Aminoglycoside Conjugation for RNA Targeting: Antimicrobials and Beyond

Natural aminoglycosides are therapeutically useful antibiotics and very efficient RNA ligands. They are oligosaccharides that contain several ammonium groups able to interfere with the translation process in prokaryotes upon binding to bacterial ribosomal RNA (rRNA), and thus, impairing protein synthesis. Even if aminoglycosides are commonly used in therapy, these RNA binders lack selectivity and are able to bind to a wide number of RNA sequences/structures. This is one of the reasons for their toxicity and limited applications in therapy. At the same time, the ability of aminoglycosides to bind to various RNAs renders them a great source of inspiration for the synthesis of new binders with improved affinity and specificity toward several therapeutically relevant RNA targets. Thus, a number of studies have been performed on these complex and highly functionalized compounds, leading to the development of various synthetic methodologies toward the synthesis of conjugated aminoglycosides. The aim of this review is to highlight recent progress in the field of aminoglycoside conjugation, paying particular attention to modifications performed toward the improvement of affinity and especially to the selectivity of the resulting compounds. This will help readers to understand how to introduce a desired chemical modification for future developments of RNA ligands as antibiotics, antiviral, and anticancer compounds.     

MCSS-based predictions of RNA binding sites

The diversity of RNA tertiary structures provides the basis for specific recognition by proteins or small molecules. To investigate the structural basis and the energetics which control RNA-ligand interactions, favorable RNA binding sites are identified using the MCSS method, which has been employed previously only for protein receptors. Two different RNAs for which the structures have been determined by NMR spectroscopy were examined: two structures of the TAR RNA which contains an arginine binding site, and the structure of the 16S rRNA which contains an aminoglycoside binding site (paromomycin). In accord with the MCSS methodology, the functional groups representing the entire ligand or only part of it (one residue in the case of the aminoglycosides) are first replicated and distributed with random positions and orientations around the target and then energy minimized in the force field of the target RNA. The Coulombic term and the dielectric constant of the force field are adjusted to approximate the effects of solvent-screening and counterions. Optimal force field parameters are determined to reproduce the binding mode of arginine to the TAR RNA. The more favorable binding sites for each residue of the aminoglycoside ligands are then calculated and compared with the binding sites observed experimentally. The predictability of the method is evaluated and refinements are proposed to improve its accuracy. Received: 24 April 1998 / Accepted: 4 August 1998 / Published online: 7 December 1998     

Discovery of aminoglycoside mimetics by NMR-based screening of Escherichia coli A-site RNA

A method is described for the NMR-based screening for the discovery of aminoglycoside mimetics that bind to Escherichia coli A-site RNA. Although aminoglycosides are clinically useful, they exhibit high nephrotoxicity and ototoxicity, and their overuse has led to the development of resistance to important microbial pathogens. To identify a new series of aminoglycoside mimetics that could potentially overcome the problems associated with toxicities and resistance development observed with the aminoglycosides, we have prepared large quantities of E. coli 16 S A-site RNA and conducted an NMR-based screening of our compound library in search for small-molecule RNA binders against this RNA target. From these studies, several classes of compounds were identified as initial hits with binding affinities in the range of 70 microM to 3 mM. Lead optimization through synthetic modifications of these initial hits led to the discovery of several small-molecule aminoglycoside mimetics that are structurally very different from the known aminoglycosides. Structural models of the A-site RNA/ligand complexes were prepared and compared to the three-dimensional structures of the RNA/aminoglycoside complexes.     

Aminoglycosides modified by resistance enzymes display diminished binding to the bacterial ribosomal aminoacyl-tRNA site

Understanding the basic principles that govern RNA binding by aminoglycosides is important for the design of new generations of antibiotics that do not suffer from the known mechanisms of drug resistance. With this goal in mind, we examined the binding of kanamycin A and four derivatives (the products of enzymic turnovers of kanamycin A by aminoglycoside-modifying enzymes) to a 27 nucleotide RNA representing the bacterial ribosomal A site. Modification of kanamycin A functional groups that have been directly implicated in the maintenance of specific interactions with RNA led to a decrease in affinity for the target RNA. Overall, the products of reactions catalyzed by aminoglycoside resistance enzymes exhibit diminished binding to the A site of bacterial 16S rRNA, which correlates well with a loss of antibacterial ability in resistant organisms that harbor these enzymes.     

