Antibiotic bacitracin induces hydrolytic degradation of nucleic acids.

BACKGROUND: Bacitracin is a polypeptide antibiotic active against Gram-positive bacterial strains. Its mechanism of action postulates disturbing the cell wall synthesis by inhibiting dephosphorylation of the lipid carrier. We have discovered that bacitracin induces degradation of nucleic acids, being particularly active against RNA. METHODS: In the examination of the nucleolytic activity of bacitracin several model RNA and DNA oligomers were used. The oligomers were labeled at their 5' ends with (32)P radioisotope and following treatment with bacitracin the cleavage sites and efficiency were determined. RESULTS AND CONCLUSIONS: Bacitracin induces degradation of RNA at guanosine residues, preferentially in single-stranded RNA regions. Bacitracin is also able to degrade DNA to some extent but comparable effects to those observed with RNA require its 10-fold higher concentration. The sites of degradation in DNA are very infrequent and preferentially occur near cytidine residues. Free radicals are not involved in the reaction, and which probably proceeds via a hydrolytic mechanism. The phosphate groups at the cleavage sites are present at the 3' ends of RNA products and at the 5' ends of DNA fragments. Importantly, the presence of EDTA does not influence RNA degradation but completely inhibits the degradation of DNA. For DNA degradation divalent metal ions like Mg(2+), Mn(2+) or Zn(2+) are absolutely necessary. GENERAL SIGNIFICANCE: The ability of bacitracin to degrade nucleic acids via a hydrolytic mechanism was a surprising observation, and it is of interest whether these properties can contribute to its mechanisms of action during antibiotic treatment.

High affinity of copper(II) towards amoxicillin, apramycin and ristomycin. Effect of these complexes on the catalytic activity of HDV ribozyme.

Three representatives of the distinct antibiotics groups: amoxicillin, apramycin and ristomycin A were studied regarding their impact on hepatitis D virus (HDV) ribozyme both in the metal-free form and complexed with copper(II) ions. Hence the Cu(II)-ristomycin A complex has been characterized by means of NMR, EPR, CD and UV-visible spectroscopic techniques and its binding pattern has been compared with the coordination modes estimated previously for Cu(II)-amoxicillin and Cu(II)-apramycin complexes. It has thus been found that all three antibiotics bind the Cu(II) ion in a very similar manner, engaging two nitrogen and two oxygen donors into coordination with the square planar symmetry in physiological conditions. All three tested antibiotics were able to inhibit the HDV ribozyme catalysis. However, in the presence of the complexes, the catalytic reactions were almost completely inhibited. It was important therefore to check whether the complexes used in lower concentrations could inhibit the HDV ribozyme catalytic activity, thus creating opportunities for their practical application. It turned out that the complexes used in the concentrations of 50µM influenced the catalysis much less effectively comparing to the 200 micromolar concentration. The kobs values were lower than those observed in the control reaction, in the absence of potential inhibitors: 2-fold for amoxicillin, ristomycin A and 3.3-fold for apramycin, respectively.

The impact of isomers of hemiaminal-1,2,4-triazole conjugates differently substituted in the phenyl ring and their Cu2+ complexes on the catalytic activity of the antigenomic delta ribozyme.

