glmS Riboswitch Research Articles

Phosphonate and Thiasugar Analogues of Glucosamine-6-phosphate: Activation of the glmS Riboswitch and Antibiotic Activity.

The glmS riboswitch is a motif found in 5'-untranslated regions of bacterial mRNA that controls the synthesis of glucosamine-6-phosphate (GlcN6P), an essential building block for the bacterial cell wall, by a feedback mechanism. Activation of the glmS riboswitch by GlcN6P mimics interferes with the ability of bacteria to synthesize its cell wall. Accordingly, GlcN6P mimics acting as glmS activators are promising candidates for future antibiotic drugs that may overcome emerging bacterial resistance against established antibiotics. We describe the synthesis of a series of phosphonate mimics of GlcN6P as well as the thiasugar analogue of GlcN6P. The phosphonate mimics differ in their pKa value to answer the question of whether derivatives with a pKa matching that of GlcN6P would be efficient glmS activators. We found that all derivatives activate the riboswitch, however, less efficiently than GlcN6P. This observation can be explained by the missing hydrogen bonds in the case of phosphonates and is valuable information for the design of future GlcN6P mimics. The thiasugar analogue of GlcN6P on the other hand turned out to be a glmS riboswitch activator with the same activity as the natural metabolite GlcN6P. The nonphosphorylated thiasugar displayed antimicrobial activity against certain bacilli. Therefore, the compound is a promising lead structure for the development of future antibiotics with a potentially novel mode of action.

Carba-Sugar Analogs of Glucosamine-6-Phosphate: New Activators for the glmS Riboswitch.

Riboswitches are 5'-untranslated mRNA regions mostly found in bacteria. They are promising drug targets to overcome emerging bacterial resistance against commonly used antibiotics. The glmS riboswitch is unique among the family of riboswitches as it is a ribozyme that undergoes self-cleavage upon binding to glucosamine-6-phosphate (GlcN6P). Previously, we showed that carba glucosamine-6-phosphate (carba-GlcN6P) induces self-cleavage of the riboswitch with a potency similar to that of GlcN6P. Here, we report a synthetic approach to a new class of carba-GlcN6P derivatives with an alkoxy substituent in the carba position. Key features of the synthesis are a ring closing metathesis followed by a hydroboration. The strategy gives access to libraries of carba-GlcN6P derivatives. Ribozyme cleavage assays unraveled new activators for the glmS riboswitch from Listeria monocytogenes and Clostridium difficile.

Targeting glmS Ribozyme with Chimeric Antisense Oligonucleotides for Antibacterial Drug Development.

Due to the steady rise of multidrug-resistant pathogenic bacteria worldwide, it is critical to develop novel antibacterial drugs. This article presents chimeric antisense oligonucleotides that inhibit the bacterial growth of Staphylococcus aureus, one of the most frequent causes of hospital-acquired infections. The chimeric antisense oligonucleotides have a combination of first- and second-generation chemical modification. To deliver the antisense oligonucleotides into a cell, we apply a cell-penetrating oligopeptide attached to them. We have performed complete bioinformatics analyses of the glmS ribozyme present in S. aureus and its essential role in the biochemical pathway of glucosamine-6-phosphate synthesis. Besides, we have analyzed the bacteria for alternative metabolic pathways, such as the nagA gene. The first antisense oligonucleotide explicitly targets the glmS riboswitch, while the second explicitly targets the nagA mRNA. We have evaluated that combined, the antisense oligonucleotides block the synthesis of glucosamine-6-phosphate entirely and inhibit the bacterial growth of S. aureus. However, the glmS riboswitch targeting the antisense oligonucleotide is sufficient to inhibit the growth of S. aureus with a MIC80 of 5 μg/mL. The glmS ribozyme is a very suitable target for antibacterial drug development with antisense oligonucleotides.

Staphylococcusaureus Transcriptome Data and Metabolic Modelling Investigate the Interplay of Ser/Thr Kinase PknB, Its Phosphatase Stp, the glmR/yvcK Regulon and the cdaA Operon for Metabolic Adaptation.

