that would allow rapid screening for c-di-GMP analogues by in vitro selection of a fused allosteric ribozyme consisting of a c-di-GMP-I riboswitch aptamer and a hammerhead ribozyme and a short bridging sequence.58 Another riboswitch that enables screening of chemical libraries in a straightforward manner is the class of glmS riboswitches. These RNAs are unique, since they regulate gene expression through a ligand-activated self-cleavage mechanism. Thus, the glmS riboswitch acts as an enzyme utilizing Dglucosamine 6-phosphate (GlcN6P) as a cofactor. The scission process yields two fragments, one with a 2′,3′-cyclic phosphate and the other bearing a 5′-OH group.32,59 The cleavage products can be labeled either radioactively or fluorescently to be identified, e.g., by different retention times during polyacrylamide gel electrophoresis, changes in fluorescence polarization, or FRET, whereas the last two are compatible with characteristics of high-throughput approaches.46,47 However, both assays did not identify new compounds but confirmed GlcN as an activator of the glmS riboswitch, albeit 50-fold higher concentrations compared to GlcN6P are required. These findings reflect the highly discriminative characteristics of the glmS riboswitch, in which ligand recognition and high affinity binding are accomplished through multiple hydrogen bonds to the OH groups, the ring oxygen and predominantly through interaction with one Mg2+ ion coordinating the phosphate group of the metabolite.31 Nevertheless high-throughput screening (HTS) approaches could close the gap between the chemical space which is accessible through a large number of synthetic libraries and potential artificial riboswitch ligands that must meet high requirements, reflected by restricted space and the limited accessibility of the metabolite-binding pocket. HTS is imaginable for all riboswitch classes, though traditional druglike compound libraries may not yield potent activators.8 Rather, novel chemically focused libraries that are inspired by the metabolite-core structure will apply and yield novel riboswitch activators. In silico screening may especially increase the diversity of riboswitch compounds based on the cognate ligand as a first step in lead structure discovery.60 As already stated in the previous paragraph, HTS approaches mainly focusing on low Kd values as sole hit criteria do not guarantee success in finding active and potentially antimicrobial compounds because of numerous requirements that particularly C


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value of 2−50 μM) more reasonable. However, further studies on the binding of four hits resulting from the fragment-based screening show rearrangement of the pyrophosphate sensor helix distinct from that of the complex of the TPP riboswitch with its cognate ligand or thiamine.68 A correct induced fit mechanism of these fragments and the ability to induce subsequent conformational changes in the expression platform and, thus, the ability to regulate gene expression could not be tested. Therefore, fragment-based screening does not primarily aim to find new riboswitch activators. Rather promising binding motifs could be identified which would help to design more sophisticated molecules (>250 g/mol) to address the complex requirements for ligand binding and riboswitch activation, which requires a pyrophosphate group.

aim at an in vivo application. One crucial criterion that could already be implemented in screening approaches is binding kinetics of compounds to the aptamer domain and conformational changes of the riboswitch upon compound recognition while transcription is ongoing. This would also result in hit structures that not only bind but also activate the appropriate riboswitch. The importance of temporal aspects on riboswitch regulation is excellently reviewed.8,44,61,62 Chinnappan et al. utilized a dual molecular beacon assay to monitor transcription regulation upon binding of different artificial ligands of the pbuE adenine riboswitch during the transcriptional process.63

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FRAGMENT-BASED SCREENING Fragment-based screening approaches utilize libraries of small molecules with a molecular weight of less than 250 g/mol and have already been used for the identification of artificial protein ligands.64,65 Using libraries with less complex compounds allows efficient access to chemical space and could also be advantageous considering the limited access within specific binding pockets. This approach would yield lead compounds with low millimolar to high micromolar affinity that can then be optimized by increasing complexity.65,66 A broad spectrum of methods, e.g., NMR, isothermal titration calorimetry (ITC), and Thermofluor are available for measuring ligand binding to riboswitches.67 Equilibrium dialysis was used to identify ligands recognizing the TPP riboswitch within a library of ∼1300 fragments. These were screened for their ability to displace thiamine from the riboswitch.66 The TPP riboswitch represents an interesting target, since it regulates gene expression of genes that code for proteins involved in transport and synthesis of thiamine in bacteria. This riboswitch is an OFF switch, regulating gene expression by either transcription attenuation or inhibition of translation initiation in bacteria. The binding affinity of TPP to the riboswitch was measured through in-line probing experiments yielding Kd values between 600 nM (thiM riboswitch) and 100 nM (thiC riboswitch).13 The riboswitch harshly discriminates between TPP and thiamine by a factor of ∼200. Ligand recognition and binding are mediated through two flexible sensor helices, the pyrophosphate sensor helix and the pyrimidine sensor helix. Together they form a clamp, which is closed upon binding of TPP and is stabilized by new hydrogen bonds between both helices.37 This metaboliteinduced conformational change in the aptamer region of the riboswitch is supported by SAXS analysis by Baird et al.50 The pyrophosphate group is recognized through direct hydrogen bonds between the aptamer and pyrophosphate and further through coordination of Mg2+ ions that bridge the pyrophosphate moiety with the RNA backbone.37 The aromatic pyrimidine ring is bound by a second sensor helix through stacking interactions, where base pairing with a guanine and a hydrogen bond with the 2′-OH group of another guanine occurs. The central thiazole ring of TPP could be excluded to be important for ligand discrimination.49 This explains why the well-known antibiotic PTPP acts on the TPP riboswitch with almost the same affinity as the natural metabolite TPP.54 Fragment-based screening of Cressina et al. resulted in 20 hit compounds with dissociation constants ranging from 20 to 280 μM, and 10 of these were specific for the TPP riboswitch.48 The lower affinity compared to TPP is evident, as mainly the pyrimidine sensor helix will be addressed by these fragments, since the readout of the primary screening was competition of thiamine, which binds in the pyrimidine sensor helix. This makes a comparison to the binding affinity of thiamine (Kd

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STRUCTURE-GUIDED LIGAND DESIGN In the past decade most of the research in the riboswitch field has dealt with the identification of new riboswitch classes, basic research on the recognition of metabolites, and the mode of gene regulation. These research endeavors yielded many highresolution crystal structures of riboswitches bound to their corresponding metabolites, e.g., lysine, purines, TPP, GlcN6P, or cyclic-di-GMP. In most cases the structure of the isolated aptamer domain was obtained and provides a starting point for ligand design, synthesis, and testing. In this way, crystal structures excellently describe the recognition of the metabolite engaged in functional groups, aromatic ring systems, electrostatic contacts, or even hydrophobic interactions. This is exemplified by the interaction of the glmS riboswitch with GlcN6P, whereas 12 direct interactions were observed consisting of hydrogen bonds of the OH groups, the ring oxygen and predominantly through interaction with Mg2+ ions coordinating the phosphate group of the metabolite within the RNA structure.69 This shows that almost every side chain and functional group of a metabolite are engaged in RNA binding, accounting for the high affinity and specificity of riboswitches. Similarly, the ligand-binding pocket of purine riboswitches is highly conserved among this class45 and seven hydrogen bonds were observed to be formed between the RNA and its purine ligand (Figure 3).70 Of these connections two nucleotides, namely, U51 and Y74, are critical for ligand binding and, thus, are of special interest for the development of metabolite analogues.45,70 The pyrimidine residue (Y74) establishes a base pair with the ligand and defines the specificity of the riboswitch for either adenine (U74) or guanine (C74). The available structures reveal that space for chemical variation in riboswitches with tight binding pockets as with glmS or purine riboswitches might be very limited but could nevertheless allow insight and access to distinct sites for synthesizing metabolite analogues that still retain riboswitch binding and activating properties. This has been disclosed in the case of lysine riboswitches, in which free space was available next to C4 (Figure 4) and an access next to the carboxyl group.35 Likewise, the lack of recognition of the central thiazole in the TPP riboswitch can be exploited to design and synthesize TPP analogues with various modifications or substitutions of the central thiazole moiety, as exemplified by PTPP and triazolethiamine pyrophosphate (TTPP), which still bind and activate the TPP riboswitch.49,54 High-resolution crystal structures also form the basis for structure-guided design of artificial riboswitch ligands by probing of different positions and functional groups in a ligand. This has been done in a comprehensive manner for GlcN6P, in D