The ability of four stable hemiaminals differently substituted in the phenyl ring and their complexes with Cu(2+) ions to inhibit catalytic cleavage of the antigenomic delta ribozyme was compared. The hemiaminals were novel chiral derivatives of 1,2,4-triazole [i.e. (2,4-dinitrophenyl)(4H-1,2,4-triazol-4-ylamino) methanol (2,4-dnbald), (2-nitrophenyl)(4H-1,2,4-triazol-4-ylamino) methanol (2-nbald), (3-nitrophenyl)(4H-1,2,4-triazol-4-ylamino) methanol (3-nbald) and (4-nitrophenyl)(4H-1,2,4-triazol-4-ylamino) methanol (4-nbald)]. The complexes of nbalds with Cu(2+) were characterized using UV and EPR methods and additionally, the formation of 2,4-dnbald-Cu(2+) complex with CuL(2) stoichiometry was confirmed by mass spectrometry. The data suggest that there are two ways in which nbalds and their Cu(2+) complexes can influence catalytic cleavage of antigenomic delta ribozyme. The coordinated Cu(2+) ions may play the role of new cationic ligands increasing the affinity of the complexes to the ribozyme. Such situation occurs in the case of 2- and 2,4-nbald. Their Cu(2+) complexes decrease ribozyme cleavage rates twice more efficiently than uncomplexed compounds. Moreover, the Cu(2+) complexes displace the catalytic divalent metal ions from their strong binding sites located in the ribozyme J4/2 region as shown by the Pb(2+)-induced cleavage approach. On the other hand, 3- and 4-nbald inhibit catalysis more strongly as compared to 2-nbald and 2,4-dnbald but the ribozyme cleavage rates are changed only slightly upon Cu(2+) complexation. The mechanism of ribozyme inhibition by interfering with the formation of a correct ribozyme tertiary structure seems to operate in this case.

The Cu(II)-fluconazole complex revisited. Part I: Structural characteristics of the system.

Protonation equilibria and Cu(II) binding processes by an antifungal agent fluconazole, α-(2,4-difluorophenyl)-α-(1H-1,2,4-triazol-1-yl-methyl)-1H-1,2,4-triazole-1-ethanol, were studied using the UV-Vis, EPR and NMR spectroscopic techniques. The protonation constant of fluconazole was determined from NMR titration and attributed to N4' nitrogen atoms using the DFT methods. The spectroscopic data suggest that at pH as low as 0.4 the first complex is formed, in which one or two Cu(II) ions are bound to one of the nitrogen atoms (N4') from triazole rings. Above pH 1.5 each Cu(II) ion is surrounded by two nitrogen atoms (also N4') from two different ligand molecules, forming primary monomeric complexes and above pH=5, both dimeric or oligomeric species occur, which is well registered by the EPR technique. The mixture of Cu(NO(3))(2) with fluconazole in a 1:1 molar ratio in a water (pH=4.5)/ethanol solution gave crystals of [Cu(2)(H(2)O){(C(6)H(3)-2,4-F(2))(CH(2)N(3)C(2)H(2))(2)C-OH}{(C(6)H(3)-2,4-F(2))(CH(2)N(3)C(2)H(2))(2)C-O}(NO(3))](NO(3))(2)·9(H(2)O). This complex is the first example of a cupric 3D polymeric structure with a fluconazole ligand coordinated via both N2' and N4' atoms from the same triazole rings. At higher pH values, we obtained a binuclear complex [Cu(2)(L)(2)(H(2)O)(2)(NO(3))(2)], in which the copper(II) atoms were bridged by the oxygen atoms of the deprotonated OH group of fluconazole. The hypothetical oxidative properties of this system were also examined, however it failed to generate either reactive oxygen species or DNA scission products.

Capreomycin--a polypeptide antitubercular antibiotic with unusual binding properties toward copper(II).

Capreomycin is an important therapeutic agent having intriguing and diverse molecular features. Its polypeptidic structure rich in nitrogen donors makes the drug a promising chelating agent for a number of transition metal ions, especially for copper(II). The results of the model investigational studies suggest that capreomycin anchors Cu(2+) ion with an amino function of the α,ß-diaminopropionic acid residue at pH around 5. At physiological pH copper(II) ion is coordinated by two deprotonated amide nitrogen atoms of the α,ß-diaminopropionic acid, the serine residue as well as the amino function deriving from the ß-lysine. Above that pH value we observe a rearrangement within the coordination sphere leading to movement of Cu(2+) to the center of the peptide ring with concurrent coordination of four nitrogen donors. Spin-lattice relaxation enhancements and potentiometric measurements clearly indicate that deprotonated amide nitrogen atom from the ß-ureidodehydroalanine moiety is the fourth donor atom.

Coordination pattern, solution structure and DNA damage studies of the copper(II) complex with the unusual aminoglycoside antibiotic hygromycin B.

