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Riboswitches As Drug Targets for Antibiotics antibiotics Review Riboswitches as Drug Targets for Antibiotics Vipul Panchal * and Ruth Brenk * Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5020 Bergen, Norway * Correspondence: [email protected] (V.P.); [email protected] (R.B.) Abstract: Riboswitches reside in the untranslated region of RNA and regulate genes involved in the biosynthesis of essential metabolites through binding of small molecules. Since their discovery at the beginning of this century, riboswitches have been regarded as potential antibacterial targets. Using fragment screening, high-throughput screening and rational ligand design guided by X-ray crystallography, lead compounds against various riboswitches have been identified. Here, we review the current status and suitability of the thiamine pyrophosphate (TPP), flavin mononucleotide (FMN), glmS, guanine, and other riboswitches as antibacterial targets and discuss them in a biological context. Further, we highlight challenges in riboswitch drug discovery and emphasis the need to develop riboswitch specific high-throughput screening methods. Keywords: antibacterial drug target; riboswitch; structure-based drug design; fragment screening; high-throughput screening 1. Introduction The emergence of bacterial resistance to antibiotics has become an urgent and serious threat to global public health. Resistant bacteria are expected to cause nearly 10 million deaths each year globally by 2050 [1]. As many pathogenic bacteria are evolving to persist against all existing antibiotics, it is feared that the health system will become incapacitated against serious bacterial infections [2,3]. Indeed, to address this concern, the World Health Citation: Panchal, V.; Brenk, R. Organization (WHO) has released a priority list of bacterial pathogens that need immediate Riboswitches as Drug Targets for attention, to focus antibiotic drug discovery efforts [4]. Consequently, a need for a new Antibiotics. Antibiotics 2021, 10, 45. generation of antibiotics with novel mechanisms of action against resistant bacteria is https://doi.org/10.3390/antibiotics 10010045 increasingly being recognized [5] and current drug discovery programs are exploring both proteins, as well as nucleic acid targets [6,7]. Received: 30 November 2020 Riboswitches are a novel antibacterial drug target class that could deliver urgently Accepted: 29 December 2020 needed antibiotics via a new mechanism of action. They occur almost exclusively in bacteria Published: 5 January 2021 and regulate the biosynthesis and transport of amino acids and essential metabolites, such as coenzymes, nucleobases and their derivatives by binding small molecules [8,9]. Residing 0 Publisher’s Note: MDPI stays neu- in the 5 untranslated region (UTR) of mRNA, these cis-regulatory elements are structured tral with regard to jurisdictional clai- non-coding RNAs that adopt alternative 3D-conformations to function as genetic switches ms in published maps and institutio- (“ribo-switching”) [10–12]. Riboswitches are comprised of two domains; an aptamer nal affiliations. domain that selectively binds the cognate ligands and an expression platform that translates the presence of ligands into expression or repression of downstream genes (Figure1). When the concentration of a cognate ligand in the intracellular milieu increases beyond a threshold, its binding to the aptamer domain induces a conformational change. In most Copyright: © 2021 by the authors. Li- cases, this ligand-induced alternate conformation leads to transcriptional attenuation by the censee MDPI, Basel, Switzerland. formation of a terminator or inhibition of translation by sequestering the ribosomal binding This article is an open access article distributed under the terms and con- site (RBS) or both, followed by switching off the downstream gene or operon [13–15]. These ditions of the Creative Commons At- riboswitch elements are termed “OFF-switches” (Figure1a). In other cases, the ligand- tribution (CC BY) license (https:// induced alternate conformation leads to the formation of an anti-terminator stem and/or creativecommons.org/licenses/by/ release of the RBS from the terminator stem followed by activation of the downstream 4.0/). target gene(s). Accordingly, these are referred to as “ON-switches” (Figure1b). Antibiotics 2021, 10, 45. https://doi.org/10.3390/antibiotics10010045 https://www.mdpi.com/journal/antibiotics Antibiotics 2021, 10, x FOR PEER REVIEW 2 of 24 Antibiotics 2021, 10, 45 2 of 22 of the downstream target gene(s). Accordingly, these are referred to as “ON-switches” (Figure 1b). Figure 1. Riboswitch-mediated gene regulation. Bacterial riboswitches regulate the expression of Figureimmediate 1. Riboswitch-mediated downstream genes by modulating gene