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findings, D-glucosamine activates the riboswitch but to a much lesser extent. Furthermore, tolerated modifications to the hydroxyl groups were investigated, showing minor decreases in activation due to removal of the 3-OH group, whereas the 4OH group is critical.40,71 The anomeric center is of particular interest, as the position of the 1-OH relative to the 2-amine is critical for enzymatic activity. It has been shown that only the α-anomer is active. This explains why L-serine, which possesses the same relative conformation between 1-OH and 2-amine, is active while D-serine is not.72 Lünse et al. were the first to identify the pseudo-sugar variant of GlcN6P analogue, named carba-GlcN6P (Figure 5a). This variant is able to activate the

Figure 3. Recognition of guanine by the xpt guanine riboswitch (PDB code 1Y27). Seven hydrogen bonds (dashed lines) are formed between uridine, cytidin, and the ligand. Residues C74 and U51 are marked, since they define specificity of the RNA toward guanine. Parts of the riboswitch that are not directly engaged in binding to guanine are removed in this illustration. Figure is based on Kim et al., 2009.70

Figure 5. (a) The natural (GlcN6P) and the most active artificial metabolite (carba-GlcN6P) of the glmS riboswitch. Arrows indicate groups that are important for ligand recognition, e.g., forming hydrogen bonds whose modification usually leads to a dramatic decrease of affinity/activity. (b) Scheme of a linear 12-step synthesis yielding carba-GlcN6P.

glmS riboswitch almost as efficiently as the natural metabolite. The EC50 of carba-GlcN6P is 6.2 μM compared to 4 μM of GlcN6P (S. aureus). Similar to hydroxyl groups, the ring oxygen is also capable of forming hydrogen bonds to adjacent nucleotides. This is obviously lost in the carba analogue, explaining the slightly lower affinity/activity. The carba position might represent a valuable starting point to generate novel pseudo-sugar variants that cope with the loss of RNA interactions at this site. However, the synthesis of carbaGlcN6P analogues is extremely complex, as it takes at least 12 steps of linear organic synthesis combined with costly purification steps (RP-HPLC) (Figure 5b). Illustrated by PTPP, the thiazole ring of TPP represents a flexible position to develop novel analogues activating the TPP riboswitches. Variants containing triazole moieties instead of a thiazole are synthetically accessible through a so-called “click” chemistry approach. This also allows the introduction of modified pyrophosphates, which are necessary for maintaining efficient riboswitch activation. For example, compound 20, bearing a methylenediphosphonate group instead of pyrophosphate (Figure 6), has been shown to be the most potent TPP analogue with an affinity (Kd value of 9 nM) very close to the one observed by TPP.49 This approach points in the direction

Figure 4. Binding pocket of the lysine riboswitch from Thermotoga maritima (PDB code 3D0U) with bound lysine shown as a cross section through the structure. The opening next to the carboxyl group of lysine is indicated (red arrow) together with free space around the C4 position (red curve). Figure is based on Serganov et al.34,35

which the conformations of most of the hydroxyl groups were changed, the amino group was modified or deleted, and the ring oxygen was substituted by a methylene group leading to a pseudo-sugar.40,47 Through this approach the contributions of different functional groups of the metabolite to maintain affinity for RNA binding and induction of cleavage have been investigated and tolerated or incompatible modifications were elucidated. The amine function has been shown to be irreplaceable. DGlucose 6-phosphate, as the closest related compound missing the amine, was used to obtain crystal structures of the noncleaved riboswitch.31 In many biochemical studies the importance of the phosphate group in maintaining high affinity binding to the RNA has been elucidated. In line with these E


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Figure 6. Natural and artificial metabolites of the TPP riboswitches. PTPP is a validated antibacterial drug acting at least partially on TPP riboswitches. Gray areas mark positions where artificial modifications lead to active analogues. Arrows indicate groups that are important for ligand recognition, e.g., forming hydrogen bonds whose modifications lead to a dramatic decrease of affinity/activity.

of compounds that could be independent of bacterial transportation/phosphorylation systems (vide infra). A starting point for research on new metabolites of lysine riboswitches is the antibacterial analogue L-aminoethylcysteine (AEC), for which binding to the lysC riboswitch was found.20 Screening of a small library of L-lysine analogues identified structural motifs, which are important for recognition and the regulative activity of this metabolite. The length of the alkyl chain, the carboxylic group, the α-amino group, and the 6amino group are important factors in ligand binding and discrimination. AEC differs from L-lysine only by a sulfur group at position 4 of the alkyl chain, similar to L-4-oxalysine or DLtrans-2,6-diamino-4-hexenoic acid, where a formal oxygen or a double bond substitution at this position was made respectively (Figure 7).55 These analogues reveal affinities comparable to the natural ligand, and Kd values ranging from 1 to 13 μM were determined. Another position that gave yield to interesting analogues of L-lysine is position N6. L-Homoarginine, exhibiting a substitution of the ε-NH2 group with a guanidinium moiety, binds the riboswitch with relatively high affinity (Kd value of 7 μM) as compared to the cognate ligand.35 Many artificial ligands of purine riboswitches have been identified and investigated based on pyrimidine and purine chemical frameworks. Several purine analogues were identified emanating from cocrystal structures of the aptamer domains of four members of the purine riboswitch family in complex with the natural metabolite or derivatives thereof.21,33,45,73,74 Modifications near C2 and C6 (Figure 8A) were predicted to be well tolerated due to space availability near these groups as revealed by crystal structures solved of the aptamer region of the xpt-pbuX riboswitch bound to guanine.70 C2- and C6modified guanines were analyzed by Kim et al., resulting in ligands with similar or even a higher affinity compared to the natural metabolite guanine, which binds the riboswitch with a Kd value of 5 nM. For example, the C2-modified compound G4 recognizes the riboswitch with a Kd value of 0.5 nM, whereas the C6-modified compound G7 revealed a lower affinity (Kd value of 20 nM).70 Advantageously, the small library used in the study could be synthesized from commercially available starting materials, e.g., 2-bromohypoxanthine (C2-modified) and 2-

Figure 7. Natural and artificial metabolites of the lysine riboswitch. Gray areas mark positions where artificial modifications lead to active analogues. Arrows indicate groups that are important for ligand recognition, e.g., forming hydrogen bonds whose modification usually leads to a dramatic decrease of affinity/activity.

amino-6-chloropurine (C6-modified) in a one-step nucleophilic substitution reaction (Figure 8a).

