RNA As a Drug Target: Chemical, Modelling, and Evolutionary Tools Thomas Hermann and Eric Westhof
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66 RNA as a drug target: chemical, modelling, and evolutionary tools Thomas Hermann and Eric Westhof Dramatic technical progress in RNA synthesis and structure ment [SELEX]) [1] and high-resolution NMR structure determination has allowed several dif®culties inherent to the determination, together with improved methods for the preparation, handling and structural analysis of RNA to be combinatorial synthesis of therapeutic agents [2,3•,4,5•] overcome, and this has led to a wealth of information about have opened the road for drug discovery in the ®eld RNA structure and its relationship with biological function. It of RNA-targeted effectors [6,7•,8]. Here, we will review is now fully recognized that RNA molecules intervene at all the recent progress made in the study of the interaction stages of cell life, not only because of key sequence motifs of small molecules with functional RNA by means but also because of intricate three-dimensional folds. This of chemical, modelling and evolutionary tools. More realization has promoted RNA to a potential therapeutic extensive reviews, especially on antibiotic and metal target. As in protein motifs recognizing nucleic acids, groups complex binding, were recently written by Wallis and of the molecule interacting with RNA contribute to speci®c Schroeder [9] and by Chow and Bogdan [10]. Among the binding through de®ned hydrogen bonds and van der numerous arti®cial RNA molecules (aptamers) that have Waals docking, while other parts contribute to the driving been selected by in vitro evolution for speci®c binding to force of binding via less speci®c electrostatic interactions a target molecule, we will focus on RNA aptamers that accompanied by water and ion displacement. speci®cally bind antibiotics. The recent work on all kinds of aptamers has been summarized in several excellent reviews [11±13]. Due to its therapeutical importance, we Addresses will discuss separately in the last section recent efforts Institut de Biologie Moleculaire et Cellulaire du CNRS, UPR 9002, 15 rue Rene Descartes, F-67084 Strasbourg, France in targeting RNA components of the replication process Correspondence: Eric Westhof; e-mail: [email protected] of HIV. Current Opinion in Biotechnology 1998, 9:66±73 Targeting ribosomal RNA http://biomednet.com/elecref/0958166900900066 The 16S RNA component of the prokaryotic ribosome was Current Biology Ltd ISSN 0958-1669 the ®rst RNA to be identi®ed as a target for small molecule Abbreviations effectors [14]. Binding of a variety of different antibiotics, HDV hepatitis delta virus such as aminoglycosides (Figure 1), to functional sites RRE Rev-response element of the bacterial ribosomal RNA leads to miscoding TAR trans-activating region during the translation process. Mutational and chemical probing experiments [15•,16], along with ¯uorescence measurements [17], on the interaction of aminoglycosides Introduction with model oligonucleotides that mimic the antibiotic Because of its key and versatile roles in biological target in the A site of the 16S rRNA decoding region processes, such as protein synthesis, mRNA splicing, (an asymmetric internal loop of three and four unpaired transcriptional regulation and retroviral replication, RNA bases) have revealed structural features important for the is a prime target for natural compounds as well as speci®c interaction of aminoglycosides with RNA. The for chemically designed drugs. Indeed, the binding of structure of a 27 nucleotide A-site model RNA complexed molecules to a speci®c RNA target might in¯uence with the aminoglycoside paromomycin (Figure 1) has the biological activity of the RNA by preventing the been determined by NMR spectroscopy [18••], showing binding of the biologically relevant macromolecule (either that the antibiotic binds to the loop region in the protein or RNA), by inhibiting RNA catalysis, or by deep groove of the RNA within a pocket opened by a forcing an alternative conformation on the RNA. The single bulged adenine and an A±A base pair. Amino and three-dimensional folding of RNA gives rise to complex hydroxyl substituents of rings A and B, conserved among structures allowing the highly speci®c binding of effector aminoglycosides that bind to the A site, are required for molecules. As with proteins, the structural features of speci®c interactions of paromomycin with the ribosomal the potential RNA targets that determine the selective RNA, while rings C and D, relatively disordered in the interaction with drugs depend on the source organism. NMR structure [18••], contribute unspeci®cally to the Compared to DNA, RNA displays a greater structural complex formation by additional electrostatic and van der diversity and lacks repair mechanisms, a fact that enhances Waals interactions. The principle of providing a moiety the impact of therapeutics directed at RNA. that conveys binding speci®city by recognizing internal bulges or asymmetric