New validated cellular targets are needed to reinvigorate antibacterial drug discovery. This need could potentially be filled by riboswitches-messenger RNA (mRNA) structures that regulate gene expression in bacteria. Riboswitches are unique among RNAs that serve as drug targets in that they have evolved to form structured and highly selective receptors for small drug-like metabolites. In most cases, metabolite binding to the receptor represses the expression of the gene(s) encoded by the mRNA. If a new metabolite analog were designed that binds to the receptor, the gene(s) regulated by that riboswitch could be repressed, with a potentially lethal effect to the bacteria. Recent work suggests that certain antibacterial compounds discovered decades ago function at least in part by targeting riboswitches. Herein we will summarize the experiments validating riboswitches as drug targets, describe the existing technology for riboswitch drug discovery and discuss the challenges that may face riboswitch drug discoverers.

Lysine riboswitches are bacterial RNA structures that sense the concentration of lysine and regulate the expression of lysine biosynthesis and transport genes. Members of this riboswitch class are found in the 5' untranslated region of messenger RNAs, where they form highly selective receptors for lysine. Lysine binding to the receptor stabilizes an mRNA tertiary structure that, in most cases, causes transcription termination before the adjacent open reading frame can be expressed. A lysine riboswitch conceivably could be targeted for antibacterial therapy by designing new compounds that bind the riboswitch and suppress lysine biosynthesis and transport genes. As a test of this strategy, we have identified several lysine analogs that bind to riboswitches in vitro and inhibit Bacillus subtilis growth, probably through a mechanism of riboswitch-mediated repression of lysine biosynthesis. These results indicate that riboswitches could serve as new classes of antibacterial drug targets.

A powerful approach to understanding protein enzyme catalysis is to examine the structural context of essential amino acid side chains whose deletion or modification negatively impacts catalysis. In principle, this approach can be even more powerful for RNA enzymes, given the wide variety and subtlety of functionally modified nucleotides now available. Numerous recent success stories confirm the utility of this approach to understanding ribozyme function. An anomaly, however, is the hammerhead ribozyme, for which the structural and functional data do not agree well, preventing a unifying view of its catalytic mechanism from emerging. To delineate the hammerhead structure-function comparison, we have evaluated and distilled the large body of biochemical data into a consensus set of functional groups unambiguously required for hammerhead catalysis. By examining the context of these functional groups within available structures, we have established a concise set of disagreements between the structural and functional data. The number and relative distribution of these inconsistencies throughout the hammerhead reaffirms that an extensive conformational rearrangement from the fold observed in the crystal structure must be necessary for cleavage to occur. The nature and energetic driving force of this conformational isomerization are discussed.

Riboswitches in messenger RNAs carry receptor domains called aptamers that can bind to metabolites and control expression of associated genes. The Gram-positive bacterium Bacillus subtilis has two representatives of a class of riboswitches that bind flavin mononucleotide (FMN). These riboswitches control genes responsible for the biosynthesis and transport of riboflavin, a precursor of FMN. We found that roseoflavin, a chemical analog of FMN and riboflavin that has antimicrobial activity, can directly bind to FMN riboswitch aptamers and downregulate the expression of an FMN riboswitch-lacZ reporter gene in B. subtilis. A role for the riboswitch in the antimicrobial mechanism of roseoflavin is supported by our observation that some previously identified roseoflavin-resistant bacteria have mutations within an FMN aptamer. Riboswitch mutations in these resistant bacteria disrupt ligand binding and derepress reporter gene expression in the presence of either riboflavin or roseoflavin. If FMN riboswitches are a major target for roseoflavin antimicrobial action, then future efforts to develop compounds that trigger FMN riboswitch function could lead to the identification of new antimicrobial drugs.

Riboswitches are structured RNA domains that can bind directly to specific ligands and regulate gene expression. These RNA elements are located most commonly within the noncoding regions of bacterial mRNAs, although representatives of one riboswitch class have been discovered in organisms from all three domains of life. In several Gram positive species of bacteria, riboswitches that selectively recognize guanine regulate the expression of genes involved in purine biosynthesis and transport. Because these genes are involved in fundamental metabolic pathways in certain bacterial pathogens, guanine-binding riboswitches may be targets for the development of novel antibacterial compounds. To explore this possibility, the atomic-resolution structure of a guanine riboswitch aptamer from Bacillus subtilis was used to guide the design of several riboswitch-compatible guanine analogs. The ability of these compounds to be bound by the riboswitch and repress bacterial growth was examined. Many of these rationally designed compounds are bound by a guanine riboswitch aptamer in vitro with affinities comparable to that of the natural ligand, and several also inhibit bacterial growth. We found that one of these antimicrobial guanine analogs (6-N-hydroxylaminopurine, or G7) represses expression of a reporter gene controlled by a guanine riboswitch in B. subtilis, suggesting it may inhibit bacterial growth by triggering guanine riboswitch action. These studies demonstrate the utility of a three-dimensional structure model of a natural aptamer to design ligand analogs that target riboswitches. This approach also could be implemented to design antibacterial compounds that specifically target other riboswitch classes.