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ACTIVITY OF ARTIFICIAL RIBOSWITCH LIGANDS There are advanced requirements for potential riboswitch targeting drugs that need to be investigated depending on the riboswitch class, compound uptake, and the type of regulation that would lead to an antimicrobial effect in the bacteria in question. In this context, the gene that is regulated by a riboswitch needs to be essential or necessary for bacterial growth or its virulence. This prerequisite causes the observation that among the 24 classes of riboswitches known today, probably fewer are predictive of developing antibiotics. Another advanced requirement for a potential antibiotic is the incapability of substituting the natural ligand in bacterial metabolic processes to maintain activity and prevent potential off-target effects. Compounds that act on riboswitches not only need to bind to a particular riboswitch but, in the case of OFFswitches, have to activate it to exert antibacterial activity. Thus, upon binding they should also induce conformational changes within the riboswitch structure that ultimately lead to inhibition of gene expression and, thereby, loss of protein function. Depending on the method employed for compound identification, riboswitch ligands may not necessarily guarantee activation of a particular riboswitch in vivo. Most approaches rely on riboswitch binding data in vitro, e.g., fragment-based screening aimed at thiamine competitors,66 and only rarely imply induction of structural changes or even regulation of gene expression in the first place. The glmS riboswitch represents an exception in this regard, since the available assays for compound identification in vitro utilizes this riboswitchs’ unique RNA self-cleavage activity.40 The special regulation F


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Figure 8. (a) Synthesis scheme of guanine analogues starting from 2-bromohypoxanthine (C-2-modified) and 2-amino-6-chloropurine (C-6modified) in one step by nucleophilic substitution reactions. (b) Natural and artificial metabolites of the guanine riboswitch. Gray areas mark positions where artificial modifications lead to active analogues. Arrows indicate groups that are important for ligand recognition. (c) Instability of guanine analogue PC1 through self-condensation via oxidation in the presence of air forming colored pyrimido[5,4-g]pteridines. (d) One-step synthesis scheme of PC1 starting from 2,6-diamino-5-nitroso-4-pyrimidinol.

described for the FMN riboswitch.75 Subsequent studies in bacteria using riboswitch-dependent reporter genes, e.g., βgalactosidase or fluorescent reporter proteins, garner insight on the impact of compounds on riboswitch activation.56 The proof that a ligand analogue of a certain riboswitch is able to induce switching and consecutively regulate gene expression is still far away from yielding an antimicrobial drug. In many cases the riboswitch does not control all biochemical pathways of biosynthesis for the corresponding metabolite; therefore, the bacterium would survive by non-riboswitch controlled pathways.76 Even with all pathways of the respective metabolite synthesis down-regulated by one riboswitch class, the bacterial cell could still survive by the use of transporter proteins and the uptake of the metabolite if it is present in the surrounding media.55 Ultimately, the effect on bacterial growth of promising compounds is determined. This has already been demonstrated for artificial compounds acting on purine, TPP, and FMN riboswitches.54,56,70,77,78 Interestingly, the compound with the highest affinity toward the guanine riboswitch identified by Kim et al., named G4, was not able to inhibit bacterial cell growth whereas the weaker binding compound G7 inhibited growth of B. subtilis, and a minimal inhibitory concentration (MIC) of 260 μM was observed.70 In contrast, Mulhbacher et al. identified the pyrimidine-related compound 2,5,6-triaminopyrimidin-4-one, named PC1 (Figure 8b), which interacts with guanine riboswitches (Kd value of ∼100 nM) and inhibits growth of S. aureus in rich cation-adjusted Mü ller−Hinton broth

mechanism described for the lysine riboswitch lysC allows the testing of artificial ligands independently on either inhibition of translation initiation or exposition of RNase E cleavage sites followed by mRNA decay.10 Currently, the latter mechanism of gene regulation lacks proof with artificial activators but enables future testing of promising compounds in a similar straightforward way as for the glmS riboswitch. The Rho depending gene regulation is related, as it also utilizes an external protein factor and is described for the FMN riboswitch ribB from E. coli that regulate transcription by promoting a conformation that stimulates Rho-dependent termination.11 Again the use of this mechanism, as a method to verify artificial ligand activity, is not described, but it has to be kept in mind to identify promising drug candidates for this class of riboswitches. In-line probing assays, as they have been used to test compounds, e.g., on lysine riboswitches35 or guanine riboswitches,56 not only address binding of a potential ligand but allow for the investigation of conformational changes of the RNA construct. Comparison of in-line probing profiles of artificial analogues with those obtained for the natural metabolite allows prediction of the activity of artificial compounds in regard to riboswitch activation. In this context temporal aspects for riboswitches under “kinetic control” come into play, as there might be large differences between the Kd of the isolated aptamer domain and the concentration that is actually required to trigger half maximal regulatory response (T50) during transcription assays.44 The discrepancy between in vitro observations and the transfer of assays to in vivo conditions is comprehensibly G


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Journal of Medicinal Chemistry (CAMHB) with a MIC of 4 mM.56 In a subsequent study Ster et al. showed that the MIC could be lowered to 14 μM by adding 1.5 mM DTT, preventing self-condensation via oxidation in the presence of air (Figure 8C).79−81 Moreover, bactericidal activity of PC1 was observed in rich medium and in mammalian infection models.79 Compared to guanine, PC1 lacks one aromatic ring, whereas two amino groups at positions 5 and 6 substitute the pyrimidine-core structure, respectively. Important attributes of the natural metabolite that are conserved through this modification are the planarity, which preserves stacking interactions, and the possibility of forming two hydrogen bounds between the two amino groups, and the sensing nucleobases of the guanine riboswitch. Gram quantities of PC1 can be easily obtained, as it is commercially available from several sources, or it can be chemically synthesized in a one-step protocol from 2,6-diamino-5-nitroso-4-pyrimidinol allowing simple access to PC1 derivatives (Figure 8d).82 Lysine analogues substituted at position C4 (Figure 7) were also shown to inhibit growth of B. subtilis in chemical defined medium. MICs ranging from 26 to 940 μM were observed, whereas the N6-guanidium analogue L-homoarginine (Figure 7) did not inhibit bacterial growth despite its low Kd value. It rather supported growth of the B. subtilis strain probably because of metabolization and, thus, functioning as a lysine precursor.55 High affinity of the introduced lysine analogues and the potential antibacterial activity are not necessarily connected with each other, as Ataide et al. identified the lysyltRNA-synthetase (LysRS) as the primary target of AEC.83 Nevertheless antibiotic resistance according to AEC treatment is maintained by mutations in the riboswitch sequence. This can be explained by an indirect effect, since the loss of repression of lysC expression would increase the cellular level of lysine, which in the next step leads to competition of lysine with AEC for binding to LysRS.

