Deciphering the origins of observed heat capacity changes for aminoglycoside binding to prokaryotic and eukaryotic ribosomal RNA a-sites: a calorimetric, computational, and osmotic stress study

Isothermal titration calorimetry (ITC), computational, and osmotic stress techniques have been used to characterize the changes in heat capacity, solvent-accessible surface, and hydration that accompany the binding of the aminoglycoside paromomycin to both prokaryotic and eukaryotic rRNA A-site model oligonucleotides. Regarded as a whole, the results of these studies suggest that the intrinsic heat capacity change (DeltaC(p)) for the binding of paromomycin to each rRNA A-site is near zero, with the negative DeltaC(p) observed for the binding of the drug to the prokaryotic rRNA A-site being dictated by the coupled destacking of the adenine residues at positions 1492 and 1493. In this connection, DeltaC(p) provides a useful calorimetric signature for assessing the relative impacts of novel and existing A-site targeting ligands on rRNA conformation, which, in turn, should provide a useful analytical tool for facilitating the drug design process, since aminoglycoside-induced destacking of A1492 and A1493 is thought to be a determining factor in the mistranslational and antimicrobial activities of the drugs.     

Aminoglycoside-induced reduction in nucleotide mobility at the ribosomal RNA A-site as a potentially key determinant of antibacterial activity

Steady-state and time-resolved fluorescence techniques have been used to characterize the energetics and dynamics associated with the interaction of an E. coli 16 S rRNA A-site model oligonucleotide and four aminoglycoside antibiotics that exhibit a broad range of antibacterial activity. The results of these characterizations suggest that aminoglycoside-induced reduction in the mobility of an adenine residue at position 1492 of the rRNA A-site is a more important determinant of antibacterial activity than drug affinity for the A-site. This observation is consistent with a recently proposed model for the mechanism of protein synthesis inhibition by aminoglycosides that invokes a drug-induced alteration in the conformational equilibrium of the rRNA A-site (centered around the conserved adenine residues at positions 1492 and 1493), which, in turn, promotes an enhanced interaction between the rRNA and the minihelix formed by the tRNA anticodon and the mRNA codon, even when the anticodon is noncognate. Regarded as a whole, the results reported here indicate that the rational design of antibiotics that target the 16 S rRNA A-site requires consideration of not only the structure and energetics of the drug-RNA complex but also the dynamics associated with that complex.     

Molecular dynamics study of the ribosomal A-site

Many aminoglycosidic antibiotics target the A-site of 16S RNA in the small ribosomal subunit and affect the fidelity of protein translation in bacteria. Upon binding, aminoglycosides displace two adenines (A1492 and A1493 for E. coli numbering) that are involved in tRNA anticodon loop recognition. The major difference in the aminoglycosidic binding site between the prokaryota and eukaryota is an adenine into guanine substitution in the position 1408. This mutation likely affects the dynamics of near A1492 and A1493 and hinders the binding of aminoglycosides to eukaryotic ribosomes. With multiple 20 ns long all-atom molecular dynamics simulations, we study the flexibility of a 22 nucleotide RNA fragment which mimics the aminoglycosidic binding site. Simulations are carried out for both native and A1408G mutated RNA as well as for their complexes with aminoglycosidic representative paromomycin. We observe intra- and extrahelical configurations of A1492 and A1493, which differ between the prokaryotic and the mutated structure. We obtain configurations of the A-site that are also observed in the NMR and crystal structures. Our studies show the differences in the internal mobility of the A-site, as well as that in ion and water density distributions inside of the binding cleft, between the prokaryotic and mutated RNA. We also compare the performance of two force field parameters for RNA, Amber and Charmm.     

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.     

Crystal structure of a complex between the aminoglycoside tobramycin and an oligonucleotide containing the ribosomal decoding a site

Aminoglycoside antibiotics target the decoding aminoacyl site (A site) on the 16S ribosomal RNA and induce miscoding during translation. Here, we present the crystal structure, at 2.54 A resolution, of an RNA oligonucleotide containing the A site sequence complexed to the 4,6-disubstituted 2-deoxystreptamine aminoglycoside tobramycin. The three aminosugar rings making up tobramycin interact with the deep-groove atoms directly or via water molecules and stabilize a fully bulged-out conformation of adenines A(1492) and A(1493). The comparison between this structure and the one previously solved in the presence of paromomycin confirms the importance of the functional groups on the common neamine part of these two antibiotics for binding to RNA. Furthermore, the analysis of the present structure provides a molecular explanation to some of the resistance mechanisms that have spread among bacteria and rendered aminoglycoside antibiotics inefficient.     

