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Mycobacterium tuberculosis ( Mtb ) contains potential G-quadruplex (PGQ) motifs in the genes espK and cyp51 , which are crucial for the bacteria's virulence within host cells. Aminoglycoside molecules are commonly used as antibiotics for ribosomal targets. This study provides insight into the interactions between these aminoglycosides and Mtb-PGQ sequences ( espK and cyp51 ), shedding light on the structural and thermodynamic dynamics of their binding. This study demonstrates the stability, affinity, and conformation of Mtb-PGQ in the presence of neomycin and streptomycin. Ultraviolet-visible spectroscopy (UV-vis), circular dichroism spectroscopy (CD), CD thermal melting, isothermal titration calorimetry (ITC), and fluorescence intercalator displacement (FID) assays were used to comprehensively examine these interactions. Our results reveal that neomycin with Mtb-PGQ exhibits hypochromism accompanied by a 4-5 nm red shift in the UV-visible absorption titration, whereas streptomycin exhibits a hypochromic shift without changes in the maximum wavelength. Notably, neomycin shows a nonlinear binding isotherm, suggesting the involvement of more than one binding process in the formation of neomycin. Mtb-PGQ complexes. Scatchard plot analysis indicates higher binding affinity values for neomycin compared with weaker affinity of streptomycin. CD studies reveal that neomycin decreases the ellipticity of Mtb-PGQ with a red shift while retaining the parallel topology, ultimately enhancing the thermal stability of both espK and cyp51 . In contrast, streptomycin destabilizes the cells. ITC analysis reveals that neomycin exhibits the strongest binding affinity for cyp51 , with the relative order being NEO- cyp51 > NEO- espk > STR- cyp51 > STR- espk. Moreover, thermodynamic analysis reveals that neomycin possesses a unique dual mode of binding through grooves as well as stacking. FID studies further confirm a lower DC 50 value for neomycin than for streptomycin, suggesting that neomycin is a strong displacer of thiazole orange. Thus, the results show that neomycin with amino groups selectively recognizes the grooves of cyp51 over espK .

DNA adopts a number of conformations that can affect its binding to other macromolecules. The conformations (A, B, Z) can be sequence- and/or solution-dependent. While AT-rich DNA sequences generally adopt a Canonical B-form structure, GC-rich sequences are more promiscuous. Recognition of GC-rich nucleic acids by small molecules has been much more challenging than the recognition of AT-rich duplexes. Spectrophotometric and calorimetric techniques were used to characterize the binding of neomycin-class aminoglycosides to a GC-rich DNA duplex, G 4 C 4 , in various ionic and pH conditions. Our results reveal that binding enhances the thermal stability of G 4 C 4 , with thermal enhancement decreasing with increasing pH and/or Na + concentration. Although G 4 C 4 bound to aminoglycosides demonstrated a mixed A- and B-form conformation, circular dichroism studies indicate that binding induces a conformational shift toward A-form DNA. Isothermal titration calorimetry studies reveal that aminoglycoside binding to G 4 C 4 is linked to the uptake of protons at pH = 7.0 and that this uptake is pH-dependent. Increased pH and/or Na + concentration results in a decrease in G 4 C 4 affinity for the aminoglycosides. The binding affinities of the aminoglycosides follow the expected hierarchy: neomycin > paromomycin > ribostamycin. The salt dependence of DNA binding affinities of aminoglycosides is consistent with at least two drug NH 3 + groups participating in electrostatic interactions with G 4 C 4 . These studies further embellish our understanding of the many factors facilitating recognition of GC-rich DNA structures as guided by their optimum charge and shape complementarity for small-molecule amino sugars.

Cationic lipids (CLs) have gained significant attention among nonviral gene delivery vectors due to their ease of synthesis and functionalization with multivalent moieties. In particular, there is an increasing request for multifunctional CLs having gene delivery capacity and antibacterial activity. Herein, we describe the design and synthesis of a novel class of aminoglycoside (AG)-based multifunctional vectors with high transfection efficiency and noticeable antibacterial properties. Specifically, cationic amphiphiles were built on a triazine scaffold, allowing for an easy derivatization with up to three potentially different substituents, such as neomycin (Neo) that serves as the polar head and one or two lipophilic tails, namely stearyl (ST) and oleyl (OL) alkyl chains and cholesteryl (Chol) tail. With the aim to shed more light on the effect of different types and numbers of lipophilic moieties on the ability of CLs to condense and transfect cells, the performance of Neo-triazine-based derivatives as gene delivery vectors was evaluated and compared. The ability of Neo-triazine-based derivatives to act as antimicrobial agents was evaluated as well. Neo-triazine-based CLs invariably exhibited excellent DNA condensation ability, even at a low charge ratio (CR, +/-). Besides, each derivative showed very good transfection performance at its optimal CR on two different cell lines, along with negligible cytotoxicity. CLs bearing symmetric two-tailed OL proved to be the most effective in transfection. Interestingly, Neo-triazine-based derivatives, used as either free lipids or lipoplexes, exhibited strong antibacterial activity against Gram-negative bacteria, especially in the case of CLs bearing one or two aliphatic chains. Altogether, these results highlight the potential of Neo-triazine-based derivatives as effective multifunctional nonviral gene delivery vectors.