Short RNA hairpins represent one tool to achieve this, as these molecular probes can easily be obtained by in vitro selection procedures85,86 or design87 and maintain high affinity and specific binding to one expression domain. In this way, Mayer et al. selected two RNA hairpins binding the thiM riboswitch.88 One hairpin has been shown to target the aptamer domain, while the other specifically recognizes the expression platform with a Kd value of 34 nM. The latter RNA hairpin is able to sense conformational changes that occur upon TPP recognition in the full-length riboswitch and more essentially in the expression platform.89 These sensor RNA hairpins can be used as reporter molecules in so-called aptamer displacement screens. In these, compound libraries are screened for those representatives that are able to compete, either directly or allosterically, with the RNA hairpin for target binding.90 This approach will allow the identification of compounds with a new mode of action on riboswitches.88

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CONFLICT BETWEEN SIMILARITY AND DISSIMILARITY OF ARTIFICIAL RIBOSWITCH LIGANDS Emanating from the mere native ligand or available crystal structures of certain classes of riboswitches, rational design of metabolite-analogues without a doubt allows access to an excellent understanding of the molecular structure−activity relationship of a riboswitch. In this way, the natural metabolite represents a point of reference, as the riboswitch’s binding motif is perfectly adapted to the requirements of its cognate ligand. This, of course, supports the aim to design an artificial ligand as similar as possible to the natural activator. The use of a similar compound is appealing, as it would most likely fit into the riboswitch’s binding pocket. Synthetic approaches to design a small library of analogues would be tangible; however, these compounds bear the risk to interact with bacterial metabolic processes. At this point, the dissimilarity aspect comes into play, as substitution of the natural ligand in any other role in bacterial cellular processes, except the activation of the riboswitch, should be ruled out to minimize potential off-target effects and to generate the ultimate selective compound. Unfortunately, dissimilarity gives an unsatisfactory and imprecise idea of the strategy to implement it in synthetically produced compounds. Appropriate screening approaches could close the gap between “similarity” and “dissimilarity”. In this context “similarity” is related to high affinity binding, activation of gene regulation by the riboswitch, upon binding under consideration of temporal factors for riboswitches under kinetic control. Screening of diverse chemical libraries especially combined with in silico screening focused on ligand binding motifs that would not be predictable through rational design could add an adequate amount of dissimilarity to hit structures. Fragment-based screening in particular already imply a certain degree of dissimilarity, as they screen libraries of compounds smaller than the cognate ligand of the riboswitch. This approach is advantageous, as hit structures of moderate binding affinity could function as leads that may be tuned to meet further ligand requirements that result from continuing insights into the world of riboswitches. Promising starting points are the hit compounds resulting from the fragment-based screening by Cressina et al. and the following insight into their binding behavior to the TPP riboswitch.48,68 To merge the almost equally contributing factors of a promising artificial drug candidate, namely, “similarity” and “dissimilarity” to “(dis)-

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EXPRESSION PLATFORM AS A “DRUGGABLE” SITE? The common mechanism through which riboswitches regulate gene expression is (i) recognition of a metabolite and (ii) conformational changes that occur upon metabolite binding and which are relayed to the expression platform. Except for the glmS riboswitch, all known bacterial riboswitches act in this manner and changes in the expression platform structure cause either transcription attenuation or inhibition of translation initiation. Obviously, the aptamer domain represents the logical target site for the time being to develop synthetic compounds acting on riboswitches. These domains are structurally very well described. Ligands that are bound by the aptamer domains are already known and serve as a starting point, inspiring synthesis and testing of metabolite analogues, which represent the vast majority of yet known compounds.84 Targeting the expression platform is also conceivable, though less straightforward. It can be imagined that compounds binding to the expression platforms also induce alterations in the RNA structure and thus changes in gene regulation patterns. A disadvantage of expression platform targeting might be the lower degree of conservation of these domains among a class of riboswitch. For example, different representatives of the TPP riboswitches act on the level transcription attenuation (thiC) or on the level inhibition of translation (thiM), therefore requiring completely different expression platforms while the aptamer domains are highly conserved.36,37 Also the identification of specific ligands that bind to expression platform modules is a challenging task. H


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able to identify chemical synthesized diphosphate-mimicking, metal-chelating triazolethiamine analogues that are indeed independent of ThiK and show transport-independent activity.98 These compounds are a good example of metabolite analogues that reveal sophisticated affinity through similarity but chemical modification adds a significant level of dissimilarity, since the triazole group is not likely to act as a coenzyme in pyruvate decarboxylation.99 Nevertheless, inhibition with enzymes that are involved in thiamine metabolism might be possible.100 Very recently Fei et al. were able to realize a similar approach for artificial activators of the glmS riboswitch by introducing phosphate mimicking groups at the 6-position of GlcN6P-analogues. 101 The most potent compounds containing either a malonyl ether or phosphonate substitution show ∼7-fold reduced activity toward riboswitch activation compared to GlcN6P. However, both modifications substantially increase the level of dissimilarity in these compounds, since phosphatase resistance is achieved by these nonhydrolyzable functionalities. PC1 represents the most comprehensively investigated compound to date that binds and activates guanine riboswitches and, most importantly, has been proven to inhibit bacterial growth in vivo.56,79 This compound may serve as a good starting point for analogues with higher activity, enhanced stability against oxidation, and lower MIC of therapeutically relevant levels. Moreover, one could imagine applying combination therapies combining PC1 with other well-known antibiotics. Some riboswitches require phosphorylated (glmS) or diphosphorylated (TPP) metabolites for activation; therefore, artificial compounds that act on these riboswitches may require in vivo activation, e.g., pyrithiamine or moieties that allow crossing bacterial membranes or cell walls and subsequent mimicry of riboswitch binding. This complicates synthesis efforts and adds further steps toward active synthetic compounds. Nonetheless, targeting riboswitches with artificial compounds is a promising research area that could lead to a selective antibacterial therapy but still is in its infancy. Since many riboswitch classes are only found in a distinct number of bacteria, this could lower the toxicity to probiotic bacteria and lead to more thorough use of antibiotics.8 Another interesting pathway in discovery of potential riboswitch activators is mining of natural compound libraries. It might be anticipated that if riboswitches are suitable targets for antibiotics, nature might have already evolved such compounds within its almost exhaustless library of substances. The finding that roseoflavin, a natural compound, activates the FMN riboswitch and exerts antibacterial activity already points into this direction.77,78,102,103 Roseoflavin belongs to the class of FMN analogues, thus, representing a similar compound. It will be intriguing to screen the entire repository of natural compounds to dig for dissimilar compounds that activate riboswitches and thereby exert antibacterial activity.

similarity”, the contribution of organic chemistry is inevitable but versatile.

