Molecular mechanisms for dynamic regulation of N1 riboswitch by aminoglycosides.

A synthetic riboswitch N1, inserted into the 5'-untranslated mRNA region of yeast, regulates gene expression upon binding ribostamycin and neomycin. Interestingly, a similar aminoglycoside, paromomycin, differing from neomycin by only one substituent (amino versus hydroxyl), also binds to the N1 riboswitch, but without affecting gene expression, despite NMR evidence that the N1 riboswitch binds all aminoglycosides in a similar way. Here, to explore the details of structural dynamics of the aminoglycoside-N1 riboswitch complexes, we applied all-atom molecular dynamics (MD) and temperature replica-exchange MD simulations in explicit solvent. Indeed, we found that ribostamycin and neomycin affect riboswitch dynamics similarly but paromomycin allows for more flexibility because its complex lacks the contact between the distinctive 6' hydroxyl group and the G9 phosphate. Instead, a transient hydrogen bond of 6'-OH with A17 is formed, which partially diminishes interactions between the bulge and apical loop of the riboswitch, likely contributing to riboswitch inactivity. In many ways, the paromomycin complex mimics the conformations, interactions, and Na+ distribution of the free riboswitch. The MD-derived interaction network helps understand why riboswitch activity depends on aminoglycoside type, whereas for another aminoglycoside-binding site, aminoacyl-tRNA site in 16S rRNA, activity is not discriminatory.

Single lipoaminoglycoside promotes efficient intracellular antibody delivery: A comprehensive insight into the mechanism of action.

Delivery of biologically active proteins into cells is emerging as important strategy for many applications. Previous experiments have shown that lipoaminoglycosides were capable of delivery of the anti-cytokeratin8 antibody (anti-K8) but only when formulated with lipid helpers potentially leading to toxicity from excess lipids. Here, we optimized anti-K8 delivery with various lipoaminoglycosides in the absence of a lipid helper. Results led to the identification of the aminoglycoside lipid dioleyl phosphoramido ribostamycin (DOPRI) as a potent intracellular delivery system for anti-K8. Electron microscopy revealed that delivered anti-K8 molecules were bound to intermediate filaments in cells. Anti-K8 was bound to the surface of DOPRI vesicles without perturbing lipid organization. Macropinocytosis and caveolin mediated endocytosis contributed to anti-K8 internalization and to filament labeling with a major contribution being made by the caveolin pathway. The results showed that the unique properties of DOPRI were sufficient for efficient intracellular protein delivery without requiring lipid helpers.

Abundance and distribution of antibiotic resistance genes in a full-scale anaerobic-aerobic system alternately treating ribostamycin, spiramycin and paromomycin production wastewater.

The occurrence of antibiotic-resistant bacteria and antibiotic resistance genes (ARGs) has been intensively investigated for wastewater treatment systems treating single class of antibiotic in recent years. However, the impacts of alternately occurring antibiotics in antibiotic production wastewater on the behavior of ARGs in biological treatment systems were not well understood yet. Herein, techniques including high-capacity quantitative PCR and quantitative PCR (qPCR) were used to investigate the behavior of ARGs in an anaerobic-aerobic full-scale system. The system alternately treated three kinds of antibiotic production wastewater including ribostamycin, spiramycin and paromomycin, which referred to stages 1, 2 and 3. The aminoglycoside ARGs (52.1-79.3%) determined using high-capacity quantitative PCR were the most abundant species in all sludge samples of the three stages. The total relative abundances of macrolide-lincosamide-streptogramin (MLS) resistance genes and aminoglycoside resistance genes measured using qPCR were significantly higher (P < 0.05) in aerobic sludge than in sewage sludge. However, the comparison of ARGs acquired from three alternate stages revealed that MLS genes and the aminoglycoside ARGs did not vary significantly (P > 0.05) in both aerobic and anaerobic sludge samples. In aerobic sludge, one acetyltransferase gene (aacA4) and the other three nucleotidyltransferase genes (aadB, aadA and aadE) exhibited positive correlations with intI1 (r 2 = 0.83-0.94; P < 0.05), implying the significance of horizontal transfer in their proliferation. These results and facts will be helpful to understand the abundance and distribution of ARGs from antibiotic production wastewater treatment systems.

