Coupling of drug protonation to the specific binding of aminoglycosides to the A site of 16 S rRNA: elucidation of the number of drug amino groups involved and their identities

2-Deoxystreptamine (2-DOS) aminoglycoside antibiotics bind specifically to the central region of the 16S rRNA A site and interfere with protein synthesis. Recently, we have shown that the binding of 2-DOS aminoglycosides to an A site model RNA oligonucleotide is linked to the protonation of drug amino groups. Here, we extend these studies to define the number of amino groups involved as well as their identities. Specifically, we use pH-dependent 15N NMR spectroscopy to determine the pK(a) values of the amino groups in neomycin B, paromomycin I, and lividomycin A sulfate, with the resulting pK(a) values ranging from 6.92 to 9.51. For each drug, the 3-amino group was associated with the lowest pK(a), with this value being 6.92 in neomycin B, 7.07 in paromomycin I, and 7.24 in lividomycin A. In addition, we use buffer-dependent isothermal titration calorimetry (ITC) to determine the number of protons linked to the complexation of the three drugs with the A site model RNA oligomer at pH 5.5, 8.8, or 9.0. At pH 5.5, the binding of the three drugs to the host RNA is independent of drug protonation effects. By contrast, at pH 9.0, the RNA binding of paromomycin I and neomycin B is coupled to the uptake of 3.25 and 3.80 protons, respectively, with the RNA binding of lividomycin A at pH 8.8 being coupled to the uptake of 3.25 protons. A comparison of these values with the protonation states of the drugs predicted by our NMR-derived pK(a) values allows us to identify the specific drug amino groups whose protonation is linked to complexation with the host RNA. These determinations reveal that the binding of lividomycin A to the host RNA is coupled to the protonation of all five of its amino groups, with the RNA binding of paromomycin I and neomycin B being linked to the protonation of four and at least five amino groups, respectively. For paromomycin I, the protonation reactions involve the 1-, 3-, 2'-, and 2"'-amino groups, while, for neomycin B, the binding-linked protonation reactions involve at least the 1-, 3-, 2', 6'-, and 2"'-amino groups. Our results clearly identify drug protonation reactions as important thermodynamic participants in the specific binding of 2-DOS aminoglycosides to the A site of 16S rRNA.     

Aminoglycoside binding to human and bacterial A-Site rRNA decoding region constructs

The 16S bacterial ribosomal A-site decoding rRNA region is thought to be the pharmacological target for the aminoglycoside antibiotics. The clinical utility of aminoglycosides could possibly depend on the preferential binding of these drugs to the prokaryotic A-site versus the corresponding A-site from eukaryotes. However, quantitative aminoglycoside binding experiments reported here on prokaryotic and eukaryotic A-site RNA constructs show that there is little in the way of differential binding affinities of aminoglycosides for the two targets. The largest difference in affinity is 4-fold in the case of neomycin, with the prokaryotic A-site construct exhibiting the higher binding affinity. Mutational studies revealed that decoding region constructs retaining elements of non-Watson-Crick (WC) base pairing, specifically bound aminoglycosides with affinities in the muM range. These studies are consistent with the idea that aminoglycoside antibiotics can specifically bind to RNA molecules as long as the latter have non-A form structural elements allowing access of aminoglycosides to the narrow major groove.     

Aminoglycoside antibiotics bound to aminoglycoside-detoxifying enzymes and RNA adopt similar conformations

Conformations of ribostamycin and isepamicin, aminoglycoside antibiotics, bound to an aminoglycoside antibiotic, 3′-phosphotransferase, were determined by transferred nuclear Overhauser effect spectroscopy and molecular modeling. Two major conformers of enzyme-bound ribostamycin, a neomycin-group aminoglyeoside were observed. The 3′- and 5″-OH groups (reactive hydroxyl groups) in the conformers are placed in approximate locations. One of the conformers is similar to the structure of paromomycin bound to a 27-nucleotide piece of ribosomal RNA that represents the A-site of the small ribosomal subunit, where rings A and C are in an orthogonal arrangement. Isepamicin, a kanamycin-group aminoglycoside antibiotic, also showed two major enzyme-bound conformations. Both conformations were similar to those observed for bound isepamicin in the active site of an aminoglycoside(6′)-acetyl transferase-Ii. Conformations of other RNA-bound kanamycin-group aminoglycosides were also similar to the enzyme-bound conformations of isepamicin. These observations suggest that aminoglycosides may adopt similar conformations when bound to RNA and protein targets. This may have significant implications in the design of enzyme inhibitors and/or antibiotics.     