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A CHEMIST’S PERSPECTIVE ON RIBOSWITCH LIGAND DRUG DESIGN From a chemical point of view, riboswitches provide an interesting novel starting point for developing antibacterial compounds, disclosing a fundamental new mode of action. The specific targeting of RNA structures is a challenging task, often limited because of unspecific interactions of hit compounds. Besides traditional antibiotics as, for example, aminoglycosides or macrolides, which target mainly rRNA, only a few other small molecules are known to target specific RNA structures and exert druglike properties.91−94 However, riboswitches specifically interact with metabolites, revealing that even RNA recognizes small molecules with unprecedented selectivity. These RNA elements went through a natural selection and evolution process that took millions of years, and thus, recognition of a cognate ligand by a riboswitch has been perfectly optimized and adapted. These natural ligands serve as valuable starting point for developing metabolite analogues. Indeed, most of the currently available synthetic compounds that have been developed to target riboswitches belong to the same class of compounds as the respectively natural metabolite and engage almost the same sites of interactions with the RNA as the natural precursor.47,55,95 This approach bears the inherent risk of interfering not only with riboswitch activities but also with enzymes that are involved in a metabolite’s catabolism or anabolism.55 Nevertheless, these compounds already prove that riboswitch targeting might be a valuable strategy to inhibit bacterial growth. Notably, the compounds identified thus far, e.g., targeting guanine, lysine, or TPP riboswitches, reveal MICs in the high micromolar to low millimolar range, and consequently further improvement is necessary to reach therapeutically relevant values. One route will be synthesizing novel derivatives, emanating from active riboswitch targeting metabolite mimics. These derivatives may engage in novel interactions with the cognate riboswitch structures and reveal enhanced biophysical properties. Available structural and dynamic data on the binding pockets will further support these activities, as it is the case for the preQ- and guaninesensing riboswitches.30,33,96 This branch focuses on the dissimilarity of the targeting compound, an important aspect for avoiding potential drugs to be metabolized by the pathogen or cross-reactivity with enzymes that naturally interact with the respective metabolite. From a synthesis viewpoint this approach is interesting, certainly feasible as modern organic chemistry equips researchers with powerful tools to design and synthesize diverse sets of compounds. Lafontaine and co-workers used molecular docking to screen a virtual library, actually finding PC1 binding to the xpt guanine riboswitch, which can be performed nowadays with online tools.97 This approach is yet underrepresented in possible compound design recognizing riboswitches probably because it is hard to foresee which artificial modifications will be tolerated or even engaged by the RNA. Thus, Chen et al. synthesized a comprehensive library of thiamine pyrophosphate analogues from which they identified the triazole-containing analogue of TPP as potent TPP riboswitch activator in vitro.49 In this context, Lünse et al. further investigated the dependency of triazole analogues, thiamine and pyrithiamine, on metabolic activation by thiamine kinase (ThiK) and thiamine transporters. Thereby they were

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +49 (0)228 734808. Home Page: www.mayerlab.de. Notes

The authors declare no competing financial interest. Biographies Daniel Matzner studied chemistry at the University of Bonn, Germany, and did his diploma thesis in Chemistry in the field of I


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(7) Deigan, K. E.; Ferre-D’Amare, A. R. Riboswitches: discovery of drugs that target bacterial gene-regulatory RNAs. Acc. Chem. Res. 2011, 44, 1329−1338. (8) Penchovsky, R.; Stoilova, C. C. Riboswitch-based antibacterial drug discovery using high-throughput screening methods. Expert Opin. Drug Discovery 2013, 8, 65−82. (9) Breaker, R. R. Prospects for riboswitch discovery and analysis. Mol. Cell 2011, 43, 867−879. (10) Caron, M. P.; Bastet, L.; Lussier, A.; Simoneau-Roy, M.; Masse, E.; Lafontaine, D. A. Dual-acting riboswitch control of translation initiation and mRNA decay. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, E3444−E3453. (11) Hollands, K.; Proshkin, S.; Sklyarova, S.; Epshtein, V.; Mironov, A.; Nudler, E.; Groisman, E. A. Riboswitch control of Rho-dependent transcription termination. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 5376−5381. (12) Loh, E.; Dussurget, O.; Gripenland, J.; Vaitkevicius, K.; Tiensuu, T.; Mandin, P.; Repoila, F.; Buchrieser, C.; Cossart, P.; Johansson, J. A trans-acting riboswitch controls expression of the virulence regulator PrfA in Listeria monocytogenes. Cell 2009, 139, 770−779. (13) Winkler, W.; Nahvi, A.; Breaker, R. R. Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature 2002, 419, 952−956. (14) 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. 1998, 30, 737−749. (15) Winkler, W. C.; Nahvi, A.; Sudarsan, N.; Barrick, J. E.; Breaker, R. R. An mRNA structure that controls gene expression by binding Sadenosylmethionine. Nat. Struct. Biol. 2003, 10, 701−707. (16) Corbino, K. A.; Barrick, J. E.; Lim, J.; Welz, R.; Tucker, B. J.; Puskarz, I.; Mandal, M.; Rudnick, N. D.; Breaker, R. R. Evidence for a second class of S-adenosylmethionine riboswitches and other regulatory RNA motifs in alpha-proteobacteria. Genome Biol. 2005, 6, R70. (17) Lu, C.; Smith, A. M.; Fuchs, R. T.; Ding, F.; Rajashankar, K.; Henkin, T. M.; Ke, A. Crystal structures of the SAM-III/S(MK) riboswitch reveal the SAM-dependent translation inhibition mechanism. Nat. Struct. Mol. Biol. 2008, 15, 1076−1083. (18) Poiata, E.; Meyer, M. M.; Ames, T. D.; Breaker, R. R. A variant riboswitch aptamer class for S-adenosylmethionine common in marine bacteria. RNA 2009, 15, 2046−2056. (19) Winkler, W. C.; Cohen-Chalamish, S.; Breaker, R. R. An mRNA structure that controls gene expression by binding FMN. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 15908−15913. (20) 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. 2003, 17, 2688−2697. (21) Batey, R. T.; Gilbert, S. D.; Montange, R. K. Structure of a natural guanine-responsive riboswitch complexed with the metabolite hypoxanthine. Nature 2004, 432, 411−415. (22) Mandal, M.; Breaker, R. R. Gene regulation by riboswitches. Nat. Rev. Mol. Cell Biol. 2004, 5, 451−463. (23) Mandal, M.; Breaker, R. R. Adenine riboswitches and gene activation by disruption of a transcription terminator. Nat. Struct. Mol. Biol. 2004, 11, 29−35. (24) Lee, E. R.; Baker, J. L.; Weinberg, Z.; Sudarsan, N.; Breaker, R. R. An allosteric self-splicing ribozyme triggered by a bacterial second messenger. Science 2010, 329, 845−848. (25) Sudarsan, N.; Lee, E. R.; Weinberg, Z.; Moy, R. H.; Kim, J. N.; Link, K. H.; Breaker, R. R. Riboswitches in eubacteria sense the second messenger cyclic di-GMP. Science 2008, 321, 411−413. (26) Nelson, J. W.; Sudarsan, N.; Furukawa, K.; Weinberg, Z.; Wang, J. X.; Breaker, R. R. Riboswitches in eubacteria sense the second messenger c-di-AMP. Nat. Chem. Biol. 2013, 9, 834−839. (27) Winkler, W. C.; Nahvi, A.; Roth, A.; Collins, J. A.; Breaker, R. R. Control of gene expression by a natural metabolite-responsive ribozyme. Nature 2004, 428, 281−286.

aptamer templated synthesis at the University of Bonn in 2011. He is currently working on his Ph.D. in the group of G. Mayer, and his research focuses on the synthesis of carbasugars as artifical activators of the glmS riboswitch. Günter Mayer received his Ph.D. in Chemistry in 2001 from University of Bonn, Germany. In 2001 he joined the biotechnology company NascaCell, which he cofounded, as head of the combinatorial biotechnology department. After rejoining academia in 2004, he finished his Habilitation in 2009 and became Reader of Translational Biology at the University of Strathclyde, U.K. In 2010 he was appointed Associate Professor of Chemical Biology and Genetics at the University of Bonn and promoted to Full Professor at the LIMES Institute, University of Bonn. His research is focused on light control of aptamer activity, synthesis and characterization of riboswitch targeting compounds, and cell targeting/manipulation with aptamers.