The aminoglycosidic antibiotic hygromycin B presents a peculiar chemical structure, characterized by two sugar rings joined via a spiro connection. The Cu(ii) complex of hygromycin B in water solution was characterized by (1)H-NMR, UV-Vis, EPR and CD spectroscopy, combined with potentiometric measurements. The spin-lattice relaxation enhancements were interpreted by the Solomon-Bloembergen-Morgan theory, allowing us to calculate copper-proton distances that were used to build a model of the complex by molecular mechanics and dynamics calculations. The fidelity of the proposed molecular model was checked by ROESY maps. Moreover DNA damage by the Cu(ii)-hygromycin B system was also investigated, showing single and double strand scissions exerted by the complex at concentrations in the range 1-5 mM. Addition of either hydrogen peroxide or ascorbic acid to each sample resulted in the shift of the cleavage potency towards lower concentrations of the complex.

DNA oxidative cleavage induced by the novel peptide derivatives of 3-(quinoxalin-6-yl)alanine in combination with Cu(II) or Fe(II) ions.

Three model dipeptides containing 3-(2,3-di(pyridin-2-yl)quinoxalin-6-yl)alanine, 3-(dipyrido[3,2-a:2,3-c]phenazin-11-yl)alanine, and 3-(2,3-diphenylquinoxalin-6-yl)alanine were studied with respect to their ability to bind selected transition metal ions, such as Cu(II), Fe(II), Ni(II), Co(II), Mn(II), and Cr(III). It was found that only Cu(II) and Fe(II) ions could form stable complex species with the studied compounds. The ability to form the complexes correlated well with DNA damage experiments. Only the ferrous and cupric complexes are capable of generating both single- and double-strand scissions. However, double-strand breakages appear to be dominating lesions in the presence of hydrogen peroxide, especially for copper(II) containing systems. The quantity of breakage products in the presence of N-(3-(dipyrido[3,2-a:2,3-c]phenazine-11-yl)alanyl)glycine complexes was the highest as compared to the complexes of the remaining compounds. Moreover, this ligand was the only one that cleaved DNA in the absence of either Cu(II) or Fe(II) ions.

Characterization of copper(II) interactions with sinefungin, a nucleoside antibiotic: combined potentiometric, spectroscopic and DFT studies.

Interactions between sinefungin and copper(II) ions were investigated. Stoichiometry and stability constants of the metal-free system and two mononuclear complexes present in solution were determined on the basis of potentiometric data analysis. The results were compared to the Cu(II)-ornithine system due to structural similarities between both molecules. Combined spectroscopic and theoretical studies allowed for determination of coordination pattern for the Cu(II)-sinefungin complexes. At acidic pH, copper is bound in "glycine-like" coordination mode, identical with that of ornithine. This involves alpha-amino group and the carboxyl oxygen. At higher pH, a "bis-complex" is formed by two sinefungin molecules. The second ligand binds in equatorial position displacing two water molecules, what results in the stable {2N,2O} coordination. Both axial positions are supposed to be occupied by N1 nitrogen donors of adenine moiety, what is confirmed by DFT calculations. They interact indirectly with copper(II) through water molecules as the result of dominant syn conformation of purine.

Inhibition of the catalytic activity of trans-acting antigenomic delta ribozyme by selected antibiotics and their Cu2+ complexes.

The effect of selected 10 antibiotics and their complexes with Cu(2+) ions on the catalytic activity of the trans-acting antigenomic delta ribozyme was investigated. Sisomicin, vancomycin, and actinomycin D displayed weak inhibitory properties. However, much stronger effects were detected with complexes of these antibiotics with Cu(2+) ions. The strongest inhibition was observed with actinomycin D-Cu(2+) complex, for which the calculated K(i) value was reduced ca. 35-fold upon metal ion complexation. We postulate that the antibiotic-Cu(2+) complexes are guided to the ribozyme metal ion binding site(s) presumably displacing the catalytically important metal ion(s). Moreover, we assume that, once positioned in appropriate distances to RNA phosphate groups and bases, the coordinated Cu(2+) ions become positively charged factors that enhance the affinity of the antibiotics to the ribozyme. These observations indicate that coordination of metal ions to antibiotics substantially changes their properties which might also have a biological relevance inside the cell.