TINS, target immobilized NMR screening: an efficient and sensitive method for ligand discovery

We propose a ligand screening method, called TINS (target immobilized NMR screening), which reduces the amount of target required for the fragment-based approach to drug discovery. Binding is detected by comparing 1D NMR spectra of compound mixtures in the presence of a target immobilized on a solid support to a control sample. The method has been validated by the detection of a variety of ligands for protein and nucleic acid targets (K(D) from 60 to 5000 muM). The ligand binding capacity of a protein was undiminished after 2000 different compounds had been applied, indicating the potential to apply the assay for screening typical fragment libraries. TINS can be used in competition mode, allowing rapid characterization of the ligand binding site. TINS may allow screening of targets that are difficult to produce or that are insoluble, such as membrane proteins.     

Two-dimensional combinatorial screening identifies specific aminoglycoside-RNA internal loop partners

Herein is described the identification of RNA internal loops that bind to derivatives of neomycin B, neamine, tobramycin, and kanamycin A. RNA loop-ligand partners were identified by a two-dimensional combinatorial screening (2DCS) platform that probes RNA and chemical spaces simultaneously. In 2DCS, an aminoglycoside library immobilized onto an agarose microarray was probed for binding to a 3 x 3 nucleotide RNA internal loop library (81,920 interactions probed in duplicate in a single experiment). RNAs that bound aminoglycosides were harvested from the array via gel excision. RNA internal loop preferences for three aminoglycosides were identified from statistical analysis of selected structures. This provides consensus RNA internal loops that bind these structures and include: loops with potential GA pairs for the neomycin derivative, loops with potential GG pairs for the tobramycin derivative, and pyrimidine-rich loops for the kanamycin A derivative. Results with the neamine derivative show that it binds a variety of loops, including loops that contain potential GA pairs that also recognize the neomycin B derivative. All studied selected internal loops are specific for the aminoglycoside that they were selected to bind. Specificity was quantified for 16 selected internal loops by studying their binding to each of the arrayed aminoglycosides. Specificities ranged from 2- to 80-fold with an average specificity of 20-fold. These studies show that 2DCS is a unique platform to probe RNA and chemical space simultaneously to identify specific RNA motif-ligand interactions.     

Studying aminoglycoside antibiotic binding to HIV-1 TAR RNA by electrospray ionization mass spectrometry

The recognition of the aminoglycosides neomycin and streptomycin by HIV-1 TAR RNA was studied by electrospray ionization mass spectrometry (ESI-MS). Members of the aminoglycoside family of antibiotics are known to target a wide variety of RNA molecules. Neomycin and streptomycin inhibit the formation of the Tat protein–TAR RNA complex, an assembly that is believed to be necessary for HIV replication. The noncovalent complexes formed by the binding of aminoglycosides to TAR RNA and the Tat–TAR complex were detected by ESI-MS. Neomycin has a maximum binding stoichiometry of three and two to TAR RNA and to the Tat–TAR complex, respectively. Data from the ESI-MS experiments suggest that a high affinity binding site of neomycin is located near the three-nucleotide bulge region of TAR RNA. This is consistent with previous solution phase footprinting measurements [H.-Y. Mei et al., Biochemistry 37 (1998) 14204]. Neomycin has a higher affinity toward TAR RNA than streptomycin, as measured by ESI-MS competition binding experiments. A noncovalent complex formed between a small molecule inhibitor of TAR RNA, which has a similar solution binding affinity as the aminoglycosides, and TAR RNA is much less stable than the RNA–aminoglycoside complexes to collisional dissociation in the gas phase. It is believed that the small molecule inhibitor interacts with TAR RNA via hydrophobic interactions, whereas the aminoglycosides bind to RNAs through electrostatic forces. This difference in gas phase stabilities may prove useful for discerning the types of noncovalent forces holding complexes together.     

Conformational constraint as a means for understanding RNA-aminoglycoside specificity

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.     

Fluorescent HIV-1 Dimerization Initiation Site: design, properties, and use for ligand discovery