Neomycin, a multicomponent aminoglycoside antibiotic, is mainly utilized in livestock husbandry and feed additives in animals. The antimicrobial potency of the main product neomycin B is higher than that of its stereoisomer neomycin C. However, the content of neomycin C as an impurity in the high-producing strain is relatively high, and its isolation or removal from neomycin B is quite difficult, which influences the widespread application of neomycin. In this work, the essential genes responsible for neomycin biosynthesis were evaluated and overexpressed to reduce the content of neomycin C. Among them, neoG and neoH are two novel regulatory genes for neomycin biosynthesis, aphA is a resistance gene, neoN encoding a radical SAM-dependent epimerase is responsible for the conversion of neomycin C to B using SAM as the cofactor, and metK is a SAM synthetase coding gene. We demonstrated that the reconstitution and overexpression of a mini-gene-cluster (P kasO * - neoN - metK -P kasO * - neoGH - aphA ) could effectively reduce the accumulation of neomycin C from 19.1 to 12.7% and simultaneously increase neomycin B by ∼13.1% in the engineered strain Sf/pKCZ04 compared with the wild-type strain (Sf). Real-time quantitative polymerase chain reaction analysis revealed the remarkable up-regulation of the neoE , neoH , neoN , and metK genes situated in the mini-gene-cluster. The findings will pave a new path for component optimization and the large-scale industrial production of significant commercial antibiotics.

There is significant interest in developing analytical methods to characterize molecular recognition events between proteins and phosphoinositides, which are a medically important class of carbohydrate-functionalized lipids. Within this scope, one area of high priority involves quantitatively evaluating drug candidates that pharmacologically inhibit protein-phosphoinositide interactions. As full-length proteins are often difficult to produce, establishing methods to study these interactions with shorter, bioactive peptides would be advantageous. Herein, we report an atomic force microscopy (AFM)-based force spectroscopic approach to detect the specific interaction between an amphipathic, α-helical (AH) peptide derived from the hepatitis C virus NS5A protein and its biological target, the phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P 2 ] phosphoinositide receptor. After optimization of the peptide tethering strategy and measurement parameters, the binding specificity of AH peptide for PI(4,5)P 2 receptors was comparatively evaluated across a panel of phosphoinositides and the influence of ionic strength on AH-PI(4,5)P 2 binding strength was tested. Importantly, these capabilities were translated into the development of a novel experimental methodology to determine the inhibitory activity of a small-molecule drug candidate acting against the AH-PI(4,5)P 2 interaction, and extracted kinetic parameters agree well with literature values obtained by conventional biochemical methods. Taken together, our findings provide a nanomechanical basis for explaining the high binding specificity of the NS5A AH to PI(4,5)P 2 receptors, in turn establishing an analytical framework to study phosphoinositide-binding viral peptides and proteins as well as a broadly applicable approach to evaluate candidate inhibitors of protein-phosphoinositide interactions.

By the forced unfolding of polyglutamine and polyalanine homopeptides in competing α-helix and β-hairpin secondary structures, we disentangle equilibrium free energetics from nonequilibrium dissipative effects. We find that α-helices are characterized by larger friction or dissipation upon unfolding, regardless of whether they are free energetically preferred over β-hairpins or not. Our analysis, based on MD simulations for atomistic peptide models with explicit water, suggests that this difference is related to the internal friction and mostly caused by the different number of intrapeptide hydrogen bonds in the α-helix and β-hairpin states.

Both multicomponent reactions and diversity oriented synthesis are indispensable tools for the modern medicinal chemist. However, their employment for the synthesis of multivalent glycomimetics has not been exploited so far although the importance that such compounds play in exploring multivalency on glycoside inhibition. Herein, we report the combinatorial synthesis of diversity oriented hetero di- and trivalent glycomimetics through a multicomponent domino process. The process is high yielding and very general, working efficiently with easily accessible sugar starting materials such as glycosylamines, glycosylazides, and glycosylisothiocyanates, having the reactive functional groups tethered either directly to the anomeric carbon, through a suitable linker, or to the primary 6 position of hexoses (or 5 position of pentoses), leading, in the latter case, to glycomimetics with artificial enzymatically stable backbone. The process has been also exploited for the multicomponent synthesis of aminoglycoside (neomycin) conjugates.

The aminoglycoside N3 acetyltransferase-IIIb (AAC) is responsible for conferring bacterial resistance to a variety of aminoglycoside antibiotics. Nuclear magnetic resonance spectroscopy and dynamic light scattering analyses revealed a surprising result; the dynamics of the ternary complex between AAC and its two ligands, an antibiotic and coenzyme A, are dependent upon the order in which the ligands are bound. Additionally, two structurally similar aminoglycosides, neomycin and paromomycin, induce strikingly different dynamic properties when they are in their ternary complexes. To the best of our knowledge, this is the first example of a system in which two identically productive pathways of forming a simple ternary complex yield significant differences in dynamic properties. These observations emphasize the importance of the sequence of events in achieving optimal protein-ligand interactions and demonstrate that even a minor difference in molecular structure can have a profound effect on biochemical processes.