Influence of the physiologically widespread substances on the oxidative activity of copper(II) complexes with sinefungin, a nucleoside antibiotic.

The oxidation-promoting reactivity of the Cu(II)-sinefungin complex in the presence of hydrogen peroxide was studied at pH 7.4, using N,N-dimethyl-p-nitrosoaniline (NDMA), as well as plasmid DNA as target molecules. Mixture of the complex with H(2)O(2) was found to be an efficient oxidant, bleaching NDMA solution, and generating single- and double-strand breaks in DNA. The oxidative DNA damage was investigated also in the presence of varying amounts of glutathione, histidine, Gly-Gly-His peptide, H2A histone, and ascorbic acid, showing diverse influence of those substances on the cleavage extension.

Acid-base, coordination and oxidative properties of systems containing ATP, L-histidine and Ni(II) ions.

Potentiometric measurements of ATP-His system proved an existence of five adducts in the solution with stoichiometries ranging from H(5)(ATP)(His) to H(ATP)(His)(4-). Their formation is a consequence of electrostatic interactions only. In the ternary Ni(II)-ATP-His system, two complex species NiH(ATP)(His)(2-) and Ni(ATP)(His)(3-), were found. In the former, stacking interaction between the aromatic moiety of ATP and the imidazole ring of l-histidine is crucial to the adduct stability. All studied systems are able to generate single strand lesions of plasmid DNA in the presence of hydrogen peroxide. However, only binary systems produce linear form of DNA, which is a consequence of the accumulation of the single-stranded breaks.

tRNAPhe cleavage by aminoglycosides is triggered off by formation of an abasic site.

This communication reports the characteristics of the mechanism of highly specific tRNA(Phe) cleavage, which occurs in the anticodon loop in the presence of aminoglycoside antibiotic-neomycin B. The data prove that the cleavage requires previous depurination of the polynucleotide chain at position 37, which is occupied by a hypermodified guanine base-wybutine. The results suggest that the phenomenon, previously considered as selective with respect to the presence of tRNA hypermodification, may concern far more RNA molecules, namely the ones carrying abasic sites.

Identification of copper(II) binding sites in actinomycin D, a cytostatic drug--correlation of coordination with DNA damage.

Actinomycin D (AD) is a potent anticancer drug widely applied in therapy, which however exhibits very high toxicity in humans. As the character of donors present in the AD molecule seems to be very favorable for Cu(II) ions, we undertook the coordination study on the Cu(II)-AD system. Potentiometric experiments proved a formation of very stable complexes and with the use of spectroscopic methods the identification of the binding sites was made. The values of potential energy minima, provided by theoretical modeling, confirmed the feasibility of formation of the complexes in water solution. We also demonstrated a significant effect of Cu(II) ions on AD interactions with DNA. The strand-nicking activity was observed. This process could be correlated with the speciation of complex forms. We also found out that in the presence of H2O2, low levels of Cu(II)-AD complexes induce the formation of considerable amounts of linearised plasmid. In consequence, the hypothesis is proposed that the physiologically available cupric ions may participate in the drug-induced toxic effects.

ATP, histidine or magnesium ions can protect DNA against sisomicin-induced damage, following stray Cu(II) binding.

The oxidative DNA damage by the cupric complexes of sisomicin was investigated in the presence of varying amounts of histidine, ATP, Mg(II) ions or phosphates. We found that by very low concentrations, the amino acid is able to inhibit the cleavage totally. This occurs both by its competition with antibiotic for copper(II) binding, what was proved by spectroscopic measurements, as well as by ROS scavenging by the imidazole ring. ATP and magnesium also exert an influence on the yield of the DNA destruction by decreasing the amount of the single strand breaks, however only their significant excess is able to break this process. The influence of ATP on the plasmid damage has in this case a similar chemical mechanism to that one observed for histidine. Mg(II) ions, however, interact with DNA and thus prevent the complex binding. Only phosphate anions, in the range of their physiological concentrations, exert no influence on the cleavage process.