Calorimetric and spectroscopic studies of aminoglycoside binding to AT-rich DNA triple helices

Calorimetric and fluorescence techniques were used to characterize the binding of aminoglycosides-neomycin, paromomycin, and ribostamycin, with 5′-dA₁₂-x-dT₁₂-x-dT₁₂-3′ intramolecular DNA triplex (x = hexaethylene glycol) and poly(dA)·2poly(dT) triplex. Our results demonstrate the following features: (1) UV thermal analysis reveals that the T_m for triplex decreases with increasing pH value in the presence of neomycin, while the T_m for the duplex remains unchanged. (2) The binding affinity of neomycin decreases with increased pH, although there is an increase in observed binding enthalpy. (3) ITC studies conducted in two buffers (sodium cacodylate and MOPS) yield the number of protonated drug amino groups (Δn) as 0.29 and 0.40 for neomycin and paromomycin interaction with 5′-dA₁₂-x-dT₁₂-x-dT₁₂-3′, respectively. (4) The specific heat capacity change (ΔC_p) determined by ITC studies is negative, with more negative values at lower salt concentrations. From 100 mM to 250 mM KCl, the ΔC_p ranges from −402 to −60 cal/(mol K) for neomycin. At pH 5.5, a more positive ΔC_p is observed, with a value of −98 cal/(mol K) at 100 mM KCl. ΔC_p is not significantly affected by ionic strength. (5) Salt dependence studies reveal that there are at least three amino groups of neomycin participating in the electrostatic interactions with the triplex. (6) FID studies using thiazole orange were used to derive the AC₅₀ (aminoglycoside concentration needed to displace 50% of the dye from the triplex) values. Neomycin shows a seven fold higher affinity than paromomycin and eleven fold higher affinity than ribostamycin at pH 6.8. (7) Modeling studies, consistent with UV and ITC results, show the importance of an additional positive charge in triplex recognition by neomycin. The modeling and thermodynamic studies indicate that neomycin binding to the DNA triplex depends upon significant contributions from charge as well as shape complementarity of the drug to the DNA triplex Watson–Hoogsteen groove.     

Efficient Stabilization of Phosphodiester (PO), Phosphorothioate (PS), and 2'-O-Methoxy (2'-OMe) DNA·RNA Hybrid Duplexes by Amino Sugars

Antisense strategies that target DNA·RNA hybrid structures offer potential for the development of new therapeutic drugs. The α-sarcin loop region of the 16S rRNA domain has been shown to be a high value target for such strategies. Herein, aminoglycoside interaction with three RNA·DNA α-sarcin targeted duplexes (rR·dY, rR·S-dY, and rR·2'OMe-rY) have been investigated to determine the overall effect of aminoglycoside interaction on the stability, affinity, and conformation of these hybrid duplexes. To this end, UV thermal denaturation, circular dichroism spectroscopy, fluorescence intercalator displacement, and ITC as well as DSC calorimetry experiments were carried out. The results suggest the following. (1) Of all the aminoglycosides studied, neomycin confers the highest thermal stability on all three hybrid duplexes studied. (2) There is no appreciable difference in aminoglycoside-induced thermal stability between the unmodified rR·dY and phophorothioate modified rR·S-dY duplexes. (3) The rR·2'OMe-rY duplexes thermal stability is slightly less than the other two hybrids. (4) In all three duplexes, aminoglycoside-induced thermal stability decreased as the number of amino groups decreased. (5) CD scans revealed similar spectra for the rR·dY and rR·S-dY duplexes as well as a more pronounced A-form signal for the rR·2'OMe-rY duplex. (6) FID assays paralleled the CD results, yielding similar affinity values between the rR·dY and rR·S-dY duplexes and higher affinities with the rR·2'OMe-rY duplex. (7) The overall affinity trend between aminoglycosides and the three duplexes was determined to be neomycin > paromomycin > neamine > ribostamycin. (8) ITC K(a) values revealed similar binding constants for the rR·dY and rR·S-dY duplexes with rR·dY having a K(1) of (1.03 ± 0.58) × 10(7) M(-1) and K(2) of (1.13 ± 0.07) × 10(5) M(-1) while rR·S-dY produced a K(1) of (1.17 ± 0.54) × 10(7) M(-1) and K(2) of (1.27 ± 0.69) × 10(5) M(-1). (8) The rR·2'OMe-rY produced a slightly higher binding constant values with a K(1) of (1.25 ± 0.24) × 10(7) M(-1) and K(2) of (3.62 ± 0.18) × 10(5) M(-1). (9) The ΔT(m)-derived K(Tm) of 3.81 × 10(7) M(-1) for rR·S-dY was in relative agreement with the corresponding K(1) of 1.17 × 10(7) M(-1) derived constant from the fitted ITC. These results illustrate that the increased DNA·RNA hybrid duplex stability in the presence of aminoglycosides can help extend the roles of aminoglycosides in designing modified ODNs for targeting RNA.     