A series of neomycin dimers have been synthesized using "click chemistry" with varying functionality and length in the linker region to target the human immunodeficiency virus type 1 (HIV-1) TAR RNA region of the HIV virus. The TAR (Trans-Activation Responsive) RNA region, a 59 bp stem-loop structure located at the 5'-end of all nascent viral transcripts, interacts with its target, a key regulatory protein, Tat, and necessitates the replication of HIV-1. Neomycin, an aminosugar, has been shown to exhibit multiple binding sites on TAR RNA. This observation prompted us to design and synthesize a library of triazole-linked neomycin dimers using click chemistry. The binding between neomycin dimers and TAR RNA was characterized using spectroscopic techniques, including FID (fluorescent intercalator displacement), a FRET (fluorescence resonance energy transfer) competitive assay, circular dichroism (CD), and UV thermal denaturation. UV thermal denaturation studies demonstrate that binding of neomycin dimers increases the melting temperature (T(m)) of the HIV TAR RNA up to 10 °C. Ethidium bromide displacement (FID) and a FRET competition assay revealed nanomolar binding affinity between neomycin dimers and HIV TAR RNA, while in case of neomycin, only weak binding was detected. More importantly, most of the dimers exhibited lower IC(50) values toward HIV TAR RNA, when compared to the fluorescent Tat peptide, and show increased selectivity over mutant TAR RNA. Cytopathic effects investigated using MT-2 cells indicate a number of the dimers with high affinity toward TAR show promising anti-HIV activity.

Biosynthetically inspired manipulation of the antibiotic paromomycin led, in six high-yielding steps, to a ring A harboring an α,β-unsaturated 6'-aldehyde and an allylic 3'-methylcarbonate group. Tsuji deoxygenation in the presence of 5 mol % Pd(2)(dba)(3) and Bu(3)P granted access to a novel series of 3',4'-dideoxy-4',5'-dehydro ring A hybrids. The neomycin-sisomicin hybrid exhibited superior in vitro antibacterial activity to the parent compound neomycin.

Recognition of nucleic acids is important for our understanding of nucleic acid structure as well as for our understanding of nucleic acid-protein interactions. In addition to the direct readout mechanisms of nucleic acids such as H-bonding, shape recognition of nucleic acids is being increasingly recognized as playing an equally important role in DNA recognition. Competition dialysis, UV, flourescent intercalator displacement (FID), computational docking, and calorimetry studies were conducted to study the interaction of neomycin with a variety of nucleic acid conformations (shapes). At pH 5.5, the results suggest the following. (1) Neomycin binds three RNA structures [16S A site rRNA, poly(rA)·poly(rA), and poly(rA)·poly(rU)] with high affinities (K(a) ~ 10(7) M(-1)). (2) The binding of neomycin to A-form GC-rich oligomer d(A(2)G(15)C(15)T(2))(2) has an affinity comparable to those of RNA structures. (3) The binding of neomycin to DNA·RNA hybrids shows a 3-fold variance that can be attributed to their structural differences [for poly(dA)·poly(rU), K(a) = 9.4 × 10(6) M(-1), and for poly(rA)·poly(dT), K(a) = 3.1 × 10(6) M(-1)]. (4) The interaction of neomycin with DNA triplex poly(dA)·2poly(dT) yields a binding affinity (K(a)) of 2.4 × 10(5) M(-1). (5) Poly(dA-dT)(2) shows the lowest association constant for all nucleic acids studied (K(a) < 10(5)). (6) Neomycin binds to G-quadruplexes with K(a) values of ~10(4)-10(5) M(-1). (7) Computational studies show that the decrease in major groove width in the B to A transition correlates with increasing neomycin affinity. Neomycin's affinity for various nucleic acid structures can be ranked as follows: RNAs and GC-rich d(A(2)G(15)C(15)T(2))(2) structures > poly(dA)·poly(rU) > poly(rA)·poly(dT) > T·A-T triplex, G-quadruplex, B-form AT-rich, or GC-rich DNA sequences. The results illustrate the first example of a small molecule-based "shape readout" of different nucleic acid conformations.