regulation. their conformation Bacterial in response riboswitches to a change regulate in the the expression of immediateconcentration downstream of their cognate genes ligan byds (L). modulating (Strands involved their conformation in the formation in of response anti-antitermina- to a change in the con- centrationtor/anti-antisequester of their cognate (cyan and ligands magenta) (L). and (Strands terminator/sequester involved inthe (orange formation and yellow) of anti-antiterminator/anti- are color- coded). In most cases, binding of ligand leads to transcriptional and/or translational termination of antisequester (cyan and magenta) and terminator/sequester (orange and yellow) are color-coded). In downstream gene expression (open reading frame (ORF), marked in yellow). These riboswitches mostare known cases, as binding “OFF-switches” of ligand (a). leads In other to transcriptionalcases, ligand binding and/or leads translational to their expression termination of down- of downstream genestream expression genes and these (open riboswitches readingframe are called (ORF), “ON-switches” marked in(b). yellow). In the case These of translation riboswitches regu- are known as “OFF-switches”lation, the RBS (green (a). box) Inother is either cases, sequestered ligand (a binding) or released leads (b) upon to their ligand expression binding. In of the downstream case genes of transcriptional regulation, the RNA polymerase is either stalled by a terminator (a) or moves andpast thesethe antiterminator riboswitches (b) areupon called ligand “ON-switches” binding. RBS: ribosomal (b). In binding the case site. of translation regulation, the RBS (green box) is either sequestered (a) or released (b) upon ligand binding. In the case of transcriptional regulation,Riboswitches the RNA are polymerase widespread is in either bacteria. stalled Currently, by a terminator 28 experimentally (a) or moves validated past the ri- antiterminator (bboswitch) upon ligandclasses binding.are known, RBS: which ribosomal are distribu bindingted across site. more than 6000 bacterial species [16,17]. Riboswitches are categorized based on their cognate ligand and the fold of their Riboswitches are widespread in bacteria. Currently, 28 experimentally validated riboswitch classes are known, which are distributed across more than 6000 bacterial species [16,17]. Riboswitches are categorized based on their cognate ligand and the fold of their aptamer domain (Table1). Biochemically, these natural ligands span a wide spectrum of biomolecules, namely coenzymes, nucleotides and their derivatives, ions, amino acids, phosphorylated sugars and guanidine. Some riboswitches are widely present across hu- man bacterial pathogens and therefore, are suitable targets for broad-spectrum antibiotics, while less widespread riboswitches are potential targets for selective antibacterial drugs. The thiamine pyrophosphate (TPP) riboswitch is the most prevalent riboswitch and is the only one present in eukaryotes, including algae [18], fungi [19–21] and plants [22,23]. Antibiotics 2021, 10, x FOR PEER REVIEW 3 of 24 Antibiotics 2021, 10, x FOR PEER REVIEW 3 of 24 Antibiotics 2021, 10, x FOR PEER REVIEW 3 of 24 Antibiotics 2021, 10, x FOR PEER REVIEWaptamer domain (Table 1). Biochemically, these natural ligands span a wide spectrum3 of of24 aptamer domain (Table 1). Biochemically, these natural ligands span a wide spectrum of biomolecules, namely coenzymes, nucleotides and their derivatives, ions, amino acids, biomolecules,aptamer domain namely (Table coenzyme 1). Biochemically,s, nucleotides thes eand natural their ligands derivatives, span aions, wide amino spectrum acids, of phosphorylated sugars and guanidine. Some riboswitches are widely present across hu- phosphorylatedbiomolecules, namely sugars coenzymeand guanidine.s, nucleotides Some riboswitches and their derivatives, are widely presentions, amino across acids, hu- man bacterial pathogens and therefore, are suitable targets for broad-spectrum antibiotics, aptamermanphosphorylated bacterial domain pathogens (Tablesugars 1). andand Biochemically, guanidine.therefore, are Some thes suitable eriboswitches natural targets ligands for are broad-spectrum widelyspan a widepresent spectrum antibiotics, across hu-of while less widespread riboswitches are potential targets for selective antibacterial drugs. biomolecules,whileman bacterial less widespread namelypathogens coenzymeriboswitches and thers,efore, nucleotides are arepotential suitable and targets targetstheir forderivatives, for selective broad-spectrum antibacterialions, amino antibiotics, drugs.acids, The thiamine pyrophosphate (TPP) riboswitch is the most prevalent riboswitch and is the phosphorylatedThewhile thiamine less widespread pyrophosphate sugars andriboswitches guanidine. (TPP)
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