Which aminoglycoside ring is most important for binding? A hydropathic analysis of gentamicin, paromomycin, and analogues

The NMR structures of gentamicin and paromomycin in complex with the A-site of Escherichia coli 16S ribosomal RNA were modified with molecular modeling to 12 analogues. The intermolecular interactions between these molecules and RNA were examined using the HINT (Hydropathic INTeractions) computational model to obtain interaction scores that have been shown previously to be related to free energy. The calculations correlated well with experimental binding data, and the interaction scores were used to analyze the specific structural features of each aminoglycoside that contribute to the overall binding with the 16S rRNA. Our calculations indicate that, while ring I binds to the main binding pocket of the rRNA A-site, ring IV of paromomycin-based aminoglycosides contributes significantly to the overall binding.     

A molecular dynamics simulation study of an aminoglycoside/A-site RNA complex: conformational and hydration patterns

Aminoglycoside antibiotics interfere with the translation mechanism by binding to the tRNA decoding site of the 16S ribosomal RNA. Crystallographic structures of aminoglycosides bound to A-site systems clarified many static aspects of RNA-ligand interactions. To gain some insight on the dynamic aspects of recognition phenomena, we conducted molecular dynamics simulations of the aminoglycoside paromomycin bound to a eubacterial ribosomal decoding A-site oligonucleotide. Results from 25 ns of simulation time revealed that: (i) the neamine part of the antibiotic represents the main anchor for binding, (ii) additional sugar rings provide limited and fragile contacts, (iii) long-resident water molecules present at the drug/RNA interface are involved in the recognition phenomena. The combination of MD simulations together with systematic structural information offers striking insights into the molecular recognition processes underlying RNA/aminoglycoside binding. Important methodological considerations related to the use of medium resolution starting structures and associated sampling problems are thoroughly discussed.     

Structural origins of aminoglycoside specificity for prokaryotic ribosomes

Aminoglycoside antibiotics, including paromomycin, neomycin and gentamicin, target a region of highly conserved nucleotides in the decoding region aminoacyl-tRNA site (A site) of 16 S rRNA on the 30 S subunit. Change of a single nucleotide, A1408 to G, reduces the affinity of many aminoglycosides for the ribosome; G1408 distinguishes between prokaryotic and eukaryotic ribosomes. The structures of a prokaryotic decoding region A-site oligonucleotide free in solution and bound to the aminoglycosides paromomycin and gentamicin C1a were determined previously. Here, the structure of a eukaryotic decoding region A-site oligonucleotide bound to paromomycin has been determined using NMR spectroscopy and compared to the prokaryotic A-site-paromomycin structure. A conformational change in three adenosine residues of an internal loop, critical for high-affinity antibiotic binding, was observed in the prokaryotic RNA-paromomycin complex in comparison to its free form. This conformational change is not observed in the eukaryotic RNA-paromomycin complex, disrupting the binding pocket for ring I of the antibiotic. The lack of the conformational change supports footprinting and titration calorimetry data that demonstrate approximately 25-50-fold weaker binding of paromomycin to the eukaryotic decoding-site oligonucleotide. Neomycin, which is much less active against Escherichia coli ribosomes with an A1408G mutation, binds non-specifically to the oligonucleotide. These results suggest that eukaryotic ribosomal RNA has a shallow binding pocket for aminoglycosides, which accommodates only certain antibiotics.     