A dimeric neomycin-neomycin conjugate 3 with a flexible linker, 2,2'-(ethylenedioxy)bis(ethylamine), has been synthesized and characterized. Dimer 3 can selectively bind to AT-rich DNA duplexes with high affinity. Biophysical studies have been performed between 3 and different nucleic acids with varying base composition and conformation by using ITC (isothermal calorimetry), CD (circular dichroism), FID (fluorescent intercalator displacement), and UV (ultraviolet) thermal denaturation experiments. A few conclusions can be drawn from this study: (1) FID assay with 3 and polynucleotides demonstrates the preference of 3 toward AT-rich sequences over GC-rich sequences. (2) FID assay and UV thermal denaturation experiments show that 3 has a higher affinity for the poly(dA)·poly(dT) DNA duplex than for the poly(dA)·2poly(dT) DNA triplex. Contrary to neomycin, 3 destabilizes poly(dA)·2poly(dT) triplex but stabilizes poly(dA)·poly(dT) duplex, suggesting the major groove as the binding site. (3) UV thermal denaturation studies and ITC experiments show that 3 stabilizes continuous AT-tract DNA better than DNA duplexes with alternating AT bases. (4) CD and FID titration studies show a DNA binding site size of 10-12 base pairs/drug, depending upon the structure/sequence of the duplex for AT-rich DNA duplexes. (5) FID and ITC titration between 3 and an intramolecular DNA duplex [d(5'-A(12)-x-T(12)-3'), x = hexaethylene glycol linker] results in a binding stoichiometry of 1:1 with a binding constant ∼10(8) M(-1) at 100 mM KCl. (6) FID assay using 3 and 512 hairpin DNA sequences that vary in their AT base content and placement also show a higher binding selectivity of 3 toward continuous AT-rich than toward DNA duplexes with alternate AT base pairs. (7) Salt-dependent studies indicate the formation of three ion pairs during binding of the DNA duplex d[5'-A(12)-x-T(12)-3'] and 3. (8) ITC-derived binding constants between 3 and DNA duplexes have the following order: AT continuous, d[5'-G(3)A(5)T(5)C(3)-3'] > AT alternate, d[5'-G(3)(AT)(5)C(3)-3'] > GC-rich d[5'-A(3)G(5)C(5)T(3)-3']. (9) 3 binds to the AT-tract-containing DNA duplex (B* DNA, d[5'-G(3)A(5)T(5)C(3)-3']) with 1 order of magnitude higher affinity than to a DNA duplex with alternating AT base pairs (B DNA, d[5'-G(3)(AT)(5)C(3)-3']) and with almost 3 orders of magnitude higher affinity than a GC-rich DNA (A-form, d[5'-A(3)G(5)C(5)T(3)-3'])., (© 2011 American Chemical Society)

Synthesis of a novel perylene-neomycin conjugate (3) and the properties of its binding to human telomeric G-quadruplex DNA, 5'-d[AG3(T2AG3)3] (4), are reported. Various spectroscopic techniques were employed to characterize the binding of conjugate 3 to 4. A competition dialysis assay revealed that 3 preferentially binds to 4, in the presence of other nucleic acids, including DNA, RNA, DNA-RNA hybrids, and other higher-order structures (single strands, duplexes, triplexes, other G-quadruplexes, and the i-motif). UV thermal denaturation studies showed that thermal stabilization of 4 increases as a function of the increasing concentration of 3. The fluorescence intercalator displacement (FID) assay displayed a significantly tighter binding of 3 with 4 as compared to its parent constituents [220-fold stronger than neomycin (1) and 4.5-fold stronger than perylene diamine (2), respectively]. The binding of 3 with 4 resulted in pronounced changes in the molar ellipticity of the DNA absorption region as confirmed by circular dichroism. The UV-vis absorption studies of the binding of 3 to 4 resulted in a red shift in the spectrum of 3 as well as a marked hypochromic change in the perylene absorption region, suggesting that the ligand-quadruplex interaction involves stacking of the perylene moiety. Docking studies suggest that the perylene moiety serves as a bridge that end stacks on 4, making contacts with two thymine bases in the loop, while the two neomycin moieties branch into the grooves of 4.

Thermodynamic studies on the interactions between intercalator-neomycin conjugates and a DNA polynucleotide triplex [poly(dA).2poly(dT)] were conducted. To draw a complete picture of such interactions, naphthalene diimide-neomycin (3) and anthraquinone-neomycin (4) conjugates were synthesized and used together with two other analogues, previously synthesized pyrene-neomycin (1) and BQQ-neomycin (2) conjugates, in our investigations. A combination of experiments, including UV denaturation, circular dichroism (CD) titration, differential scanning calorimetry (DSC), and isothermal titration calorimetry (ITC), revealed that all four conjugates (1-4) stabilized poly(dA).2poly(dT) much more than its parent compound, neomycin. UV melting experiments clearly showed that the temperature (T(m3-->2)) at which poly(dA).2poly(dT) dissociated into poly(dA).poly(dT) and poly(dT) increased dramatically (>12 degrees C) in the presence of intercalator-neomycin conjugates (1-4) even at a very low concentration (2 muM). In contrast to intercalator-neomycin conjugates, the increment of T(m3-->2) of poly(dA).2poly(dT) induced by neomycin was negligible under the same conditions. The binding preference of intercalator-neomycin conjugates (1-4) to poly(dA).2poly(dT) was also confirmed by competition dialysis and a fluorescent intercalator displacement assay. Circular dichroism titration studies revealed that compounds 1-4 had slightly larger binding site size ( approximately 7-7.5) with poly(dA).2poly(dT) as compared to neomycin ( approximately 6.5). The thermodynamic parameters of these intercalator-neomycin conjugates with poly(dA).2poly(dT) were derived from an integrated van't Hoff equation using the T(m3-->2) values, the binding site size numbers, and other parameters obtained from DSC and ITC. The binding affinity of all tested ligands with poly(dA).2poly(dT) increased in the following order: neomycin < 1 < 3 < 4 < 2. Among them, the binding constant [(2.7 +/- 0.3) x 10(8) M(-1)] of 2 with poly(dA).2poly(dT) was the highest, almost 1000-fold greater than that of neomycin. The binding of compounds 1-4 with poly(dA).2poly(dT) was mostly enthalpy-driven and gave negative DeltaC(p) values. The results described here suggest that the binding affinity of intercalator-neomycin conjugates for poly(dA).2poly(dT) increases as a function of the surface area of the intercalator moiety.