loops and another moiety that Recent advances in RNA synthesis, in vitro evolution supports binding strength by non-speci®c interactions is (systematic evolution of ligands by exponential enrich- frequently exploited by RNA-binding molecules, as we RNA as a drug target: chemical, modelling, and evolutionary tools Hermann and Westhof 67 will see later for other examples of RNA±drug interactions. Figure 1 The speci®city of aminoglycoside binding to the 16S rRNA is highlighted by the fact that aminoglycoside (a) + antibiotics act preferentially on prokaryotic organisms due 6' X Neomycin B (6' X = H3N ) to a much higher af®nity of prokaryotic 16S rRNA for these Paromomycin (6' X = HO) antibiotics compared to eukaryotic ribosomal RNA. The HO O A NMR analysis of the A-site RNA±paromomycin complex HO •• + H N [18 ] has revealed the structural basis for the difference: 2' H3N 2 3 O ®rstly, a disruption of the A±A base pair in eukaryotes, B which have a G in place of one of the adenines; and, sec- O NH3+ 1 ondly, a mispairing at positions 1409±1491, which provide HO OH the ¯oor of the antibiotic binding pocket in the prokaryotic O A site. In eukaryotes, a G±A base pair equivalent to C the A±A pair in prokaryotes cannot be formed due to geometrical reasons and, thus, the RNA deep groove is NH + 6"' O 3 not opened, preventing aminoglycoside binding to the OH •• eukaryotic A site [18 ]. Finally, the NMR structure of O D the A site RNA±paromomycin complex has uncovered + OH 2'" H3N the molecular basis of resistance development against OH speci®c aminoglycosides by methylation of nucleotides within the 16S rRNA that border the aminoglycoside (b) Tobramycin •• + binding pocket [18 ]. 2' H3N It has been speculated that aminoglycosides, exerting HO O their antibiotic effects by inducing miscoding during A + H2N 3 the translation process, may stabilize a high af®nity 2' H3N O conformation of rRNA for the aminoacyl-tRNA for which B •• proofreading is impeded [18 ]. Likewise, it has been HO NH3+ 1 suggested that binding of the pyrrolidine antibiotic O anisomycin to 28S rRNA induces a conformational change OH • in ribosomal RNA [19 ], a system where the action C O of the antibiotic on the ribosomal RNA activates a HO OH + stress-activated protein kinase and, thus, giving a response 2" H3N similar to that induced by the enzyme ribotoxins α-sarcin Current Opinion in Biotechnology and ricin. The example of anisomycin nicely illustrates how a low-molecular-weight effector targeted at a speci®c Structures of some aminoglycoside antibiotics discussed in the binding site in RNA is able to trigger a complex process text. (a) Neomycin B and paromomycin, which belong to the such as the activation of a protein kinase. 4,5-disubstituted deoxystreptamine class. (b) Tobramycin, which belongs to the 4,6-disubstituted deoxystreptamines. Targeting catalytic RNAs As well as being among compounds that bind to the 16S of group I introns [28] has been performed to construct rRNA, the aminoglycoside antibiotics also act as inhibitors model complexes useful in rationalizing the differences of catalytic RNAs (reviewed in [20]), such as the group I in inhibition behaviour between these antibiotics [27,29]. intron [21], the hammerhead ribozyme [22], and the Another class of compounds that affect the self-splicing ribozymes from hepatitis delta virus (HDV) [23]. Group I of group I introns is L-arginine and its derivatives, which introns are attractive targets for therapeutics because competitively inhibit the cleavage step but stimulate they are interspersed in key genes of several pathogenic the following ligation reaction [25]. Recently, a ribozyme microorganisms, while they are not found in the human system has been developed from group I introns and genome [21,24,25]. Self-splicing of group I intron RNA was used to rapidly identify small molecule inhibitors is inhibited by aminoglycosides non-competitively with targeting catalytic introns in high-throughput screening respect to the binding of the guanosine cofactor, indicating assays [24,30•]. that these antibiotics do not interfere with the G-binding site [26]. In contrast, the macrocyclic peptide antibiotics The in¯uence of antibiotics on another large catalytic of the tuberactinomycin family, for example, viomycin, RNA, M1 RNA, the RNA component of RNase P, is compete with the guanosine cofactor for the G-binding not well documented. In contrast to group I introns site within helix P7, part of a pseudoknot which is located and small ribozymes, M1 RNA is not inhibited by in the conserved core of the intron [27]. Docking of both aminoglycoside antibiotics but only by puromycin, a aminoglycosides and tuberactinomycins to a 3D model nucleoside derivative [31]. The fact that M1 RNA is 68 Analytical biotechnology catalytically active in vitro only under high magnesium obtained when high-stringency conditions were imposed concentration certainly contributes to the absence of during the in vitro selection [38,40,41,42•], while a wide inhibition by known cationic inhibitors of catalytic RNAs.