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ACKNOWLEDGMENTS

We thank Jeffrey Hannam, Anna Schüller, and Shannon Smith for critical reading of the manuscript. This work was made possible by funds provided by the Deutsche Forschungsgemeinschaft (Grants FOR 854, Ma 3442/2-2).

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ABBREVIATIONS USED AEC, L-aminoethylcysteine; AMR, antimicrobial resistance; CAMHB, cation-adjusted Müller−Hinton broth; EC50, halfmaximal effective concentration; EPR, electron paramagnetic resonance; FMN, flavin mononucleotide; FRET, fluorescence resonance energy transfer; GlcN6P, D-glucosamine 6-phosphate; HAI, healthcare-associated infection; HTS, highthroughput screening; ITC, isothermal titration calorimetiry; MIC, minimal inhibitory concentration; MRSA, methicillinresistant Staphylococcus aureus; NMR, nuclear magnetic resonance; PTPP, pyrithiamine pyrophosphate; RP-HPLC, reverse-phase high perfomance liquid chromatography; SAM, small-angle X-ray scattering (SAXS), S-adenosylmethione; TPP, thiamine pyrophosphate; TTPP, triazolethiamine pyrophosphate

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REFERENCES

(1) Centers for Disease Control and Prevention (CDC). Antibiotic resistance threats in the United States, 2013. http://www.cdc.gov/ drugresistance/threat-report-2013/pdf/ar-threats-2013-508.pdf (accessed Dec 18, 2014). (2) Roberts, R. R.; Hota, B.; Ahmad, I.; Scott, R. D., 2nd; Foster, S. D.; Abbasi, F.; Schabowski, S.; Kampe, L. M.; Ciavarella, G. G.; Supino, M.; Naples, J.; Cordell, R.; Levy, S. B.; Weinstein, R. A. Hospital and societal costs of antimicrobial-resistant infections in a Chicago teaching hospital: implications for antibiotic stewardship. Clin. Infect. Dis. 2009, 49, 1175−1184. (3) European Centre for Disease Prevention and Control. ECDC/ EMEA Joint technical report. The bacterial challenge: time to react, 2009. http://www.ecdc.europa.eu/en/publications/Publications/ 0909_TER_The_Bacterial_Challenge_Time_to_React.pdf (accessed Dec 18, 2014). (4) European Centre for Disease Prevention and Control. Annual epidemiological report, 2013. http://www.ecdc.europa.eu/en/ publications/Publications/annual-epidemiological-report-2013.pdf (accessed Dec 18, 2014). (5) World Health Organization. The evolving threat of antimicrobial resistance: options for action, 2012. http://whqlibdoc.who.int/ publications/2012/9789241503181_eng.pdf (accessed Dec 18, 2014). (6) Blount, K. F.; Breaker, R. R. Riboswitches as antibacterial drug targets. Nat. Biotechnol. 2006, 24, 1558−1564. J


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ligand interactions using thiamine pyrophosphate analogues. Org. Biomol. Chem. 2012, 10, 5924−5931. (50) Baird, N. J.; Ferre-D’Amare, A. R. Idiosyncratically tuned switching behavior of riboswitch aptamer domains revealed by comparative small-angle X-ray scattering analysis. RNA 2010, 16, 598−609. (51) Chen, B.; Zuo, X.; Wang, Y. X.; Dayie, T. K. Multiple conformations of SAM-II riboswitch detected with SAXS and NMR spectroscopy. Nucleic Acids Res. 2012, 40, 3117−3130. (52) Noeske, J.; Richter, C.; Grundl, M. A.; Nasiri, H. R.; Schwalbe, H.; Wohnert, J. An intermolecular base triple as the basis of ligand specificity and affinity in the guanine- and adenine-sensing riboswitch RNAs. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 1372−1377. (53) Furtig, B.; Richter, C.; Wohnert, J.; Schwalbe, H. NMR spectroscopy of RNA. ChemBioChem 2003, 4, 936−962. (54) Sudarsan, N.; Cohen-Chalamish, S.; Nakamura, S.; Emilsson, G. M.; Breaker, R. R. Thiamine pyrophosphate riboswitches are targets for the antimicrobial compound pyrithiamine. Chem. Biol. 2005, 12, 1325−1335. (55) Blount, K. F.; Wang, J. X.; Lim, J.; Sudarsan, N.; Breaker, R. R. Antibacterial lysine analogs that target lysine riboswitches. Nat. Chem. Biol. 2007, 3, 44−49. (56) Mulhbacher, J.; Brouillette, E.; Allard, M.; Fortier, L. C.; Malouin, F.; Lafontaine, D. A. Novel riboswitch ligand analogs as selective inhibitors of guanine-related metabolic pathways. PLoS Pathog. 2010, 6, e1000865. (57) Hickey, S. F.; Hammond, M. C. Structure-guided design of fluorescent S-adenosylmethionine analogs for a high-throughput screen to target SAM-I riboswitch RNAs. Chem. Biol. 2014, 21, 345−356. (58) Furukawa, R.; Gu, H. Z.; Sudarsan, N.; Hayakawa, Y.; Hyodo, M.; Breaker, R. R. Identification of ligand analogues that control c-diGMP riboswitches. ACS Chem. Biol. 2012, 7, 1436−1443. (59) Fedor, M. J. Structure and function of the hairpin ribozyme. J. Mol. Biol. 2000, 297, 269−291. (60) Eckert, H.; Bajorath, J. Molecular similarity analysis in virtual screening: foundations, limitations and novel approaches. Drug Discovery Today 2007, 12, 225−233. (61) Haller, A.; Souliere, M. F.; Micura, R. The dynamic nature of RNA as key to understanding riboswitch mechanisms. Acc. Chem. Res. 2011, 44, 1339−1348. (62) Zhang, J.; Lau, M. W.; Ferre-D’Amare, A. R. Ribozymes and riboswitches: modulation of RNA function by small molecules. Biochemistry 2010, 49, 9123−9131. (63) Chinnappan, R.; Dube, A.; Lemay, J. F.; Lafontaine, D. A. Fluorescence monitoring of riboswitch transcription regulation using a dual molecular beacon assay. Nucleic Acids Res. 2013, 41, e106. (64) Congreve, M.; Chessari, G.; Tisi, D.; Woodhead, A. J. Recent developments in fragment-based drug discovery. J. Med. Chem. 2008, 51, 3661−3680. (65) Hann, M. M.; Leach, A. R.; Harper, G. Molecular complexity and its impact on the probability of finding leads for drug discovery. J. Chem. Inf. Model. 2001, 41, 856−864. (66) Chen, L.; Cressina, E.; Leeper, F. J.; Smith, A. G.; Abell, C. A fragment-based approach to identifying ligands for riboswitches. ACS Chem. Biol. 2010, 5, 355−358. (67) Ciulli, A.; Abell, C. Biophysical tools to monitor enzyme-ligand interactions of enzymes involved in vitamin biosynthesis. Biochem. Soc. Trans. 2005, 33, 767−771. (68) Warner, K. D.; Homan, P.; Weeks, K. M.; Smith, A. G.; Abell, C.; Ferre-D’Amare, A. R. Validating fragment-based drug discovery for biological RNAs: lead fragments bind and remodel the TPP riboswitch specifically. Chem. Biol. 2014, 21, 591−595. (69) Ferre-D’Amare, A. R. The glmS ribozyme: use of a small molecule coenzyme by a gene-regulatory RNA. Q. Rev. Biophys. 2010, 43, 423−447. (70) Kim, J. N.; Blount, K. F.; Puskarz, I.; Lim, J.; Link, K. H.; Breaker, R. R. Design and antimicrobial action of purine analogues that bind guanine riboswitches. ACS Chem. Biol. 2009, 4, 915−927.