Targeting the dimerization initiation site of HIV-1 RNA with aminoglycosides: from crystal to cell

The kissing-loop complex that initiates dimerization of genomic RNA is crucial for Human Immunodeficiency Virus Type 1 (HIV-1) replication. We showed that owing to its strong similitude with the bacterial ribosomal A site it can be targeted by aminoglycosides. Here, we present its crystal structure in complex with neamine, ribostamycin, neomycin and lividomycin. These structures explain the specificity for 4,5-disubstituted 2-deoxystreptamine (DOS) derivatives and for subtype A and subtype F kissing-loop complexes, and provide a strong basis for rational drug design. As a consequence of the different topologies of the kissing-loop complex and the A site, these aminoglycosides establish more contacts with HIV-1 RNA than with 16S RNA. Together with biochemical experiments, they showed that while rings I, II and III confer binding specificity, rings IV and V are important for affinity. Binding of neomycin, paromomycin and lividomycin strongly stabilized the kissing-loop complex by bridging the two HIV-1 RNA molecules. Furthermore, in situ footprinting showed that the dimerization initiation site (DIS) of HIV-1 genomic RNA could be targeted by these aminoglycosides in infected cells and virions, demonstrating its accessibility.     

Quantitative affinity electrophoresis of RNA–small molecule interactions by cross-linking the ligand to acrylamide

We show that the affinity electrophoresis analysis of RNA–small molecule interactions can be made quantifiable by cross-linking the ligand to the gel matrix. Using an RNA–aminoglycoside model system to verify our method, we attached an acryloyl chloride molecule to the aminoglycosides paromomycin and neomycin B to synthesize an acrylamide–aminoglycoside monomer. This molecule was then used as a component in gel polymerization for affinity electrophoresis, covalently attaching an aminoglycoside molecule to the gel matrix. To test RNA binding to the cross-linked aminoglycosides, we used the aminoglycoside binding RNA molecule derived from thymidylate synthase messenger RNA (mRNA) that contains a C–C mismatch. Binding is indicated by the difference in RNA mobility between gels with cross-linked ligand, with ligand embedded during polymerization, and with no ligand present. Critically, the predicted straight line relationship between the reciprocal of the relative migration of the RNA and the ligand concentration is obtained when using cross-linked aminoglycosides, whereas a straight line is not obtained using embedded aminoglycosides. Average apparent dissociation constants are determined from the slope of the line from these plots. This method allows an easy quantitative comparison between different nucleic acid molecules for a small molecule ligand.     

Understanding the origins of bacterial resistance to aminoglycosides through molecular dynamics mutational study of the ribosomal A-site

Paromomycin is an aminoglycosidic antibiotic that targets the RNA of the bacterial small ribosomal subunit. It binds in the A-site, which is one of the three tRNA binding sites, and affects translational fidelity by stabilizing two adenines (A1492 and A1493) in the flipped-out state. Experiments have shown that various mutations in the A-site result in bacterial resistance to aminoglycosides. In this study, we performed multiple molecular dynamics simulations of the mutated A-site RNA fragment in explicit solvent to analyze changes in the physicochemical features of the A-site that were introduced by substitutions of specific bases. The simulations were conducted for free RNA and in complex with paromomycin. We found that the specific mutations affect the shape and dynamics of the binding cleft as well as significantly alter its electrostatic properties. The most pronounced changes were observed in the U1406C∶U1495A mutant, where important hydrogen bonds between the RNA and paromomycin were disrupted. The present study aims to clarify the underlying physicochemical mechanisms of bacterial resistance to aminoglycosides due to target mutations.     

Defining the basis for the specificity of aminoglycoside-rRNA recognition: a comparative study of drug binding to the A sites of Escherichia coli and human rRNA