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.     

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.     

PdCl2-induzierte umlagerung substituierter cis,trans-cyclodeca-1,5-diene

Like the unsubstituted ten-membered ring cis,trans-cyclodeca-1,5-dienes, which are substituted in the 7-, 8- or 9-position, can also be inserted into PdCl₂-complexes of the corresponding substituted cis-1,2-divinylcyclohexanes.Mono- or dimethylsubstitution in the 1-, 2-, 6- or 3,4-position makes this induced Cope-rearrangement impossible or more difficult.     

Synthetic studies with iron carbonyl complexes. Versatile synthesis of olefinic side-chains to a tricarbonyl(cyclohexadiene)iron(0) ring

Tricarbonyl(cyclohexadiene)iron(0) complexes bearing olefinic side-chains at the 2- or the 5-position have been prepared by a versatile route. Terminal olefins are isomerised to internal isomers. The presence of a methoxy substituent at the 2-position prevents isomerisation of the coordinated diene into conjugation with the olefin, so locking the side-chain at the 5-exo-position.     

Synthesis and Evaluation of Novel 5-O-(4-C-Aminoalkyl-β-D-ribofuranosyl) Apramycin Derivatives for the Inhibition of Gram-Negative Pathogens Carrying the Aminoglycoside Phosphotransferase(3′)-Ia Resistance Determinant

We report the synthesis and evaluation of two new apramycin 5-O-β-d -ribofuranosides, or apralogs, carrying aminoalkyl branches at the ribofuranose 4-position. This novel modification conveys excellent activity for the inhibition of protein synthesis by wild-type bacterial ribosomes and correspondingly high antibacterial activity against several Gram-negative pathogens. Notably, these new modifications overcome the reduction of antibacterial activity in other 2-deoxystreptamine-type aminoglycosides carrying a 5-O-ribofuranosyl moiety when challenged by the presence of an aminoglycoside phosphotransferase enzyme capable of acting on the ribose 5-position.     

Interactions between local anesthetics and Na+ channel inactivation gate peptides in phosphatidylserine suspensions as studied by 1H-NMR spectroscopy

Interactions between local anesthetics and a sodium channel inactivation gate peptide (Ac-GGQDIFMTEEQK-NH2, MP-1A), which was dissected from the cytoplasmic linker between domains III and IV of the sodium channel alpha-subunit (G1484-K1495 in rat brain type IIA), have been studied by 1H-NMR spectroscopy. Changes in 1H-NMR chemical shifts of the aromatic proton resonances of dibucaine (pH 7.0) and lidocaine (pH 6.0 and 9.0) in phosphatidylserine (PS) suspensions were observed. The effects of substitution of glutamine (F1489Q; MP-2A) or D-phenylalanine (MP-1A') for L-phenylalanine (F1489) in MP-1A and the effects of substitution of neutral amino acid residues for the corresponding acidic amino acid residues (D1487N, MP-1NA; E1492Q, MP-IQEA; E1493Q, MP-IEQA) in MP-1A, on the aromatic 1H-NMR chemical shift changes of dibucaine and lidocaine were also investigated. From these results it was concluded that: the aromatic ring of phenylalanine of MP-1A and the aromatic ring of the cationic form of dibucaine or lidocaine are interacting by pi-pi stacking; the tertiary amine nitrogen of dibucaine is interacting electrostatically with D1487, whereas that of lidocaine is interacting with E1492.     

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.     

分子动力学模拟研究A1408G突变对核糖体16S rRNA片段的影响

利用分子动力学模拟方法, 研究了原核生物核糖体小亚基中的16S rRNA片段与氨基糖苷类抗生素巴龙霉素复合物结构的柔性. 结果表明, 16S rRNA片段中的1408位点的腺嘌呤(A)突变为鸟嘌呤(G), 改变了与tRNA中反密码子环识别相关的2个腺嘌呤A1492和A1493的空间构象, 阻碍了氨基糖苷类抗生素与核糖体的结合, 从而影响原核生物蛋白转录过程. 模拟结果与实验测定的晶体结构相吻合, 可为基于核糖体16S rRNA的药物分子设计提供较可靠的结构信息.     