Recent developments have indicated that aminoglycoside binding is not limited to RNA, but to nucleic acids that, like RNA, adopt conformations similar to its A-form. We further sought to expand the utility of aminoglycoside binding to B-DNA structures by conjugating neomycin, an aminoglycoside antibiotic, with the B-DNA minor groove binding ligand Hoechst 33258. Envisioning a dual groove binding mode, we have extended the potential recognition process to include a third, intercalative moiety. Similar conjugates, which vary in the number of binding moieties but maintain identical linkages to allow direct comparisons to be made, have also been prepared. We report herein novel neomycin- and Hoechst 33258-based conjugates developed in our laboratories for exploring the recognition potential with B-DNA. Spectroscopic studies such as UV melting, differential scanning calorimetry, isothermal fluorescence titrations, and circular dichroism together illustrate the triple recognition of the novel conjugate containing neomycin, Hoechst 33258, and pyrene. This study represents the first example of DNA molecular recognition capable of minor versus major groove recognition in conjunction with intercalation.

In this work, hydrogen-deuterium exchange detected by NMR spectroscopy is used to determine the dynamic properties of the aminoglycoside phosphotransferase 3'-IIIa (APH), a protein of intense interest due to its involvement in conferring antibiotic resistance to both gram negative and gram positive microorganisms. This represents the first characterization of dynamic properties of an aminoglycoside-modifying enzyme. Herein we describe in vitro dynamics of apo, binary, and ternary complexes of APH with kanamycin A, neomycin B, and metal-nucleotide. Regions of APH in different complexes that are superimposable in crystal structures show remarkably different dynamic behavior. A complete exchange of backbone amides is observed within the first 15 h of exposure to D(2)O in the apo form of this 31 kDa protein. Binding of aminoglycosides to the enzyme induces significant protection against exchange, and approximately 30% of the amides remain unexchanged up to 95 h after exposure to D(2)O. Our data also indicate that neomycin creates greater solvent protection and overall enhanced structural stability to APH than kanamycin. Surprisingly, nucleotide binding to the enzyme-aminoglycoside complex increases solvent accessibility of a number of amides and is responsible for destabilization of a nearby beta-sheet, thus providing a rational explanation for previously observed global thermodynamic parameters. Our data also provide a molecular basis for broad substrate selectivity of APH.

Aminoglycoside antibiotics and cationic detergents constitute two classes of clinically important drugs and antiseptics. Their bacteriological and clinical efficacy, however, has decreased recently due to antibiotic resistance. We have synthesized aminoglycoside-lipid conjugates in which the aminoglycoside neomycin forms the cationic headgroup of a polycationic detergent. Our results show that neomycin-C16 and neomycin-C20 conjugates exhibit strong Gram-positive activity but reduced Gram-negative activity. The MIC of neomycin-C16 (C20) conjugates against methicillin-resistant Staphylococcus aureus (MRSA) is comparable to clinically used antiseptics.

This work describes differential effects of solvent in complexes of the aminoglycoside phosphotransferase(3')-IIIa (APH) with different aminoglycosides and the detection of change in solvent structure at specific sites away from substrates. Binding of kanamycins to APH occurs with a larger negative DeltaH in H2O relative to D2O (DeltaDeltaH(H2O-D2O) < 0), while the reverse is true for neomycins. Unusually large negative DeltaCp values were observed for binding of aminoglycosides to APH. DeltaCp for the APH-neomycin complex was -1.6 kcal x mol(-1) x deg(-1). A break at 30 degrees C was observed in the APH-kanamycin complex yielding DeltaCp values of -0.7 kcal x mol(-1) x deg(-1) and -3.8 kcal x mol(-1) x deg(-1) below and above 30 degrees C, respectively. Neither the change in accessible surface area (DeltaASA) nor contributions from heats of ionization were sufficient to explain the large negative DeltaCp values. Most significantly, 15N-1H HSQC experiments showed that temperature-dependent shifts of the backbone amide protons of Leu 88, Ser 91, Cys 98, and Leu143 revealed a break at 30 degrees C only in the APH-kanamycin complex in spectra collected between 21 degrees C and 38 degrees C. These amino acids represent solvent reorganization sites that experience a change in solvent structure in their immediate environment as structurally different ligands bind to the enzyme. These residues were away from the substrate binding site and distributed in three hydrophobic patches in APH. Overall, our results show that a large number of factors affect DeltaCp and binding of structurally different ligand groups cause different solvent structure in the active site as well as differentially affecting specific sites away from the ligand binding site.

A seven-step synthesis of orthogonally O-protected 2-deoxy-streptamine has been developed from readily available neomycin, with an overall yield of 28%. Key chemical transformations include a chemoselective glycosidic bond hydrolysis and two regioselective protective group manipulations involving acetylation and deacetylation. The synthetic route is amenable to scale-up for the production of multigram quantities of enantiopure and orthogonally O-protected 2-deoxystreptamine, a versatile scaffold for the generation of libraries of RNA-targeting ligands.