Serine/threonine kinase PknB and its corresponding phosphatase Stp are important regulators of many cell functions in the pathogen S. aureus. Genome-scale gene expression data of S. aureus strain NewHG (sigB+) elucidated their effect on physiological functions. Moreover, metabolic modelling from these data inferred metabolic adaptations. We compared wild-type to deletion strains lacking pknB, stp or both. Ser/Thr phosphorylation of target proteins by PknB switched amino acid catabolism off and gluconeogenesis on to provide the cell with sufficient components. We revealed a significant impact of PknB and Stp on peptidoglycan, nucleotide and aromatic amino acid synthesis, as well as catabolism involving aspartate transaminase. Moreover, pyrimidine synthesis was dramatically impaired by stp deletion but only slightly by functional loss of PknB. In double knockouts, higher activity concerned genes involved in peptidoglycan, purine and aromatic amino acid synthesis from glucose but lower activity of pyrimidine synthesis from glucose compared to the wild type. A second transcriptome dataset from S. aureus NCTC 8325 (sigB−) validated the predictions. For this metabolic adaptation, PknB was found to interact with CdaA and the yvcK/glmR regulon. The involved GlmR structure and the GlmS riboswitch were modelled. Furthermore, PknB phosphorylation lowered the expression of many virulence factors, and the study shed light on S. aureus infection processes.

Recent Development of Glucosamine‐6‐phosphate Derivatives as Potential Antibacterial Agents

AbstractThe glmS riboswitch (glucosamine‐6‐phosphate activated riboswitch) is located at the 5’‐ untranslated region of the glmS mRNA, which carries the gene of glucosamine‐6‐phosphate (GlcN6P) synthetase named amidotransferase. The glmS riboswitch can bind to its natural coenzyme GlcN6P and lead to its own self‐cleavage, causing the decrease in the expression of the glmS RNA and the synthesis of amidotransferase, and finally, reducing the production of GlcN6P. Since GlcN6P is of vital importance in the metabolic pathway of the bacterial cell wall synthesis, the glmS riboswitch gradually becomes a promising target of antibiotic drugs. In this review, an overview of the development of the glmS riboswitch activators as novel and potent antibacterial agents is described, which involves the structure of the glmS riboswitch and its self‐cleavage mechanism, various the glmS riboswitch activators and their structure‐activity relationships. In particular, the analogs of GlcN6P that can induce self‐cleavage of the glmS riboswitch are highlighted. The most promising compound 9 (Carba‐GlcN6P) exhibited not only excellent activity in inducing the self‐cleavage of the glmS riboswitch, but also great efficacy (kobs=0.153 min−1) at 200 μM compared to GlcN6P (kobs=0.177 min−1).

Folding to self-destruct

Protein Folding The bacterial enzyme glucosamine-6-phosphate synthase (GlmS) is essential for synthesis of the cell wall. Its expression is regulated by a structured messenger RNA (mRNA) element, the glmS riboswitch. Most riboswitches are stabilized in an “on” conformational state by binding a ligand. In GlmS, however, ligand binding leads to self-cleavage, and this, in turn, targets the mRNA for degradation. Savinov and Block used optical tweezers to measure folding dynamics and cleavage rates for the core glmS ribozyme with and without ligand. A specific duplex called P2.2 folds last and transiently. Ligand binding does not stabilize the P2.2 duplex; it is only when ligand binds this structure that cleavage occurs. A compound that stabilizes the duplex could make an antibiotic candidate. Proc. Natl. Acad. Sci. U.S.A. 115 , 11976 (2018).

Folding and Catalysis of the glmS Ribozyme Riboswitch Studied at the Single-Molecule Level

We present findings from optical trapping experiments performed on the glmS ribozyme riboswitch. Found in the glmS gene in many bacteria, the glmS riboswitch down-regulates GlmS expression in the presence of the cell wall precursor—and enzymatic product of GlmS—glucosamine-6-phosphate (GlcN6P). In response to GlcN6P, the riboswitch site-specifically cleaves itself near its 5ʹ end, which targets the mRNA for subsequent degradation1. We performed single-molecule force spectroscopy experiments on the ribozyme catalytic core, unfolding and refolding the glmS RNA under controlled mechanical loads. The force-extension curves (FECs) reveal that folding and unfolding occur through a series of intermediate states, and we observe a striking hysteresis between unfolding and refolding of the ribozyme. Analysis of the FEC data leads to a model for the major unfolding pathway, involving the sequential unfolding of a series of discrete substructures. We’ve also begun to characterize the folding energy landscape of the catalytic core, using a series of constant-force measurements. We performed self-cleavage assays with optically trapped ribozyme molecules in the presence of the enzymatic cofactor GlcN6P and its catalytically inactive analog, glucose 6-phosphate. These assays demonstrate that our experimental construct, consisting of aminimal ribozyme sequence, is enzymatically active and strictly dependent upon the presence of GlcN6P. Self-cleavage measurements under controlled loads allow us to determine the profile of the catalytic activity in response to destabilizing forces. We observe an intermediate destabilization regime where the ribozyme remains active, but displays a partial loss of function.Footnotes1 Collins, J.A., Irnov, I., Baker, S., and Winkler, W.C. (2007) Genes Dev21(24), 3356-68.