The lack of high RNA target selectivity displayed by aminoglycoside antibiotics results from both their electrostatically driven binding mode and their conformational adaptability. The inherent flexibility around their glycosidic bonds allows them to easily assume a variety of conformations, permitting them to structurally adapt to diverse RNA targets. This structural promiscuity results in the formation of aminoglycoside complexes with diverse RNA targets in which the antibiotics assume distinct conformations. Such differences suggest that covalently linking individual rings in an aminoglycoside could reduce its available conformations, thereby altering target selectivity. To explore this possibility, conformationally constrained neomycin and paromomycin analogues designed to mimic the A-site bound aminoglycoside structure have been synthesized and their affinities to the TAR and A-site, two therapeutically relevant RNA targets, have been evaluated. As per design, this constraint has minimal deleterious effect on binding to the A-site. Surprisingly, however, preorganizing these neomycin-class antibiotics into a TAR-disfavored structure has no deleterious effect on binding to this HIV-1 RNA sequence. We rationalize these observations by suggesting that the A-site and HIV TAR possess inherently different selectivities toward aminoglycosides. The inherent plasticity of the TAR RNA, coupled to the remaining flexibility within the conformationally constrained analogues, makes this RNA site an accommodating target for such polycationic ligands. In contrast, the deeply encapsulating A-site is a more discriminating RNA target. These observations suggest that future design of novel target selective RNA-based therapeutics will have to consider the inherent "structural" selectivity of the RNA target and not only the selectivity patterns displayed by the low molecular weight ligands.

Despite nearly 85 years of successful antibacterial chemotherapy, bacterial infections still pose a serious threat to human health. Continually emerging drug-resistant bacteria make existing agents less effective, and a paucity of new agents and decreased development effort from the pharmaceutical industry provide little hope of replenishing the arsenal (1, 2). Complications and mortality due to bacterial infections continue to increase, already reaching epidemic proportions in some areas of the world. If humans are to regain the upper hand in fighting bacterial infections, then innovation, investment, and new antibacterial agents will be needed. Although some of the barriers to the discovery and approval of new compounds are economic or policy related, there is also a desperate need for new targets and new mechanisms of action. A renewed effort to expand our knowledge of bacterial physiology and to translate discoveries into the clinic will be needed to address these challenges and to reinvigorate antibiotic development pipelines.Recent advances in our understanding of how bacteria maintain physiological homeostasis revealed a promising class of potential antibiotic targets called riboswitches-noncoding mRNAs that form a structured receptor (or aptamer) which can directly bind to a specific small-molecule ligand or ion and thereby regulate gene expression (3-5). Ligand binding to a riboswitch aptamer stabilizes a conformationally distinct architecture in the mRNA that modulates the expression of the adjacent coding region(s) (4-8). To date, more than 35 riboswitch classes have been discovered and characterized (7). Three of these riboswitch classes have been revealed as important cellular targets of antibacterial small molecules whose mechanism of action had not been previously defined (9-13). More recently, several publications have demonstrated that novel small molecules that bind to selected riboswitch aptamers with affinities comparable to that of the cognate ligand can be rationally identified and optimized (14-21).In some cases, synthetic or natural riboswitch ligand analogs have demonstrated potent antibacterial activity (12-15, 20, 22). For example, the phosphorylated form of roseoflavin (RoF) (

Riboswitches are newly-discovered gene control elements that are promising targets for antibacterial drug development. To facilitate the rapid discovery and development of riboswitch-targeted compounds, modern drug discovery techniques such as structure-based design and high-throughput screening will need to be applied. One promising riboswitch drug target is the glmS riboswitch, which upon binding glucosamine-6-phosphate (GlcN6P) functions as a ribozyme and catalyzes self-cleavage. Herein we report the development of a high-throughput assay for glmS ribozyme cleavage that relies on fluorescence resonance energy transfer (FRET). This assay can be used to screen for compounds that bind to and activate glmS ribozyme cleavage. To validate the screen, we demonstrate that the assay can identify the active compounds from a library of GlcN6P analogs whose affinities for ribozyme were determined by commonly used electrophoretic methods with radiolabeled RNA. Furthermore, the primary screen of a library of 960 compounds previously approved for use in humans identified five active compounds, one of which is a GlcN6P analog known to stimulate ribozyme activity. These results demonstrate that modern high-throughput screening techniques can be applied to the discovery of riboswitch-targeted drug compounds.