The HIV-1 Dimerization Initiation Site (DIS) is an intriguing, yet underutilized, viral RNA target for potential antiretroviral therapy. To study the recognition features of this target and to provide a quantitative, rapid, and real-time tool for the discovery of new binders, a fluorescence-based assay has been constructed. It relies on strategic incorporation of 2-aminopurine, an isosteric fluorescent adenosine analogue, into short hairpin RNA constructs. These oligomers self-associate to form a kissing loop that thermally rearranges into a more stable extended duplex, thereby mimicking the association and structural features of the native RNA sequence. We demonstrate the ability of two fluorescent DIS constructs, DIS272(2AP) and DIS273(2AP), to report the binding of known DIS binders via changes in their emission intensity. Binding of aminoglycosides such as paromomycin to DIS272(2AP) results in significant fluorescence enhancement, while ligand binding to DIS273(2AP) results in fluorescence quenching. These observations are rationalized by comparison to the sequence-analogous bacterial A-site, where the relative emission of the fluorescent probe is dependent on the placement of the flexible purine residues inside or outside the helical domain. Analysis of binding isotherms generated using DIS272(2AP) yields submicromolar EC50 values for paromomycin (0.5 +/- 0.2 microM) and neomycin B (0.6 +/- 0.2 microM). Other neomycin-family aminoglycosides are less potent binders with neamine, the core pharmacophore, displaying the lowest affinity of 21 +/- 1 microM. Screening of additional aminoglycosides and their derivatives led to the discovery of new, previously unreported, aminoglycoside binders of the HIV DIS RNA, among them butirosin A (5.5 +/- 0.6 microM) and apramycin (7.6 +/- 1.0 microM). A conformationally constrained neomycin B analogue displays a rather high affinity to the DIS (1.9 +/- 0.2 microM). Among a series of nucleobase aminoglycoside conjugates, only the uracil derivatives display a measurable affinity using this assay with EC50 values in the 2 microM range. In addition, similarity between the solution behavior of HIV-1 DIS and the bacterial decoding A-site has been observed, particularly with respect to the intra- and extra-helical residence of the conformationally flexible A residues within the bulge. Taken together, the observations reported here shed light on the solution behavior of this important RNA target and are likely to facilitate the design of new DIS selective ligands as potential antiretroviral agents.     

Fluorescence-based approach for detecting and characterizing antibiotic-induced conformational changes in ribosomal RNA: comparing aminoglycoside binding to prokaryotic and eukaryotic ribosomal RNA sequences

Aminoglycoside antibiotics bind specifically to a conserved sequence of the 16S ribosomal RNA (rRNA) A site and interfere with protein synthesis. One model for the mechanism underlying the deleterious effects of aminoglycosides on protein synthesis invokes a drug-induced conformational change in the rRNA that involves the destacking of two adenine residues (A1492 and A1493 in Escherichia coli) at the A site. We describe here a fluorescence-based approach for detecting and characterizing this drug-induced conformational change in the target rRNA. In this approach, we insert the fluorescent base analogue 2-aminopurine in place of A1492 in an E. coli 16S rRNA A-site model oligonucleotide (EcWT) as well as in a mutant form of this oligomer (A1408G) in which A1408 has been replaced with a guanine. The presence of guanine at 1408 instead of adenine represents one of the major sequence differences between prokaryotic and eukaryotic A sites, with the latter A sites being resistant to the deleterious effects of aminoglycosides. Binding of the aminoglycoside paromomycin to the 2AP-substituted forms of EcWT and A1408G induced changes in fluorescence quantum yield consistent with drug-induced base destacking in EcWT but not A1408G. Isothermal titration calorimetry studies reveal that paromomycin binds to the EcWT duplex with a 31-fold higher affinity than the A1408G duplex, with this differential affinity being enthalpic in origin. In the aggregate, these observations are consistent with both rRNA binding affinity and drug-induced base destacking being important determinants in the prokaryotic specificity of aminoglycosides. Combining fluorescence quantum yield and lifetime data allows for quantification of the extent of drug-induced base destacking, thereby providing a convenient tool for evaluating the relative impacts of both novel and existing A-site targeting ligands on rRNA conformation and potentially for predicting relative antibiotic activities and specificities.     

Specificity of aminoglycoside antibiotics for the A-site of the decoding region of ribosomal RNA

Background: Aminoglycoside antibiotics bind to the A-site of the decoding region of 16S RNA in the bacterial ribosome, an interaction that is probably responsible for their activity. A detailed study of the specificity of aminoglycoside binding to A-site RNA would improve our understanding of their mechanism of antibiotic activity.Results: We have studied the binding specificity of several aminoglycosides with model RNA sequences derived from the 16S ribosomal A-site using surface plasmon resonance. The 4,5-linked (neomycin) class of aminoglycosides showed specificity for wild-type A-site sequences, but the 4,6-linked class (kanamycins and gentamicins), generally showed poor specificity for the same sequences. Methylation of a cytidine in the target RNA, as found in the Escherichia coli ribosome, had negligible effects on aminoglycoside binding.Conclusions: Although both 4,5- and 4,6-linked aminoglycosides target the same ribosomal site, they appear to bind and effect antibiotic activity in different manners. The aminoglycosides might recognize different RNA conformations or the interaction might involve different RNA tertiary structures that are not equally sampled in our ribosome-free model. These results imply that models of ribosomal RNA must be carefully designed if the data are expected to accurately reflect biological activity.     