(28) Weinberg, Z.; Barrick, J. E.; Yao, Z.; Roth, A.; Kim, J. N.; Gore, J.; Wang, J. X.; Lee, E. R.; Block, K. F.; Sudarsan, N.; Neph, S.; Tompa, M.; Ruzzo, W. L.; Breaker, R. R. Identification of 22 candidate structured RNAs in bacteria using the CMfinder comparative genomics pipeline. Nucleic Acids Res. 2007, 35, 4809−4819. (29) Serganov, A.; Nudler, E. A decade of riboswitches. Cell 2013, 152, 17−24. (30) Buck, J.; Furtig, B.; Noeske, J.; Wohnert, J.; Schwalbe, H. Timeresolved NMR methods resolving ligand-induced RNA folding at atomic resolution. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 15699− 15704. (31) Klein, D. J.; Ferre-D’Amare, A. R. Structural basis of glmS ribozyme activation by glucosamine-6-phosphate. Science 2006, 313, 1752−1756. (32) Cochrane, J. C.; Lipchock, S. V.; Strobel, S. A. Structural investigation of the GlmS ribozyme bound to its catalytic cofactor. Chem. Biol. 2007, 14, 97−105. (33) Serganov, A.; Yuan, Y. R.; Pikovskaya, O.; Polonskaia, A.; Malinina, L.; Phan, A. T.; Hobartner, C.; Micura, R.; Breaker, R. R.; Patel, D. J. Structural basis for discriminative regulation of gene expression by adenine- and guanine-sensing mRNAs. Chem. Biol. 2004, 11, 1729−1741. (34) Garst, A. D.; Heroux, A.; Rambo, R. P.; Batey, R. T. Crystal structure of the lysine riboswitch regulatory mRNA element. J. Biol. Chem. 2008, 283, 22347−22351. (35) Serganov, A.; Huang, L.; Patel, D. J. Structural insights into amino acid binding and gene control by a lysine riboswitch. Nature 2008, 455, 1263−1267. (36) Serganov, A.; Polonskaia, A.; Phan, A. T.; Breaker, R. R.; Patel, D. J. Structural basis for gene regulation by a thiamine pyrophosphatesensing riboswitch. Nature 2006, 441, 1167−1171. (37) Thore, S.; Leibundgut, M.; Ban, N. Structure of the eukaryotic thiamine pyrophosphate riboswitch with its regulatory ligand. Science 2006, 312, 1208−1211. (38) Serganov, A.; Huang, L. L.; Patel, D. J. Coenzyme recognition and gene regulation by a flavin mononucleotide riboswitch. Nature 2009, 458, 233−237. (39) Peselis, A.; Serganov, A. Themes and variations in riboswitch structure and function. Biochim. Biophys. Acta 2014, 1839, 908−918. (40) Lunse, C. E.; Schmidt, M. S.; Wittmann, V.; Mayer, G. Carbasugars activate the glmS-riboswitch of Staphylococcus aureus. ACS Chem. Biol. 2011, 6, 675−678. (41) Stoddard, C. D.; Widmann, J.; Trausch, J. J.; MarcanoVelazquez, J. G.; Knight, R.; Batey, R. T. Nucleotides adjacent to the ligand-binding pocket are linked to activity tuning in the purine riboswitch. J. Mol. Biol. 2013, 425, 1596−1611. (42) Smith, K. D.; Lipchock, S. V.; Ames, T. D.; Wang, J.; Breaker, R. R.; Strobel, S. A. Structural basis of ligand binding by a c-di-GMP riboswitch. Nat. Struct. Mol. Biol. 2009, 16, 1218−1223. (43) Shanahan, C. A.; Gaffney, B. L.; Jones, R. A.; Strobel, S. A. Differential analogue binding by two classes of c-di-GMP riboswitches. J. Am. Chem. Soc. 2011, 133, 15578−15592. (44) Garst, A. D.; Batey, R. T. A switch in time: detailing the life of a riboswitch. Biochim. Biophys. Acta 2009, 1789, 584−591. (45) Porter, E. B.; Marcano-Velazquez, J. G.; Batey, R. T. The purine riboswitch as a model system for exploring RNA biology and chemistry. Biochim. Biophys. Acta 2014, 1839, 919−930. (46) Mayer, G.; Famulok, M. High-throughput-compatible assay for glmS riboswitch metabolite dependence. ChemBioChem 2006, 7, 602− 604. (47) Blount, K.; Puskarz, I.; Penchovsky, R.; Breaker, R. Development and application of a high-throughput assay for glmS riboswitch activators. RNA Biol. 2006, 3, 77−81. (48) Cressina, E.; Chen, L. H.; Abell, C.; Leeper, F. J.; Smith, A. G. Fragment screening against the thiamine pyrophosphate riboswitch thiM. Chem. Sci. 2011, 2, 157−165. (49) Chen, L.; Cressina, E.; Dixon, N.; Erixon, K.; Agyei-Owusu, K.; Micklefield, J.; Smith, A. G.; Abell, C.; Leeper, F. J. Probing riboswitchK