Synthesis of tetradialdose phosphonate nucleosides as mimics of l-nucleotides

Structurally modified nucleoside analogues are a major source of therapeutic agents and building blocks for incorporation into synthetic oligonucleotides able to interfere with information transfer or other biological functions. This work describes the synthesis of non-natural l-nucleoside phosphonate mimics containing two anomeric centers. Such compounds feature either a di- or monohydroxy tetradialdose sugar as the glycone unit, while adenine is present as nucleobase. By judicious use of protecting groups at the 2- and 3-position of commercial 1-O-acetyl-2,3,5-tri-O-benzoyl-β-d-ribofuranose, both the phosphonate and nucleobase moieties were stereoselectively introduced to provide a dihydroxylated compound with cis-configured substituents as the sole reaction product. Subsequent selective deprotection and deoxygenation at the 3′-position occurred smoothly to afford the corresponding 4′-monohydroxy tetradialdose containing analogue.     

The synthesis and electronic absorption spectra of 3-phenyl-3(4-pyrrolidino-2-substituted phenyl)-3H-naphtho[2,1-b]pyrans: further exploration of the ortho substituent effect

Introduction of a substituent into a sterically demanding 2-position of a 3-(4-pyrrolidinophenyl) ring of a 3,3-diaryl-3H-naphtho[2,1-b]pyran results in the generation of an additional short wavelength absorption band leading to organic photochromes that appear as dull shades of orange and red.     

Synthesis of Computationally Designed 2,5(6)-Benzimidazole Derivatives via Pd-Catalyzed Reactions for Potential E. coli DNA Gyrase B Inhibition

A pharmacophore model for inhibitors of Escherichia coli’s DNA Gyrase B was developed, using computer-aided drug design. Subsequently, docking studies showed that 2,5(6)-substituted benzimidazole derivatives are promising molecules, as they possess key hydrogen bond donor/acceptor groups for an efficient interaction with this bacterial target. Furthermore, 5(6)-bromo-2-(2-nitrophenyl)-1H-benzimidazole, selected as a core molecule, was prepared on a multi-gram scale through condensation of 4-bromo-1,2-diaminobenzene with 2-nitrobenzaldehyde using a sustainable approach. The challenging functionalization of the 5(6)-position was carried out via palladium-catalyzed Suzuki–Miyaura and Buchwald-Hartwig amination cross-coupling reactions between N-protected-5-bromo-2-nitrophenyl-benzimidazole and aryl boronic acids or sulfonylanilines, with yields up to 81%. The final designed molecules (2-(aminophen-2-yl)-5(6)-substituted-1H-benzimidazoles), which encompass the appropriate functional groups in the 5(6)-position according to the pharmacophore model, were obtained in yields up to 91% after acid-mediated N-boc deprotection followed by Pd-catalyzed hydrogenation. These groups are predicted to favor interactions with DNA gyrase B residues Asn46, Asp73, and Asp173, aiming to promote an inhibitory effect.     

Detecting ligand binding to a small RNA target via saturation transfer difference NMR experiments in D(2)O and H(2)O

A small RNA motif is used as a target for ligand-based NMR-screening by saturation transfer difference (STD) NMR experiments. The prerequisites for using a small RNA target in STD experiments, such as saturation time, frequency, and pulses, are discussed. We also show that it is of advantage to use D2O as solvent instead of H2O due to the reduced R1 relaxation rate in D2O. The 27-nucleotide model of the ribosomal A-site was known to bind the aminoglycoside paromomycin with high affinity. This binding interaction could be detected easily, proving the effectiveness of STD NMR experiments as a screening tool for RNA-ligand interactions.     

Interactions of designer antibiotics and the bacterial ribosomal aminoacyl-tRNA site

The X-ray crystal structures for the complexes of three designer antibiotics, compounds 1, 2, and 3, bound to two models for the ribosomal aminoacyl-tRNA site (A site) at 2.5-3.0 Angstroms resolution and that of neamine at 2.8 Angstroms resolution are described. Furthermore, the complex of antibiotic 1 bound to the A site in the entire 30S ribosomal subunit of Thermus thermophilus is reported at 3.8 Angstroms resolution. Molecular dynamics simulations revealed that the designer compounds provide additional stability to bases A1492 and A1493 in their extrahelical forms. Snapshots from the simulations were used for free energy calculations, which revealed that van der Waals and hydrophobic effects were the driving forces behind the binding of designer antibiotic 3 when compared to the parental neamine.