2-Deoxystreptamine (2-DOS) aminoglycoside antibiotics exert their antimicrobial activities by targeting the decoding region A site of the rRNA and inhibiting protein synthesis. A prokaryotic specificity of action is critical to therapeutic utility of 2-DOS aminoglycosides as antibiotics. Here, isothermal titration calorimetry (ITC) and fluorescence studies are presented that provide insight into the molecular basis for this prokaryotic specificity of action. Specifically, the rRNA binding properties of the 2-DOS aminoglycosides paromomycin and G418 (geneticin) are compared, using both human and Escherichia coli rRNA A site model oligonucleotides as drug targets. Paromomycin and G418 differ with respect to their specificities of action, with only paromomycin exhibiting a specificity for prokaryotic versus human ribosomes. G418 binds to both the human and E. coli rRNA A sites with a markedly lower affinity than paromomycin, with the affinities of both drugs for the human rRNA A site being lower than those they exhibit for the E. coli rRNA A site. Paromomycin induces the destacking of the base at position 1492 (by E. coli numbering) upon binding to the E. coli rRNA A site, but not the human rRNA A site. By contrast, the binding of G418 induces the destacking of base 1492 when either rRNA A site serves as the drug target. In the aggregate, these results suggest that binding-induced base destacking at the rRNA A site is a critical factor in determining the prokaryotic specificity of aminoglycoside action, with binding affinity for the A site being of secondary importance.     

RNA-ligand interactions: affinity and specificity of aminoglycoside dimers and acridine conjugates to the HIV-1 Rev response element

Semisynthetic aminoglycoside derivatives may provide a means to selectively target viral RNA sites, including the HIV-1 Rev response element (RRE). The design, synthesis, and evaluation of derivatives based upon neomycin B, kanamycin A, and tobramycin conjugates of 9-aminoacridine are presented. To evaluate the importance of the acridine moiety, a series of dimeric aminoglycosides as well as unmodified "monomeric" aminoglycosides have also been evaluated for their nucleic acid affinity and specificity. Fluorescence-based binding assays that use ethidium bromide or Rev peptide displacement are used to quantify the affinities of these compounds to various nucleic acids, including the RRE, tRNA, and duplex DNA. All the modified aminoglycosides exhibit a high affinity for the Rev binding site on the RRE (K(d) 相似文献   

Substrate promiscuity of an aminoglycoside antibiotic resistance enzyme via target mimicry

The misuse of antibiotics has selected for bacteria that have evolved mechanisms for evading the effects of these drugs. For aminoglycosides, a group of clinically important bactericidal antibiotics that target the A-site of the 16S ribosomal RNA, the most common mode of resistance is enzyme-catalyzed chemical modification of the drug. While aminoglycosides are structurally diverse, a single enzyme can confer resistance to many of these antibiotics. For example, the aminoglycoside kinase APH(3')-IIIa, produced by pathogenic Gram-positive bacteria such as enterococci and staphylococci, is capable of detoxifying at least 10 distinct aminoglycosides. Here we describe the crystal structures of APH(3')-IIIa in complex with ADP and kanamycin A or neomycin B. These structures reveal that the basis for this enzyme's substrate promiscuity is the presence of two alternative subsites in the antibiotic binding pocket. Furthermore, comparison between the A-site of the bacterial ribosome and APH(3')-IIIa shows that mimicry is the second major factor in dictating the substrate spectrum of APH(3')-IIIa. These results suggest a potential strategy for drug design aimed at circumventing antibiotic resistance.     

NMR resonance assignments of an engineered neomycin-sensing riboswitch RNA bound to ribostamycin and tobramycin

The neomycin-sensing riboswitch is an engineered riboswitch developed to regulate gene expression in vivo in the lower eukaryote Saccharomyces cerevisiae upon binding to neomycin B. With a size of only 27nt it is the smallest functional riboswitch element identified so far. It binds not only neomycin B but also related aminoglycosides of the 2′-deoxystreptamine class with high affinity. The regulatory activity, however, strongly depends on the identity of the aminoglycoside. As a prerequisite for the structure determination of riboswitch-ligand complexes we report here the ¹H, ¹⁵N, ¹³C and partial ³¹P chemical shift assignments for the minimal functional 27nt neomycin sensing riboswitch RNA in complex with the 4,5-linked neomycin analog ribostamycin and the 4,6-linked aminoglycoside tobramycin.     