Perspective

Journal of Medicinal Chemistry (71) Lim, J.; Grove, B. C.; Roth, A.; Breaker, R. R. Characteristics of ligand recognition by a glmS self-cleaving ribozyme. Angew. Chem., Int. Ed. 2006, 45, 6689−6693. (72) McCarthy, T. J.; Plog, M. A.; Floy, S. A.; Jansen, J. A.; Soukup, J. K.; Soukup, G. A. Ligand requirements for glmS ribozyme selfcleavage. Chem. Biol. 2005, 12, 1221−1226. (73) Delfosse, V.; Bouchard, P.; Bonneau, E.; Dagenais, P.; Lemay, J. F.; Lafontaine, D. A.; Legault, P. Riboswitch structure: an internal residue mimicking the purine ligand. Nucleic Acids Res. 2010, 38, 2057−2068. (74) Pikovskaya, O.; Polonskaia, A.; Patel, D. J.; Serganov, A. Structural principles of nucleoside selectivity in a 2′-deoxyguanosine riboswitch. Nat. Chem. Biol. 2011, 7, 748−755. (75) Wickiser, J. K.; Winkler, W. C.; Breaker, R. R.; Crothers, D. M. The speed of RNA transcription and metabolite binding kinetics operate an FMN riboswitch. Mol. Cell 2005, 18, 49−60. (76) 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 2003, 113, 577−586. (77) Mack, M.; van Loon, A. P.; Hohmann, H. P. Regulation of riboflavin biosynthesis in Bacillus subtilis is affected by the activity of the flavokinase/flavin adenine dinucleotide synthetase encoded by ribC. J. Bacteriol. 1998, 180, 950−955. (78) Lee, E. R.; Blount, K. F.; Breaker, R. R. Roseoflavin is a natural antibacterial compound that binds to FMN riboswitches and regulates gene expression. RNA Biol. 2009, 6, 187−194. (79) Ster, C.; Allard, M.; Boulanger, S.; Lamontagne Boulet, M.; Mulhbacher, J.; Lafontaine, D. A.; Marsault, E.; Lacasse, P.; Malouin, F. Experimental treatment of Staphylococcus aureus bovine intramammary infection using a guanine riboswitch ligand analog. J. Dairy Sci. 2013, 96, 1000−1008. (80) Taylor, E. C.; Loux, H. M.; Falco, E. A.; Hitchings, G. H. Pyrimidopteridines by oxidative self-condensation of aminopyrimidines. J. Am. Chem. Soc. 1955, 77, 2243−2248. (81) Prochazkova, E.; Jansa, P.; Brezinova, A.; Cechova, L.; Mertlikova-Kaiserova, H.; Holy, A.; Dracinsky, M. Compound instability in dimethyl sulphoxide, case studies with 5-aminopyrimidines and the implications for compound storage and screening. Bioorg. Med. Chem. Lett. 2012, 22, 6405−6409. (82) De Jonghe, S.; Marchand, A.; Gao, L. J.; Calleja, A.; Cuveliers, E.; Sienaert, I.; Herman, J.; Clydesdale, G.; Sefrioui, H.; Lin, Y. A.; Pfleiderer, W.; Waer, M.; Herdewijn, P. Synthesis and in vitro evaluation of 2-amino-4-N-piperazinyl-6-(3,4-dimethoxyphenyl)-pteridines as dual immunosuppressive and anti-inflammatory agents. Bioorg. Med. Chem. Lett. 2011, 21, 145−149. (83) Ataide, S. F.; Wilson, S. N.; Dang, S.; Rogers, T. E.; Roy, B.; Banerjee, R.; Henkin, T. M.; Ibba, M. Mechanisms of resistance to an amino acid antibiotic that targets translation. ACS Chem. Biol. 2007, 2, 819−827. (84) Lunse, C. E.; Schuller, A.; Mayer, G. The promise of riboswitches as potential antibacterial drug targets. Int. J. Med. Microbiol. 2014, 304, 79−92. (85) Ellington, A. D.; Szostak, J. W. In vitro selection of RNA molecules that bind specific ligands. Nature 1990, 346, 818−822. (86) Burgstaller, P.; Kochoyan, M.; Famulok, M. Structural probing and damage selection of citrulline- and arginine-specific RNA aptamers identify base positions required for binding. Nucleic Acids Res. 1995, 23, 4769−4776. (87) Rentmeister, A.; Mayer, G.; Kuhn, N.; Famulok, M. Secondary structures and functional requirements for thiM riboswitches from Desulfovibrio vulgaris, Erwinia carotovora and Rhodobacter spheroides. Biol. Chem. 2008, 389, 127−134. (88) Mayer, G.; Raddatz, M. S. L.; Grunwald, J. D.; Famulok, M. RNA ligands that distinguish metabolite-induced conformations in the TPP riboswitch. Angew. Chem., Int. Ed. 2007, 46, 557−560. (89) Rentmeister, A.; Mayer, G.; Kuhn, N.; Famulok, M. Conformational changes in the expression domain of the Escherichia coli thiM riboswitch. Nucleic Acids Res. 2007, 35, 3713−3722.

(90) Mayer, G.; Faulhammer, D.; Grattinger, M.; Fessele, S.; Blind, M. A RNA-based approach towards small-molecule inhibitors. ChemBioChem 2009, 10, 1993−1996. (91) Zapp, M. L.; Stern, S.; Green, M. R. Small molecules that selectively block RNA binding of HIV-1 Rev protein inhibit Rev function and viral production. Cell 1993, 74, 969−978. (92) Wang, Y.; Rando, R. R. Specific binding of aminoglycoside antibiotics to RNA. Chem. Biol. 1995, 2, 281−290. (93) Vicens, Q.; Westhof, E. RNA as a drug target: the case of aminoglycosides. ChemBioChem 2003, 4, 1018−1023. (94) Schroeder, R.; Waldsich, C.; Wank, H. Modulation of RNA function by aminoglycoside antibiotics. EMBO J. 2000, 19, 1−9. (95) Posakony, J. J.; Ferre-D’Amare, A. R. Glucosamine and glucosamine-6-phosphate derivatives: catalytic cofactor analogues for the glmS ribozyme. J. Org. Chem. 2013, 78, 4730−4743. (96) Santner, T.; Rieder, U.; Kreutz, C.; Micura, R. Pseudoknot preorganization of the preQ1 class I riboswitch. J. Am. Chem. Soc. 2012, 134, 11928−11931. (97) Colizzi, F.; Lamontagne, A. M.; Lafontaine, D. A.; Bussi, G. Probing riboswitch binding sites with molecular docking, focused libraries, and in-line probing assays. Methods Mol. Biol. 2014, 1103, 141−151. (98) Lunse, C. E.; Scott, F. J.; Suckling, C. J.; Mayer, G. Novel TPPriboswitch activators bypass metabolic enzyme dependency. Front. Chem. (Lausanne, Switz.) 2014, 2, 53. (99) Erixon, K. M.; Dabalos, C. L.; Leeper, F. J. Inhibition of pyruvate decarboxylase from Z. mobilis by novel analogues of thiamine pyrophosphate: investigating pyrophosphate mimics. Chem. Commun. 2007, 960−962. (100) Erixon, K. M.; Dabalos, C. L.; Leeper, F. J. Synthesis and biological evaluation of pyrophosphate mimics of thiamine pyrophosphate based on a triazole scaffold. Org. Biomol. Chem. 2008, 6, 3561− 3572. (101) Fei, X.; Holmes, T.; Diddle, J.; Hintz, L.; Delaney, D.; Stock, A.; Renner, D.; McDevitt, M.; Berkowitz, D. B.; Soukup, J. K. Phosphatase-inert glucosamine 6-phosphate mimics serve as actuators of the glmS riboswitch. ACS Chem. Biol. 2014, 9, 2875−2882. (102) Ott, E.; Stolz, J.; Lehmann, M.; Mack, M. The RFN riboswitch of Bacillus subtilis is a target for the antibiotic roseoflavin produced by Streptomyces davawensis. RNA Biol. 2009, 6, 276−280. (103) Pedrolli, D. B.; Matern, A.; Wang, J.; Ester, M.; Siedler, K.; Breaker, R.; Mack, M. A highly specialized flavin mononucleotide riboswitch responds differently to similar ligands and confers roseoflavin resistance to Streptomyces davawensis. Nucleic Acids Res. 2012, 40, 8662−8673.

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