Phosphatase-inert glucosamine 6-phosphate mimics serve as actuators of the glmS riboswitch.

The glmS riboswitch is unique among gene-regulating riboswitches and catalytic RNAs. This is because its own metabolite, glucosamine-6-phosphate (GlcN6P), binds to the riboswitch and catalytically participates in the RNA self-cleavage reaction, thereby providing a novel negative feedback mechanism. Given that a number of pathogens harbor the glmS riboswitch, artificial actuators of this potential RNA target are of great interest. Structural/kinetic studies point to the 2-amino and 6-phosphate ester functionalities in GlcN6P as being crucial for this actuation. As a first step toward developing artificial actuators, we have synthesized a series of nine GlcN6P analogs bearing phosphatase-inert surrogates in place of the natural phosphate ester. Self-cleavage assays with the Bacillus cereusglmS riboswitch give a broad SAR. Two analogs display significant activity, namely, the 6-deoxy-6-phosphonomethyl analog (5) and the 6-O-malonyl ether (13). Kinetic profiles show a 22-fold and a 27-fold higher catalytic efficiency, respectively, for these analogs vs glucosamine (GlcN). Given their nonhydrolyzable phosphate surrogate functionalities, these analogs are arguably the most robust artificial glmS riboswitch actuators yet reported. Interestingly, the malonyl ether (13, extra O atom) is much more effective than the simple malonate (17), and the “sterically true” phosphonate (5) is far superior to the chain-truncated (7) or chain-extended (11) analogs, suggesting that positioning via Mg coordination is important for activity. Docking results are consistent with this view. Indeed, the viability of the phosphonate and 6-O-malonyl ether mimics of GlcN6P points to a potential new strategy for artificial actuation of the glmS riboswitch in a biological setting, wherein phosphatase-resistance is paramount.

Signal amplification of glucosamine-6-phosphate based on ribozyme glmS

Ribozyme glmS based isothermal amplification assay is developed for the colorimetric detection of glucosamine-6-phosphate (GlcN6P). Upon binding to the metabolite target GlcN6P, self-cleavage of glmS ribozyme is initiated to release RNA fragment that can trigger the cascade signal amplification to release large amount of G-quadruplex DNAzymes as reporter for colorimetric detection. Given the importance of GlcN6P for cell wall biosynthesis, the glmS riboswitch has become a new drug target for the development of antibiotics. This assay not only offers a convenient detection of GlcN6P with high specificity and sensitivity, but also provides a platform for high-throughput screening of antibiotics based on glmS riboswitches.

An in vitro evolved glmS ribozyme has the wild-type fold but loses coenzyme dependence

Uniquely among known ribozymes, the glmS ribozyme-riboswitch requires a small-molecule coenzyme, glucosamine-6-phosphate (GlcN6P). Although consistent with its gene-regulatory function, the use of GlcN6P is unexpected because all of the other characterized self-cleaving ribozymes use RNA functional groups or divalent cations for catalysis. To determine what active site features make this ribozyme reliant on GlcN6P and to evaluate whether it might have evolved from a coenzyme-independent ancestor, we isolated a GlcN6P-independent variant through in vitro selection. Three active site mutations suffice to generate a highly reactive RNA that adopts the wild-type fold but uses divalent cations for catalysis and is insensitive to GlcN6P. Biochemical and crystallographic comparisons of wild-type and mutant ribozymes show that a handful of functional groups fine-tune the RNA to be either coenzyme or cation dependent. These results indicate that a few mutations can confer new biochemical activities on structured RNAs. Thus, families of structurally related ribozymes with divergent function may exist.

Synthesis and evaluation of glucosamine-6-phosphate analogues as activators of glmS riboswitch

The glmS riboswitch is a ribozyme found in numerous Gram-positive bacteria and responds to the cellular concentrations of glucosamine 6-phosphate (GlcN6P). Given the importance of GlcN6P for cell wall biosynthesis, the glmS riboswitch has become a new drug target for the development of antibiotics. Herein, we describe the efficient synthesis of three GlcN6P analogues and their evaluation on inducing self-cleavage of the glmS riboswitch from Bacillus subtilis. Our results provide valuable information for further elucidation of the structure–activity relationships and drug design for glmS riboswitch antibiotics.