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.

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.

Recent developments have indicated that aminoglycoside binding is limited not to RNA but to nucleic acids that, like RNA, adopt conformations similar to the A-form. We have further sought to expand the utility of aminoglycoside binding to B-DNA structures by conjugating neomycin, an aminoglycoside antibiotic, with the B-DNA minor groove binding ligand Hoechst 33258. Described herein are novel neomycin-Hoechst 33258 conjugates developed for exploring B-DNA groove recognition. We have varied the two reported conjugates in linker length and composition in an effort to improve our understanding of the spatial differences that define B-DNA binding. Spectroscopic studies such as ultraviolet (UV) melting, isothermal fluorescence titrations, differential scanning calorimetry (DSC), and circular dichroism (CD) together illustrate the mode of binding by such conjugates. Both conjugates exhibit enhanced thermal stabilization of A.T rich duplexes when compared to Hoechst 33258.

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.

[reaction: see text] A novel method for achieving the desired regioselective reduction of the N-1 azido group on a tetraazidoneamine has been developed that leads to the synthesis of both kanamycin and neomycin class antibiotics bearing N-1 modification. Both classes of aminoglycosides are active against aminoglycoside-resistant bacteria carrying APH(3')-I and AAC(6')/APH(2'').

The water-soluble (> 200 mg/mL) antibiotics tobramycin, kanamycin, and neomycin spontaneously produce rigid fibers on negatively charged surfaces (mica, graphite, DNA). Atomic force microscopy showed single strands of tobramycin on mica at pH 7 with a length of several hundred nanometers and a diameter of 0.5 nm and double helices with a diameter of 1.0 nm and a helical pitch of 7 nm. At pH 13 (NaOH) up to 15 microm long, rigid fibers with a uniform height of 2.4 nm and an apparent helical pitch of 30 nm were formed along the sodium silicate channels on the surface of mica. Kanamycin and neomycin behaved similarly. Fibers of similar length and width, but without secondary structure, were obtained from aqueous solutions at pH 7 on amorphous, hydrophilized carbon and characterized by transmission electron microscopy. Overstretched phage lambda-DNA strands with a height of 1.0 nm on mica did not interact with tobramycin coils at pH 7. After treatment with EDTA, however, the height of the magnesium-free lambda-DNA strands grew from 1.0 to 3.8 nm after treatment with tobramycin, which suggests a wrapping by the supramolecular fibers. Such fibers may interact with F-actin fibers in biological cells, which would explain the known aggressiveness of aminoglycosides toward bacterial cell membranes and their ototoxicity., (Copyright 2004 American Chemical Society)

Synthesis of a BQQ-neomycin conjugate is reported. The conjugate combines two ligands, one known to intercalate triplexes (BQQ) and another known to bind in the triplex groove (neomycin). The conjugate stabilizes T.A.T, as well as mixed base DNA triplex, better than neomycin, BQQ, or a combination of both. The conjugate selectively stabilizes the triplex (in the presence of physiological salt concentrations), with as little as 4 muM of the ligand leading to a DeltaTm of >60 degrees C. Competition dialysis studies show a clear preference for the drug binding to triplex DNA/RNA over the duplex/single strand structures. Modeling studies suggest a structure of neomycin bound to the larger W-H (Watson-Hoogsteen) groove with BQQ intercalated between the triplex bases.

Neomycin is the most effective aminoglycoside (groove binder) in stabilizing a DNA triple helix. It stabilizes TAT, as well as mixed base DNA triplexes, better than known DNA minor groove binders (which usually destabilize the triplex) and polyamines. Neomycin selectively stabilizes the triplex (in the presence of salt), without any effect on the DNA duplex. (1) Triplex stabilization by neomycin is salt dependent (increased KCl and MgCl(2) concentrations decrease neomycin's effectiveness, at a fixed drug concentration). (2) Triplex stabilization by neomycin is pH dependent (increased pH decreases neomycin's effectiveness, at a fixed drug concentration). (3) CD binding studies indicate approximately 5-7 base triplets/drug apparent binding site, depending upon the structure/sequence of the triplex. (4) Neomycin shows nonintercalative groove binding to the DNA triplex, as evident from viscometric studies. (5) Neomycin shows a preference for stabilization of TAT triplets but can also accommodate CGC(+) triplets. (6) Isothermal titration calorimetry (ITC) studies reveal an association constant of approximately 2 x 10(5) M(-)(1) between neomycin and an intramolecular triplex and a higher K(a) for polydA.2polydT. (7) Binding/modeling studies show a marked preference for neomycin binding to the larger W-H groove. Ring I/II amino groups and ring IV amines are proposed to be involved in the recognition process. (8) The novel selectivity of neomycin is suggested to be a function of its charge and shape complementarity to the triplex W-H groove, making neomycin the first molecule that selectively recognizes a triplex groove over a duplex groove.

