Aminoglycoside binding to the hammerhead ribozyme: a general model for the interaction of cationic antibiotics with RNA 1 1Edited by J. Karn

Abstract

A variety of drugs inhibit biological key processes by binding to a speci®c RNA component. We focus here on the well-analysed hammerhead ribozyme RNA that is inhibited by aminoglycoside antibiotics, a process considered as a paradigm for studying drug/RNA interactions. With insight gained from molecular dynamics simulations of the ribozyme in the presence of Mg 2 identi®ed by crystallography and of aminoglycosides in solution, a general model for aminoglycoside binding to RNA is proposed. A striking structurally based complementarity between the charged ammonium groups of the aminoglycosides and the metal binding sites in the hammerhead was uncovered. Despite dynamical exibility of the aminoglycosides, several of the intramolecular distances between the charged ammonium groups of the drugs were found to be rather constant. Intramolecular ammonium distances of the aminoglycosides span ranges similar to the interionic distances between Mg 2 in the hammerhead. Successful docking of aminoglycosides to the hammerhead ribozyme could be achieved by positioning the ammonium groups at the sites occupied by Mg 2 . The covalently linked ammonium groups of the aminoglycosides are thus able to complement in space the negative electrostatic potential created by a three-dimensional RNA fold. Consequently, it is suggested that aminoglycoside-derived sugars could constitute a basic set of yardstick synthons ideal for rational and combinatorial synthesis of drugs targeted at biologically relevant RNA folds.

Rational Drug Design and High-Throughput Techniques for RNA Targets

“…Chemically modified aminoglycosides in which amino groups had systematically been added [99] or hydroxyl groups removed one at a time [100] were analyzed for their binding and inhibitory effects on the catalytic hammerhead RNA. Binding affinities of modified aminoglycosides correlate with the number and basicity of cationic ammonium groups [61], supporting a general model for the interaction of cationic antibiotics with RNA based on structural electrostatic complementarity [60]. Similar findings were reported from screening of designed neomycin B analogs targeting the A site of eubacterial 16S rRNA [93].…”

“…This is illustrated by the examples of thiostrepton recognition in the tertiary folded GTPase center of eubacterial 23S rRNA [56][57][58], recognition of the cyclic peptide antibiotic viomycin by RNA pseudoknots [59], and aminoglycoside binding at the three-way junction of the hammerhead ribozyme [45,60,61]. In contrast, the aminoglycoside binding site in the 16S rRNA fold is located at the A site ( Fig.…”

“…Molecular recognition has been studied for RNA complexes of proteins [95,96], peptides and low-molecular weight compounds [15][16][17]97]. Strategies for the structurebased rational design of molecules targeting RNA and RNA complexes have recently been outlined [1,98]. Here, we will briefly summarize previous rational and combinatorial synthesis approaches towards target-specific RNA binders.…”

Neomycin B inhibits splicing of the td intron indirectly by interfering with translation and enhances missplicing in vivo

“…Neomycin B inhibits translation at the prokaryotic ribosome by inducing misreading of the genetic code (Davies et al+, 1965;Davies & Davis, 1968)+ Furthermore, several aminoglycosides inhibit the catalytic activity of self-splicing group I introns and of the hammerhead and HDV ribozymes in vitro (von Ahsen et al+, 1991;Stage et al+, 1995;Rogers et al+, 1996)+ Two RNA-protein interactions in HIV, RRE-Rev and TarTat, are disrupted by neomycin B in vitro (Zapp et al+, 1993;Mei et al+, 1995)+ Detailed analyses of neomycin binding sites have revealed two types of RNA target sites for aminoglycoside antibiotics+ The first type contains internal asymmetrical loops with widened major grooves, which are found in the decoding site of the 16S rRNA (Noller, 1991;Purohit & Stern, 1994;Fourmy et al+, 1996;Recht et al+, 1996), in the RRE RNA of HIV (Zapp et al+, 1993), in the P4/P5 internal loop of group I introns (von Ahsen & Noller, 1993;Cate et al+, 1996;Hoch et al+, 1998), and in several neomycin aptamers isolated via in vitro selection (Wallis et al+, 1995)+ The second type of target sites are divalent metal ion binding sites in the core of ribozymes (Clouet d'Orval et al+, 1995;Rogers et al+, 1996;Hoch et al+, 1998)+ Molecular-dynamics-simulation experiments revealed that the distances between positively charged aminogroups of aminoglycosides coincide with the distances of several divalent metal ions in the X-ray structure of the hammerhead ribozyme+ This leads to the proposal that neomycin B acts via simultaneous displacement of several essential metal ions (Hermann & Westhof, 1998)+ The inhibitory neomycin binding site in the T4 phage td intron was shown to be a type-two binding site (Hoch et al+, 1998)+ Inhibition of translation and disruption of the RRERev interaction by neomycin B has been demonstrated to occur both in vitro and in vivo (Benveniste & Davies, 1973;Zapp et al+, 1993)+ While the concentrations of neomycin B required to inhibit translation are similar in vivo and in vitro, disruption of the RRE-Rev interaction required almost 100-fold higher concentrations in vivo than in vitro+ These observations suggest that aminoglycoside binding might differ substantially depending on the intracellular conditions+ Here we examined the effects of neomycin B and several other aminoglycosides on splicing of the td intron in vivo+…”

Insights into functional modulation of catalytic RNA activity

“…It has been observed that aminoglycoside antibiotics inhibit the cleavage reaction of the ribozyme (126). Among various aminoglycosides that have been tested (such as gentamycin, kanamycin, neomycin, paramomycin, lividomycin, ribostamycin, neamine, butirosin, apramycin and streptomycin), neomycin and 5-episisomicin have been reported as the strongest inhibitors (127,128).…”