SAR by MS

RNAs have recently emerged as an exciting new target for small molecule therapeutics. Conventional HTS discovery strategies measuring disruption of RNAprotein interactions have proven unsuccessful. We describe a ligand-based drug discovery strategy that addresses the inherent difficulties RNA targets. The strategy is based on: 1) using a MS spectrometry (MS)-based assay to measure the affinity of compounds for a target; 2) performing competitive binding experiments and molecular modeling with the motifs to determine the binding site(s) of the ligands; 3) design and synthesis of derivatives of interesting binders to establish the linking sites; 4) identifying the appropriate linker group using MS; 5) fusing motifs into a more complex structure to afford higher affinity compounds. Example of applying this strategy to identify new classes of lead molecules with affinity and specificity for ribosomal RNA targets will be presented.     

20 years of DNA-encoded chemical libraries

The identification of specific binding molecules is a central problem in chemistry, biology and medicine. Therefore, technologies, which facilitate ligand discovery, may substantially contribute to a better understanding of biological processes and to drug discovery. DNA-encoded chemical libraries represent a new inexpensive tool for the fast and efficient identification of ligands to target proteins of choice. Such libraries consist of collections of organic molecules, covalently linked to a unique DNA tag serving as an amplifiable identification bar code. DNA-encoding enables the in vitro selection of ligands by affinity capture at sub-picomolar concentrations on virtually any target protein of interest, in analogy to established selection methodologies like antibody phage display. Multiple strategies have been investigated by several academic and industrial laboratories for the construction of DNA-encoded chemical libraries comprising up to millions of DNA-encoded compounds. The implementation of next generation high-throughput sequencing enabled the rapid identification of binding molecules from DNA-encoded libraries of unprecedented size. This article reviews the development of DNA-encoded library technology and its evolution into a novel drug discovery tool, commenting on challenges, perspectives and opportunities for the different experimental approaches.     

Nonionic side chains modulate the affinity and specificity of binding between functionalized polyamines and structured RNA

This Communication introduces side-chain-bearing polyamines as molecules for selective recognition of folded RNA structures. The complex folded structures associated with RNA create binding pockets for proteins, and also binding sites for small molecules. Developing organic molecules that can bind RNA with high affinity and specificity is a challenge that must be overcome for RNA to be considered a viable drug target. In this work, six polyamines with different side chains were synthesized to test for effects on binding affinity and specificity to TAR RNA and RRE RNA of HIV. Binding interactions between polyamines and RNAs were examined using two footprinting assays, based on terbium-induced cleavage and magnesium-catalyzed cleavage at higher pH. The binding constants and the binding specificity were highly dependent on the side chains of the polyamines, demonstrating that this class of molecules is a very promising starting point for development of highly selective RNA-binding ligands.     

Designer aminoglycosides: the race to develop improved antibiotics and compounds for the treatment of human genetic diseases

Aminoglycosides are highly potent, broad-spectrum antibiotics that exert their bactericidal therapeutic effect by selectively binding to the decoding aminoacyl site (A-site) of the bacterial 16 S rRNA, thereby interfering with translational fidelity during protein synthesis. The appearance of bacterial strains resistant to these drugs, as well as their relative toxicity, have inspired extensive searches towards the goal of obtaining novel molecular designs with improved antibacterial activity and reduced toxicity. In the last few years, a new, aminoglycoside dependent therapeutic approach for the treatment of certain human genetic diseases has been identified. These treatments rely on the ability of certain aminoglycosides to induce mammalian ribosomes to readthrough premature stop codon mutations. This new and challenging task has introduced fresh research avenues in the field of aminoglycoside research. Recent observations and current challenges in the design of aminoglycosides with improved antibacterial activity and the treatment of human genetic diseases are discussed.     

Design and Synthesis of Carbohydrate Mimetics: A New Strategy for Tackling the Problem of Carbohydrate-Mediated Biological Recognition

Carbohydrates have been known as poor candidates for drug development. Recent studies have, however, shown that structurally simplified small molecules as mimics of complex carbohydrates recognized by receptors can be developed as inhibitors of carbohydrate-mediated biological recognition. In addition, small molecules with higher affinity and specificity than the parent ligands can be developed by incorporating additional hydrophobic or charged groups in the carbohydrate mimetic which contains essential functional groups for receptor binding. Representative examples are illustrated in the studies of sialyl Lewis x - selectin interactions, glycosidase and glycosyltransferase reactions and aminoglycoside antibiotic - RNA interactions.     