Potentiation of Aminoglycoside Activity in Pseudomonas aeruginosa by Targeting the AmgRS Envelope Stress-Responsive Two-Component System.

A screen for agents that potentiated the activity of paromomycin (PAR), a 4,5-linked aminoglycoside (AG), against wild-type Pseudomonas aeruginosa identified the RNA polymerase inhibitor rifampin (RIF). RIF potentiated additional 4,5-linked AGs, such as neomycin and ribostamycin, but not the clinically important 4,6-linked AGs amikacin and gentamicin. Potentiation was absent in a mutant lacking the AmgRS envelope stress response two-component system (TCS), which protects the organism from AG-generated membrane-damaging aberrant polypeptides and, thus, promotes AG resistance, an indication that RIF was acting via this TCS in potentiating 4,5-linked AG activity. Potentiation was also absent in a RIF-resistant RNA polymerase mutant, consistent with its potentiation of AG activity being dependent on RNA polymerase perturbation. PAR-inducible expression of the AmgRS-dependent genes htpX and yccA was reduced by RIF, suggesting that AG activation of this TCS was compromised by this agent. Still, RIF did not compromise the membrane-protective activity of AmgRS, an indication that it impacted some other function of this TCS. RIF potentiated the activities of 4,5-linked AGs against several AG-resistant clinical isolates, in two cases also potentiating the activity of the 4,6-linked AGs. These cases were, in one instance, explained by an observed AmgRS-dependent expression of the MexXY multidrug efflux system, which accommodates a range of AGs, with RIF targeting of AmgRS undermining mexXY expression and its promotion of resistance to 4,5- and 4,6-linked AGs. Given this link between AmgRS, MexXY expression, and pan-AG resistance in P. aeruginosa, RIF might be a useful adjuvant in the AG treatment of P. aeruginosa infections.

Chemically related 4,5-linked aminoglycoside antibiotics drive subunit rotation in opposite directions.

Dynamic remodelling of intersubunit bridge B2, a conserved RNA domain of the bacterial ribosome connecting helices 44 (h44) and 69 (H69) of the small and large subunit, respectively, impacts translation by controlling intersubunit rotation. Here we show that aminoglycosides chemically related to neomycin-paromomycin, ribostamycin and neamine-each bind to sites within h44 and H69 to perturb bridge B2 and affect subunit rotation. Neomycin and paromomycin, which only differ by their ring-I 6'-polar group, drive subunit rotation in opposite directions. This suggests that their distinct actions hinge on the 6'-substituent and the drug's net positive charge. By solving the crystal structure of the paromomycin-ribosome complex, we observe specific contacts between the apical tip of H69 and the 6'-hydroxyl on paromomycin from within the drug's canonical h44-binding site. These results indicate that aminoglycoside actions must be framed in the context of bridge B2 and their regulation of subunit rotation.

The potential of protein disulfide isomerase as a therapeutic drug target.

Protein disulfide isomerase (PDI) is a multifunctional protein of the thioredoxin superfamily. PDI mediates proper protein folding by oxidation or isomerization and disrupts disulfide bonds by reduction; it also has chaperone and antichaperone activities. Although PDI localizes primarily to the endoplasmic reticulum (ER), it is secreted and expressed on the cell surface. In the ER, PDI is primarily involved in protein folding, whereas on the cell surface, it reduces disulfide bonds. The functions of PDI depend on its localization and the redox state of its active site cysteines. The ER-based functions of PDI are linked to cancer invasion and migration. Surface-associated PDI facilitates the entry of viruses, such as HIV-1, and toxins, such as diphtheria and cholera. Thus, based on its involvement in pathological events, PDI is considered a potential drug target. However, a significant challenge in the therapeutic targeting of PDI is discovering function-specific inhibitors for it. To this end, a wide range of therapeutic agents, such as antibiotics, thiol blockers, estrogenic compounds, and arsenical compounds, have been used, although few are bona fide specific inhibitors. In this review, we will describe the potential of PDI as a therapeutic drug target.

Aminoglycoside binding to Oxytricha nova telomeric DNA.