[reaction: see text] The synthesis of a novel class of aminoglycosides, pyranmycins, is reported along with the structure activity relationship (SAR) of their antibacterial activity against Escherichia coli. Two pyranmycins show prominent activity (9 microM). Pyranmycins also manifest superior stability in acidic media. The SAR information will lead to the future designs of pyranmycin against drug resistant bacteria.

Acoustic wave devices of the transverse shear-wave type are becoming increasingly important in the study of biochemical binding events at the solid-liquid interface in real time. The operation of the sensor is based on the principle that perturbations occurring at the solid-liquid interface result in changes in the propagating characteristics of the acoustic wave. The binding of the human immunodeficiency virus-type 1 Tat protein to the transactivation-responsive RNA element has been studied using this sensor. Variable acoustic signals in terms of frequency and motional resistance changes are obtained when surface-immobilized RNA is challenged by different peptide fragments derived from Tat protein. The effect of peptide concentration and mutation in addition to the inhibition of RNA-peptide binding by neomycin has been investigated. The results of this study suggest that acoustic physics offers considerable potential for the screening of small-molecule interactions with nucleic acids.

We use spectroscopic and calorimetric techniques to characterize the binding of the aminoglycoside antibiotics neomycin, paromomycin, and ribostamycin to a RNA oligonucleotide that models the A-site of Escherichia coli 16S rRNA. Our results reveal the following significant features: (i) Aminoglycoside binding enhances the thermal stability of the A-site RNA duplex, with the extent of this thermal enhancement decreasing with increasing pH and/or Na(+) concentration. (ii) The RNA binding enthalpies of the aminoglycosides become more exothermic (favorable) with increasing pH, an observation consistent with binding-linked protonation of one or more drug amino groups. (iii) Isothermal titration calorimetry (ITC) studies conducted as a function of buffer reveal that aminoglycoside binding to the host RNA is linked to the uptake of protons, with the number of linked protons being dependent on pH. Specifically, increasing the pH results in a corresponding increase in the number of linked protons. (iv) ITC studies conducted at 25 and 37 degrees C reveal that aminoglycoside-RNA complexation is associated with a negative heat capacity change (Delta C(p)), the magnitude of which becomes greater with increasing pH. (v) The observed RNA binding affinities of the aminoglycosides decrease with increasing pH and/or Na(+) concentration. In addition, the thermodynamic forces underlying these RNA binding affinities also change as a function of pH. Specifically, with increasing pH, the enthalpic contribution to the observed RNA binding affinity increases, while the corresponding entropic contribution to binding decreases. (vi) The affinities of the aminoglycosides for the host RNA follow the hierarchy neomycin > paromomycin > ribostamycin. The enhanced affinity of neomycin relative to either paromomycin or ribostamycin is primarily, if not entirely, enthalpic in origin. (vii) The salt dependencies of the RNA binding affinities of neomycin and paromomycin are consistent with at least three drug NH(3)(+) groups participating in electrostatic interactions with the host RNA. In the aggregate, our results reveal the impact of specific alterations in aminoglycoside structure on the thermodynamics of binding to an A-site model RNA oligonucleotide. Such systematic comparative studies are critical first steps toward establishing the thermodynamic database required for enhancing our understanding of the molecular forces that dictate and control aminoglycoside recognition of RNA.

The binding of paromomycin and similar antibiotics to the small (30S) ribosomal subunit has been studied using continuum electrostatics methods. Crystallographic information from a complex of paromomycin with the 30S subunit was used as a framework to develop structures of similar antibiotics in the same ribosomal binding site. Total binding energies were calculated from electrostatic properties obtained by solution of the Poisson-Boltzmann equation combined with a surface area-dependent apolar term. These computed results showed good correlation with experimental data. Additionally, calculation of the ribosomal electrostatic potential in the paromomycin binding site provided insight into the electrostatic mechanisms for aminoglycoside binding and clues for the rational design of more effective antibiotics.

The binding of aminoglycosides to RNA provides a paradigm system for the analysis of RNA-drug interactions. The electrostatic field around three-dimensional RNA folds creates localized and defined negatively charged regions which are potential docking sites for the cationic ammonium groups of aminoglycosides. To explore in RNA folds the electronegative pockets suitable for aminoglycoside binding, we used calculations of the electrostatic field and Brownian dynamics simulations of cation diffusion. We applied the technique on those RNA molecules experimentally known to bind aminoglycosides, namely, two tobramycin aptamers (Wang, Y.; Rando, R. R. Chem. Biol. 1995, 2, 281-290): the aminoglycoside-binding region in 16S ribosomal RNA (Moazed, S.; Noller, H. F. Nature 1987, 327, 389-394) and the TAR RNA from human immunodeficiency virus (Mei, H.-Y.; et al. Bioorg. Med. Chem. Lett. 1995, 5, 2755-2760). For the aptamers and ribosomal RNA, for which the binding sites of the aminoglycosides are known, a good agreement between negatively charged pockets and the binding positions of the drugs was found. On the basis of variations between neomycin-like and kanamycin-like aminoglycosides in the interaction with the electrostatic field of ribosomal RNA, we propose a model for the different binding specificities of these two classes of drugs. The spatial congruence between the electronegative pockets in RNA folds and binding positions of aminoglycosides was used to dock aminoglycosides to ribosomal and TAR RNAs. Molecular dynamics simulations were used to analyze possible RNA-drug interactions. Aminoglycosides inhibit the binding of the viral Tat protein to TAR RNA; however, the drug-binding sites are still unknown. Thus, our docking approach provides first structural models for TAR-aminoglycoside complexes. The RNA-drug interactions observed in the modeled complexes support the view that the antibiotics might lock TAR in a conformation with low affinity for the Tat protein, explaining the experimentally found aminoglycoside inhibition of the Tat-TAR interaction (Mei, H.-Y.; et al. Bioorg. Med. Chem. Lett. 1995, 5, 2755-2760).