Exploring the use of conformationally locked aminoglycosides as a new strategy to overcome bacterial resistance

The emergence of bacterial resistance to the major classes of antibiotics has become a serious problem over recent years. For aminoglycosides, the major biochemical mechanism for bacterial resistance is the enzymatic modification of the drug. Interestingly, in several cases, the oligosaccharide conformation recognized by the ribosomic RNA and the enzymes responsible for the antibiotic inactivation is remarkably different. This observation suggests a possible structure-based chemical strategy to overcome bacterial resistance; in principle, it should be possible to design a conformationally locked oligosaccharide that still retains antibiotic activity but that is not susceptible to enzymatic inactivation. To explore the scope and limitations of this strategy, we have synthesized several aminoglycoside derivatives locked in the ribosome-bound "bioactive" conformation. The effect of the structural preorganization on RNA binding, together with its influence on the aminoglycoside inactivation by several enzymes involved in bacterial resistance, has been studied. Our results indicate that the conformational constraint has a modest effect on their interaction with ribosomal RNA. In contrast, it may display a large impact on their enzymatic inactivation. Thus, the work presented herein provides a key example of how the conformational differences exhibited by these ligands within the binding pockets of the ribosome and of those enzymes involved in bacterial resistance can, in favorable cases, be exploited for designing new antibiotic derivatives with improved activity in resistant strains.     

Scanning conformational space with a library of stereo- and regiochemically diverse aminoglycoside derivatives: the discovery of new ligands for RNA hairpin sequences

A library of stereo- and regiochemically diverse aminoglycoside derivatives was screened at 1 microM using surface plasmon resonance (SPR) against RNA hairpin models of the bacterial A-site, and the HIV viral TAR and RRE sequences. In order to double the stereochemical diversity of the library, the compounds were screened against both enantiomers of each of these sequences. Remarkably, this initial screen suggested that the same four aminoglycoside derivatives bound most tightly to all three of the RNAs, suggesting that these compounds were good RNA binders which, nonetheless, discriminated poorly between the RNA sequences. The interactions between selected isomeric aminoglycoside derivatives and the RNA hairpins were then studied in more detail using an SPR assay. Three isomeric tight-binding aminoglycoside derivatives, which had been identified from the initial screen, were found to bind more tightly to the RNA hairpins (with K(D) values in the range 0.23 to 4.7 microM) than a fourth isomeric derivative (which had K(D) values in the range 6.0 to 30 microM). The magnitude of the tightest RNA-aminoglycoside interactions stemmed, in large part, from remarkably slow dissociation of the aminoglycosides from the RNA targets. The three tight-binding aminoglycoside derivatives were found, however, to discriminate rather poorly between alternative RNA sequences with, at best, around a twenty-fold difference in affinity for alternative RNA hairpin sequences. Within the aminoglycoside derivative library studied, high affinity for an RNA target was not accompanied by good discrimination between alternative RNA sequences.     

Aminoglycoside (neomycin) preference is for A-form nucleic acids,not just RNA: results from a competition dialysis study

Aminoglycosides have been at the forefront of antimicrobial therapy for the past 50 years. Their specificity is believed to lie in binding duplex RNAs (rRNA). Competition dialysis studies of various nucleic acid forms with 9-aminoacridine, quinacrine, and a neomycin-acridine conjugate were carried out. Our results suggest a strong preference for aminoglycoside binding to nucleic acids that can adopt an A-type conformation. These results challenge the common belief that aminoglycoside specificity is simply for duplex RNAs.     

Identification of Structure–Activity Relationships from Screening a Structurally Compact DNA‐Encoded Chemical Library

Methods for the rapid and inexpensive discovery of hit compounds are essential for pharmaceutical research and DNA‐encoded chemical libraries represent promising tools for this purpose. We here report on the design and synthesis of DAL‐100K, a DNA‐encoded chemical library containing 103 200 structurally compact compounds. Affinity screening experiments and DNA‐sequencing analysis provided ligands with nanomolar affinities to several proteins, including prostate‐specific membrane antigen and tankyrase 1. Correlations of sequence counts with binding affinities and potencies of enzyme inhibition were observed and enabled the identification of structural features critical for activity. These results indicate that libraries of this type represent a useful source of small‐molecule binders for target proteins of pharmaceutical interest and information on structural features important for binding.