Telomeric DNA sequences have been at the center stage of drug design for cancer treatment in recent years. The ability of these DNA structures to form four-stranded nucleic acid structures, called G-quadruplexes, has been perceived as target for inhibiting telomerase activity vital for the longevity of cancer cells. Being highly diverse in structural forms, these G-quadruplexes are subjects of detailed studies of ligand-DNA interactions of different classes, which will pave the way for logical design of more potent ligands in future. The binding of aminoglycosides was investigated with Oxytricha nova quadruplex forming DNA sequence (GGGGTTTTGGGG)(2). Isothermal titration calorimetry (ITC) determined ligand to quadruplex binding ratio shows 1:1 neomycin:quadruplex binding with association constants (K(a)) ∼ 10(5) M(-1) while paromomycin was found to have a 2-fold weaker affinity than neomycin. The CD titration experiments with neomycin resulted in minimal changes in the CD signal. FID assays, performed to determine the minimum concentration required to displace half of the fluorescent probe bound, showed neomycin as the best of the all aminoglycosides studied for quadruplex binding. Initial NMR footprint suggests that ligand-DNA interactions occur in the wide groove of the quadruplex. Computational docking studies also indicate that aminoglycosides bind in the wide groove of the quadruplex.

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.

Activity of some aminoglycoside antibiotics against true fungi, Phytophthora and Pythium species.

AIMS: To investigate the in vitro antifungal and antioomycete activities of some aminoglycosides against true fungi and Phytophthora and Pythium species and to evaluate the potential of the antibiotics against Phytophthora late blight on plants. METHODS AND RESULTS: Antifungal and antioomycete activities of aminoglycoside antibiotics (neomycin, paromomycin, ribostamycin and streptomycin) and a paromomycin-producing strain (Streptomyces sp. AMG-P1) against Phytophthora and Pythium species and 10 common fungi were measured in potato dextrose broth (PDB) and on seedlings in pots. Paromomycin was the most active against Phytophthora and Pythium species with a minimal inhibitory concentration of 1-10 microg ml(-1) in PDB, but displayed low to moderate activities towards other common fungi at the same concentration. Paromomycin also showed potent in vivo activity against red pepper and tomato late blight diseases with 80 and 99% control value, respectively, at 100 microg ml(-1). In addition, culture broth of Streptomyces sp. AMG-P1 as a paromomycin producer exhibited high in vivo activity against late blight at 500 microg freeze-dried weight per millilitre. CONCLUSIONS: Among tested aminoglycoside antibiotics, paromomycin was the most active against oomycetes both in vitro and in vivo. SIGNIFICANCE AND IMPACT OF THE STUDY: Data from this study show that aminoglycoside antibiotics have in vitro and in vivo activities against oomycetes, suggesting that Streptomyces sp. AMG-P1 may be used as a biocontrol agent against oomycete diseases.

Drug targeting of HIV-1 RNA.DNA hybrid structures: thermodynamics of recognition and impact on reverse transcriptase-mediated ribonuclease H activity and viral replication.

RNA degradation via the ribonuclease H (RNase H) activity of human immunodeficiency virus type I (HIV-1) reverse transcriptase (RT) is a critical component of the reverse transcription process. In this connection, mutations of RT that inactivate RNase H activity result in noninfectious virus particles. Thus, interfering with the RNase H activity of RT represents a potential vehicle for the inhibition of HIV-1 replication. Here, we demonstrate an approach for inhibiting the RNase H activity of HIV-1 RT by targeting its RNA.DNA hybrid substrates. Specifically, we show that the binding of the 4,5-disubstituted 2-deoxystreptamine aminoglycosides, neomycin, paromomycin, and ribostamycin, to two different chimeric RNA-DNA duplexes, which mimic two distinct intermediates in the reverse transcription process, inhibits specific RT-mediated RNase H cleavage, with this inhibition being competitive in nature. UV melting and isothermal titration calorimetry studies reveal a correlation between the relative binding affinities of the three drugs for each of the chimeric RNA-DNA host duplexes and the relative extents to which the drugs inhibit RT-mediated RNase H cleavage of the duplexes. Significantly, this correlation also extends to the relative efficacies with which the drugs inhibit HIV-1 replication. In the aggregate, our results highlight a potential strategy for AIDS chemotherapy that should not be compromised by the unusual genetic diversity of HIV-1.