The translational initiation codon for thymidylate synthase (TS) mRNA is located in a unique stem-loop structure which contains an internal cytosine-cytosine (CC) bubble. This stem-loop structure is thought to be important in the regulation of TS translation, which is itself an important target for anticancer drugs, such as 5-fluorouracil. Internal bubble or bulge structures are candidate receptors for the aminoglycoside antibiotics. It is shown here that aminoglycosides bind in a specific and saturable fashion with dissociation constants of approximately 1 microM to a TS mRNA site 1 construct and that the binding site for the aminoglycosides is located in the CC bubble region. In fact, the CC bubble, when grafted into other stem-loop structures, confers aminoglycoside binding on them. These studies reveal an additional binding domain for aminoglycosides and also suggest how novel anti-cancer drugs might be designed that affect TS mRNA translation rather than enzyme function.

Ligands capable of specific recognition of RNA structures are of interest in terms of the principles of molecular recognition as well as potential chemotherapeutic applications. We have approached the problem of identifying small molecules with binding specificity for the RNA double helix through application of the DOCK program [Kuntz, I. D., Meng, E. C., and Shoichet, B. K. (1994) Acc. Chem. Res. 27, 117-123], a structure-based method for drug discovery. A series of lead compounds was generated through a database search for ligands with shape complementarity to the RNA deep major groove. Compounds were then evaluated with regard to their fit into the minor groove of B DNA. Those compounds predicted to have an optimal fit to the RNA groove and strong discrimination against DNA were examined experimentally. Of the 11 compounds tested, 3, all aminoglycosides, exhibited pronounced stabilization of RNA duplexes against thermal denaturation with only marginal effects on DNA duplexes. One compound, lividomycin, was examined further, and shown to facilitate the ethanol-induced B to A transition in calf thymus DNA. Fluorine NMR solvent isotope shift measurements on RNA duplexes containing 5-fluorouracil provided evidence that lividomycin binds in the RNA major groove. Taken together, these results indicate that lividomycin recognizes the general features of the A conformation of nucleic acids through deep groove binding, confirming the predictions of our DOCK analysis. This approach may be of general utility for identifying ligands possessing specificity for additional RNA structures as well as other nucleic acid structural motifs.

To investigate the properties of the neomycin-hammerhead interaction, inhibition of hammerhead activity was measured as a function of magnesium concentration and pH. The data are consistent with a simple competition between magnesium and neomycin with about five magnesium ions required to displace neomycin from the hammerhead. The pH dependence of the inhibition of hammerhead cleavage by neomycin and two related aminoglycosides was also determined. The data indicate that at least three of the five positively charged ammonium ions present on neomycin are critical for inhibiting hammerhead function. Taken together, these results suggest that the neomycin-hammerhead interaction is mostly ionic in character.

Ca2+ release from sarcoplasmic reticulum during excitation--contraction coupling is likely to be mediated by conformational changes in the foot protein moiety of the triadic vesicles. As a preparative step toward the studies of dynamic conformational changes in the foot protein moiety, we have developed a new method that permits specific labeling of the foot protein moiety of the isolated membranes with a fluorophore. A novel fluorescent cleavable photoaffinity cross-linking reagent, sulfosuccinimidyl 3-((2-(7-azido-4-methylcoumarin-3-acetamido)ethyl)dithio)propionate (SAED), was conjugated with site-directing carriers, polylysine (Ca(2+)-release inducer) and neomycin (Ca(2+)-release blocker). The conjugates were allowed to bind to polylysine- and neomycin-binding sites of the heavy fraction of SR (HSR). After photolysis, the cross-linked reagent was cleaved by reduction and the fluorescently labeled HSR was separated from the carriers by centrifugation. These procedures led to specific incorporation of the methylcoumarin acetate (MCA) into the foot protein. Polylysine and neomycin bound to different sites of the foot protein, since neomycin, at release-blocking concentrations, did not interfere with polylysine binding. The fluorescence intensity of the foot protein labeled with the carrier, neomycin, showed biphasic changes as a function of ryanodine concentration (increasing up to 1 microM ryanodine and decreasing above it), while with the carrier polylysine, ryanodine induced no change in fluorescence intensity. In contrast, the fluorescence intensity of the foot protein labeled with each of the two carriers, neomycin and polylysine, showed almost identical calcium dependence (first increasing from 0.1 microM to about 3.0 microM calcium concentration, and then decreasing at higher calcium concentrations).(ABSTRACT TRUNCATED AT 250 WORDS)