Sequence-specific targeting of RNA with an oligonucleotide-neomycin conjugate

The synthesis of neomycin covalently attached at the C5-position of 2'-deoxyuridine is reported. The synthesis outlined allows for incorporation of an aminoglycoside (neomycin) at any given site in an oligonucleotide (ODN) where a thymidine (or uridine) is present. Incorporation of this modified base into an oligonucleotide, which is complementary to a seven-bases-long alpha-sarcin loop RNA sequence, leads to enhanced duplex hybridization. The increase in Tm for this duplex (DeltaTm = 6 degrees C) suggests a favorable interaction of neomycin within the duplex groove. CD spectroscopy shows that the modified duplex adopts an A-type confirmation. ITC measurements indicate the additive effects of ODN and neomycin binding to the RNA target (Ka = 4.5 x 107 M-1). The enhanced stability of the hybrid duplex from this neomycin-ODN conjugate originates primarily from the enthalpic contribution of neomycin {DeltaDeltaHobs = -7.21 kcal/mol (DeltaHneomycin conjugated - DeltaH nonconjugated)} binding to the hybrid duplex. The short linker length allows for selective stabilization of the hybrid duplex over the hybrid triplex. The results described here open up new avenues in the design and synthesis of nucleo-aminoglycoside-conjugates (N-Ag-C) where the inclusion of any number of aminoglycoside (neomycin) molecules per oligonucleotide can be accomplished.     

Structural rearrangements of HIV-1 Tat-responsive RNA upon binding of neomycin B

Binding of human immunodeficiency virus type 1 (HIV-1) transactivator (Tat) protein to Tat-responsive RNA (TAR) is essential for viral replication and is considered a promising starting point for the design of anti-HIV drugs. NMR spectroscopy indicated that the aminoglycosides neomycin B and ribostamycin bind to TAR and that neomycin is able to inhibit Tat binding to TAR. The solution structure of the neomycin-bound TAR has been determined by NMR spectroscopy. Chemical shift mapping and intermolecular nuclear Overhauser effects define the binding region of the aminoglycosides on TAR and give strong evidence for minor groove binding. Based on 15 nuclear Overhauser effect-derived intermolecular distance restraints, a model structure of the TAR-neomycin complex was calculated. Neomycin is bound in a binding pocket formed by the minor groove of the lower stem and the uridine-rich bulge of TAR, which adopts a conformation different from those known. The neamine core of the aminoglycoside (rings I and II) is covered with the bulge, explaining the inhibition of Tat by an allosteric mechanism. Neomycin reduces the volume of the major groove in which Tat is bound and thus impedes essential protein-RNA contacts.     

Structure of a eukaryotic decoding region A-site RNA

The aminoglycoside antibiotics target a region of highly conserved nucleotides in the aminoacyl-tRNA site (A site) of 16 S RNA on the 30 S subunit. The structures of a prokaryotic decoding region A-site oligonucleotide free in solution and bound to the aminoglycosides paromomycin and gentamicin C1A have been determined. Here, the structure of a eukaryotic decoding region A-site oligonucleotide has been determined using homonuclear and heteronuclear NMR spectroscopy, and compared to the unbound prokaryotic rRNA structure. The two structures are similar, with a U1406-U1495 base-pair, a C1407-G1494 Watson-Crick base-pair, and a G1408-A1493 base-pair instead of the A1408-A1493 base-pair of the prokaryotic structure. The two structures differ in the orientation of the 1408 position with respect to A1493; G1408 is rotated toward the major groove, which is the binding pocket for aminoglycosides. The structures also differ in the stacking geometry of G1494 on A1493, which could have slight long-range conformational effects.     

Characteristics of the binding of aminoglycoside antibiotics to teichoic acids. A potential model system for interaction of aminoglycosides with polyanions

The binding of the aminoglycoside antibiotic dihydrostreptomycin to defined cell-wall teichoic acids and to lipoteichoic acid isolated from various gram-positive eubacteria was followed by equilibrium dialysis. Dihydrostreptomycin was used at a wide range of concentration under different conditions of ionic strength, concentration of teichoic acid, presence of cationic molecules like Mg2+, spermidine, other aminoglycoside antibiotics (gentamicin, neomycin, paromomycin). Interaction of dihydrostreptomycin with teichoic acid was found to be a cooperative binding process. The binding characteristics seem to be dependent on structural features of teichoic acid and are influenced by cationic molecules. Mg2+, spermidine and other aminoglycosides antibiotics inhibit the binding of dihydrostreptomycin to teichoic acid competitively. The binding of aminoglycosides to teichoic acids is considered as a model system for the interaction of aminoglycoside antibiotics with cellular polyanions. Conclusions of physiological significance are drawn.