Ribostamycin inhibits the chaperone activity of protein disulfide isomerase.

In the process of screening of proteins binding to ribostamycin in bovine liver using the affinity column chromatography, we found that ribostamycin inhibited the chaperone activity of protein disulfide isomerase (PDI), but it did not inhibit the isomerase activity. PDI was identified by SDS-PAGE, Western blotting, and N-terminal amino acid sequence analysis. A 100:1 molar ratio of ribostamycin to PDI was almost sufficient to completely inhibit the chaperone activity of PDI. The binding affinity of ribostamycin to purified bovine PDI was determined by the Biacore system, which gave a K(D) value of 3.19 x 10(-4) M. This suggests that ribostamycin binds to region distinct from the CGHC motif of PDI. This is the first report to describe the inhibitor of the chaperone activity of PDI.

In vitro activity of mezlocillin, meropenem, aztreonam, vancomycin, teicoplanin, ribostamycin and fusidic acid against Borrelia burgdorferi.

The in vitro susceptibility profile of Borrelia burgdorferi is not yet well defined for several antibiotics. Our study explored the in vitro susceptibility of B. burgdorferi to mezlocillin, meropenem, aztreonam, vancomycin, teicoplanin, ribostamycin and fusidic acid. Minimal inhibitory concentrations (MICs) and minimal borreliacidal concentrations (MBCs) were measured using a standardised colorimetric microdilution method and conventional subculture experiments. MIC values were lowest for mezlocillin (MIC(90), < or =0.06 mg/l) and meropenem (MIC(90), 0.33 mg/l). Vancomycin (MIC(90), 0.83 mg/l) was less effective in vitro. Borreliae proved to be resistant to aztreonam (MIC(90), >32 mg/l), teicoplanin (MIC(90), 6.6 mg/l), ribostamycin (MIC(90), 32 mg/l), and fusidic acid (MIC(90), >4 mg/l). The mean MBCs resulting in 100% killing of the final inoculum after 72 h of incubation were lowest for mezlocillin (MBC, 0.83 mg/l). This study gathered further data on the in vitro susceptibility patterns of the B. burgdorferi complex. The excellent in vitro effectiveness of acylamino-penicillin derivatives and their suitability for the therapy of Lyme disease is emphasised.

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.

Neomycin inhibition of the hammerhead ribozyme involves ionic interactions.

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.

A novel approach to insertional mutagenesis of Haemophilus influenzae.

Insertional mutagenesis of the Haemophilus influenzae chromosome was accomplished by a novel method employing a 2.2-kbp element, TSTE. This element, consisting of the neo gene of Tn5 flanked by Haemophilus-specific uptake sequences, was ligated to circularized chromosomal fragments before transformation into the homologous strain. Eight mutants defective in the production of haemocin were detected. This strategy provides an efficient mechanism for the insertional mutagenesis of H. influenzae.

Effect of aminoglycoside antibiotics on the electrical stimulation-induced contractile response of the guinea-pig ureter.

Using electrical stimulation, we first investigated whether the movement of ureteral smooth muscle of the guinea-pig was myogenic in its control, or neurogenic. We then investigated the effects of aminoglycoside antibiotics (kanamycin, bekanamycin and ribostamycin) on the movement of ureteral smooth muscle induced by electrical stimulation. In these investigations, each ureter removed from the animal was cut into three approximately equal-sized segments, which are an upper segment (kidney side), a middle segment and a lower segment (urinary bladder side). Each segment was mounted in an organ bath filled with Krebs solution and the mechanical response induced by electrical stimulation of each segment was recorded isometrically. Each one of the upper, the middle and the lower segments was stimulated with rectangular pulses (50 volt, 0.1-3 msec durations, 40 Hz) for a period of 2 sec. Of all segments tested (ten in each group), none showed any response to the stimulation with pulses below 0.5 msec duration. While 2-3 segments out of ten of the upper, the middle and the lower ureteral segments showed a contractile response to stimulation with pulses of 1 msec duration, the rest of the segments showed no response to the same stimulation. This contractile response was not inhibited by tetrodotoxin which is known to block the nerve-mediated response. And also, all the ten samples of each upper, middle and lower segment never failed to show a contractile response to stimulation with pulses of 2 msec and 3 msec duration. Various drugs which are already known to block the nerve-mediated response (i.e., atropine, guanethidine, phentolamine, propranolol and tetrodotoxin) were tried, but none of them had an inhibitory effect on the contractile response. On the other hand, each one of kanamycin (KM), bekanamycin (AKM) and ribostamycin (RSM) in concentrations of 1 x 10(-5) g/ml-1 x 10(-3) g/ml produced a concentration-dependent decreasing effect in the magnitude of the electrical stimulation-induced contractile response of the ureteral segment. In addition, the decreasing effects of these antibiotics were also observed in the tetrodotoxin. From these results, we concluded that the contractile response of the ureter may be myogenic in its control, and the aminoglycoside antibiotics, KM, AKM and RSM, may act directly on the ureteral smooth muscle so as to decrease its contractile response.