Caged glucosamine-6-phosphate for the light-control of riboswitch activity

We have synthesized a light-activatable ("caged") derivative of glucosamine-6-phosphate (GlcN6P), which only upon irradiation becomes a cofactor for the glmS riboswitch. This glmS riboswitch maintains its activity when embedded in the 3'-untranslated region of eukaryotic mRNA molecules and caged GlcN6P reduces the amount of translated EGFP upon irradiation with light in vitro.

Noncanonical Hydrogen Bonding in Nucleic Acids. Benchmark Evaluation of Key Base–Phosphate Interactions in Folded RNA Molecules Using Quantum-Chemical Calculations and Molecular Dynamics Simulations

RNA molecules are stabilized by a wide range of noncanonical interactions that are not present in DNA. Among them, the recently classified base-phosphate (BPh) interactions belong to the most important ones. Twelve percent of nucleotides in the ribosomal crystal structures are involved in BPh interactions. BPh interactions are highly conserved and provide major constraints on RNA sequence evolution. Here we provide assessment of the energetics of BPh interactions using MP2 computations extrapolated to the complete basis set of atomic orbitals and corrected for higher-order electron correlation effects. The reference computations are compared with DFT-D and DFT-D3 approaches, the SAPT method, and the molecular mechanics force field. The computations, besides providing the basic benchmark for the BPh interactions, allow some refinements of the original classification, including identification of some potential doubly bonded BPh patterns. The reference computations are followed by analysis of some larger RNA fragments that consider the context of the BPh interactions. The computations demonstrate the complexity of interaction patterns utilizing the BPh interactions in real RNA structures. The BPh interactions are often involved in intricate interaction networks. We studied BPh interactions of protonated adenine that can contribute to catalysis of hairpin ribozyme, the key BPh interaction in the S-turn motif of the sarcin-ricin loop, which may predetermine the S-turn topology and complex BPh patterns from the glmS riboswitch. Finally, the structural stability of BPh interactions in explicit solvent molecular dynamics simulations is assessed. The simulations well preserve key BPh interactions and allow dissection of structurally/functionally important water-meditated BPh bridges, which could not be considered in earlier bioinformatics classification of BPh interactions.

GlmS Riboswitch binding to the glucosamine-6-phosphate α-anomer shifts the pKa toward neutrality.

The glmS riboswitch regulates gene expression through a self-cleavage activity. The reaction is catalyzed with the assistance of the metabolite cofactor glucosamine-6-phosphate (GlcN6P), whose amino group is proposed to serve as the general acid during the reaction. This reaction is pH-dependent with a pK(a) that is lower than the observed pK(a) for the amine of GlcN6P in solution. GlcN6P, like other pyranose sugars, undergoes spontaneous and rapid interconversion between the α and β anomers at the C1 position. Here we demonstrate by NMR that the Bacillus anthracis glmS riboswitch selectively binds the α-anomer of GlcN6P with a maximum binding affinity of 0.36 mM and that binding is pH-dependent. We also report that the anomeric ratio between α and β is pH-dependent and the pK(a)s of the two amines differ by 0.5 pH units, α being the higher of the two (pK(a)=8.3). The pH dependence of binding reveals a pK(a) of 6.7, suggesting that the glmS RNA reduces the pK(a) of the GlcN6P amine by 1.6 units in the ground state. We reevaluated previously obtained kinetic data and found the reaction pK(a) is 6.9, within error of the binding data. The data support a model where the reaction pK(a) corresponds to that of the GlcN6P amine. This observation has broader relevance for considering how the microenvironment of an RNA, despite its anionic character, can reduce the pK(a)s of functional groups for use in catalysis.

Riboswitches: Discovery of Drugs That Target Bacterial Gene-Regulatory RNAs

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

Rethinking Riboswitch Regulation

Biochemistry The enzyme glucosamine-6-phosphate (GlcN6P) synthase (GlmS) is an important point of metabolic control in the synthesis of amino sugars: essential precursors in the biosynthesis of bacterial cell walls. GlmS gene expression is regulated by the glmS riboswitch, a sequence in the 5′

The glmS riboswitch integrates signals from activating and inhibitory metabolites in vivo.

The glmS riboswitch belongs to the family of regulatory RNAs that provide feedback regulation of metabolic genes. It is also a ribozyme that self-cleaves upon binding glucosamine-6-phosphate, the product of the enzyme encoded by glmS. The ligand concentration dependence of intracellular self-cleavage kinetics was measured for the first time in a yeast model system and surprisingly revealed that this riboswitch is subject to inhibition as well as activation by hexose metabolites. Reporter gene experiments in B. subtilis confirmed that this riboswitch integrates positive and negative chemical signals in its natural biological context. Contrary to the conventional view that a riboswitch responds to just a single cognate metabolite, our new model proposes that a single riboswitch integrates information from an array of chemical signals to modulate gene expression according to the overall metabolic state of the cell.

Protonation states of the key active site residues and structural dynamics of the glmS riboswitch as revealed by molecular dynamics.

The glmS catalytic riboswitch is part of the 5'-untranslated region of mRNAs encoding glucosamine-6-phosphate (GlcN6P) synthetase (glmS) in numerous gram-positive bacteria. Binding of the cofactor GlcN6P induces site-specific self-cleavage of the RNA. However, the detailed reaction mechanism as well as the protonation state of the glmS reactive form still remains elusive. To probe the dominant protonation states of key active site residues, we carried out explicit solvent molecular dynamic simulations involving various protonation states of three crucial active site moieties observed in the available crystal structures: (i) guanine G40 (following the Thermoanaerobacter tengcongensis numbering), (ii) the GlcN6P amino/ammonium group, and (iii) the GlcN6P phosphate moiety. We found that a deprotonated G40(-) seems incompatible with the observed glmS active site architecture. Our data suggest that the canonical form of G40 plays a structural role by stabilizing an in-line attack conformation of the cleavage site A-1(2'-OH) nucleophile, rather than a more direct chemical role. In addition, we observe weakened cofactor binding upon protonation of the GlcN6P phosphate moiety, which explains the experimentally observed increase in K(m) with decreasing pH. Finally, we discuss a possible role of cofactor binding and its interaction with the G65 and G1 purines in structural stabilization of the A-1(2'-OH) in-line attack conformation. On the basis of the identified dominant protonation state of the reaction precursor, we propose a hypothesis of the self-cleavage mechanism in which A-1(2'-OH) is activated as a nucleophile by the G1(pro-R(p)) nonbridging oxygen of the scissile phosphate, whereas the ammonium group of GlcN6P acts as the general acid protonating the G1(O5') leaving group.

Antibiotic Development by Investigation of the glmS Riboswitch

Although bacterial infections have always been of significant interest to researchers and physicians, the drug-resistant bacterial strains that have recently developed are causing new concerns and are much more difficult to combat. Our current methods for treating bacterial infections include broad-spectrum antibiotics which target only a small number of bacterial processes. However, with the discovery of riboswitches, we are developing new ways to fight bacterial infections which make use of their own natural metabolic pathways, essentially causing bacteria to destroy themselves. Riboswitches are found in non-coding regions of messenger RNAs and these RNA elements bind to ligands to control the expression of nearby genes. The glucosamine-6-phosphate (glmS) riboswitch is unique in that upon binding its ligand, glucosamine-6-phosphate (GlcN6P), it undergoes self-cleavage and is therefore also a catalytic RNA. The cleavage event targets the RNA for subsequent degradation, thereby repressing further gene expression. To study the glmS riboswitch, initial experiments were performed to determine the mechanism followed upon binding of the natural ligand. Since then, analogs of the natural ligand have been obtained and are being tested for their catalytic capabilities through kinetic analyses and rate constant calculations. Once successful candidates have been determined, these non-natural ligands will be introduced into live bacterial cultures, hopefully disrupting normal cell metabolism and reproduction. If successful, these analogs could be used as novel antibiotics, offering a more specific mode of targeting a wide variety of bacterial species.

Ribozymes and riboswitches: modulation of RNA function by small molecules

Riboswitches are mRNA domains that directly bind small‐molecule metabolites and control expression of the mRNA they are part of in a ligand‐dependent manner. The glmS riboswitch is thus far unique in that it is also a catalytic RNA, or ribozyme. The self‐cleaving activity of this RNA from Gram‐positive bacteria requires binding of glucosamine‐6‐phosphate (GlcN6P), an essential precursor to the bacterial cell wall. The ribozyme is part of the mRNA for the enzyme GlcN6P synthetase. Self‐cleavage leads to mRNA destruction, and therefore, negative‐feedback regulation. Crystallographic analysis of the glmS riboswitch reveals a remarkably rigid RNA and binding of GlcN6P in contact with the scissile phosphate. This and the inactivity of the ribozyme in the absence of GlcN6P indicate that the metabolite functions as a true coenzyme. However, the RNA is not simply an inert structural scaffold. Mutational, kinetic and structural analyses reveal that an active‐site guanosine and GlcN6P mutually modulate the ability of each other to function as general acid‐base catalysts. Supported by the Damon Runyon Cancer Research Foundation, the Howard Hughes Medical Institute, the NIH and the W.M. Keck Foundation.