Comparative antimicrobial activities of ribostamycin, gentamicin, ampicillin and lincomycin in vitro and in vivo.

The antimicrobial activity of ribostamycin, a unique aminoglycoside antibiotic possessing a neutral sugar component, was compared with those of gentamicin, ampicillin and lincomycin in vitro and in vivo. Ribostamycin showed comparable or slightly weaker in vitro activity than the reference antibiotics against Gram-positive bacteria. Against Gram-negative bacteria, ribostamycin was less active than gentamicin, but comparable to or more active than ampicillin. Lincomycin was less active or inactive to Gram-negative bacteria. Ribostamycin was active against some gentamicin-resistant bacteria, especially K. pneumoniae possessing the aminoglycoside-modifying enzymes AAC(3)-l and AAD(2"). The in vivo activity of ribostamycin was weaker than that of gentamicin, but comparable to that of ampicillin and lincomycin against Gram-positive bacteria, and superior to that of ampicillin against Gram-negative bacteria. The in vivo activity of ribostamycin was characterized by (i) and ED50 value not so affected by the challenge inoculum as that of ampicillin; (ii) a lower ED50 value by bolus administration than that by divided administration of the same dosage; and (iii) a lower ED50 value than that expected from the MIC value as compared with that of ampicillin and lincomycin. These characteristics are explained by the rapid and potent bactericidal activity of ribostamycin at high inoculum and high drug concentration, assisted by high serum concentration in mice.

Simple new test for presumptive differentiation between genus Candida and genus Prototheca.

A differential test was made between genus Candida and genus Prototheca using a new and very simple differential test. A total of 59 strains of Candida and 78 strains of Prototheca were used. The basis of the test was the differential use of a disc carrying 60 mcg of Ribostamycin to which all the Candida were resistant and the Prototheca inhibited.

Voltage-dependent aminoglycoside blockade of the sarcoplasmic reticulum K+ channel.

Single-channel conductance of the K+ channel from sarcoplasmic reticulum (SR) was reduced by aminoglycoside antibiotics such as neomycin and ribostamycin and also by n-hexylamine from either side of the membrane in a dose- and voltage-dependent manner. K+ channels were incorporated into an artificial phospholipid bilayer. This inhibition follows a single-site titration curve. The voltage dependence of the inhibition is explained by assuming that these drugs bind to the open state of a single channel on one site located approximately 40% of the way through the membrane from the cis side (the side to which SR vesicles are added) when drugs are added to the cis side and bind on another site located approximately 40% of the way through the membrane from the trans side (the opposite side to the cis side) when drugs are added to the trans side.

Effects of cations, polyamines and other aminoglycosides on gentamicin C2. Binding to ribosomes from sensitive and resistant Escherichia coli strains.

Gentamicin C2 interacts cooperatively with ribosomes from a sensitive Escherichia coli strain in a multiphasic way with several classes of sites. It is shown that this binding is highly-dependent on Mg++ and natural endogenous polyamine concentrations. The differences observed between ribosomes from sensitive and resistant strains may be explained by the absence of specific cooperative gentamicin interactions with resistant ribosomes. The effects of other aminoglycoside antibiotics are discussed in terms of structure-activity relationships.