NMR and EPR structural delineation of copper(II) complexes formed by kanamycin A in water.

The complexes formed by kanamycin A at three different pH values (5.5, 7.4 and 12.0) were investigated by NMR and EPR spectroscopy. Paramagnetic relaxation contributions to proton relaxation rates were measured using a combination of the TOCSY sequence with the inversion recovery experiment in order to gain signal resolution in the bulk region. Measured contributions were converted into distances and used for structural determination by restrained simulated annealing where all possible chair and boat conformations of the rings were taken into account. The interaction of the Cu(II) ion with the nitrogen of the C ring is apparent at all pH values. At higher pH also the amino group of ring A starts to be involved in the metal coordination sphere. This is accompanied by a switch in conformation of ring C. Structures are consistent with the involvement in the coordination sphere either of the 2' or 4' hydroxyl oxygens at pH 5.5 and the 5 and the 6' hydroxyl oxygens (or the ring oxygen) at pH 12.0.

Acid-base versus structural properties of an aminoglycoside antibiotic--sisomicin: NMR and potentiometric approach.

Aminoglycoside antibiotics constitute a class of the drugs of high interest, whose therapeutic action is based upon the electrostatic interaction with the variety of RNA molecules. The positive charge of these drugs molecules, located at their amino functions, has a prevailing influence on this process. The potentiometry and (1)H NMR spectroscopy are applied hereby to achieve the characteristics of the acid-base properties of particular protonating groups. We found that the pK values of deprotonation processes cover a wide values range 6-9.8. The correlation spectra of sisomicin, both COSY and TOCSY, allowed attributing unambiguously individual signals to the corresponding protons. These spectra involve a lot of the cross-peaks originating from the B and C rings protons, while the analogous signals originating from A rings protons are less numerous. Molecular modeling provided that the methylated amino group of A ring is located too far from the protonated functions of the remaining rings to affect their pK values. The phenomena observed herein are discussed in line of strength of the analogous processes observed for other aminoglycosides. As the result, four types of amino groups consisted within these antibiotics are distinguished.

Preferences of kanamycin A towards copper(II). Effect of the resulting complexes on immunological mediators production by human leukocytes.

The widespread presence of pathogenic bacteria is a cause of permanent demand for investigating the properties of antimicrobial agents. The chemical basis of several toxic effects induced by antibiotics still remains unclear. Aminoglycosides, highly ototoxic and nephrotoxic drugs, are capable of copper(II) ions chelating. In this study we established the affinity of kanamycin A towards copper(II), in contrast with other metal ions: iron(III), nickel(II), cobalt(II) and zinc(II) by means of potentiometry. Circular dichroism spectroscopy was applied to monitor the competition of copper(II) partition between kanamycin A and human serum albumin. We show, that the drug is able to digest Cu(II) ions from HSA to some extent and comparing the stability constants for metal and antibiotic with those, obtained for the N-terminal Asp-Ala-His-Lys (DAHK) sequence, which constitutes a copper(II) binding domain within albumin, we demonstrate that the Cu(II)-kanamycin A complex formation is possible also in blood plasma. Bioassays and immunoassay were used to find out the possibility of Cu(II)-kanamycin A complexes to induce cytokines: tumor necrosis factor (TNF), interferon (IFN) and interleukin-10 (IL-10) in human peripheral blood leukocytes. The effect on the cytokines release was dose and time dependent and the interdependence between IL-10 and TNF stimulation was found. We report that Cu(II)-aminoglycoside systems can act as moderate inducers of TNF-alpha, IFN-alpha/beta and IL-10 released from human leukocytes. We have also found that these complexes are non-toxic for human A549 cells.

Oxidative activity of copper(II) complexes with aminoglycoside antibiotics as implication to the toxicity of these drugs.

The majority of aminoglycosidic antibiotics anchor Cu(ll) ions by {NH(2), O} chelates of the A and C rings of its molecule as distinct from amikacin, which belongs to the class of substituted ones. The results indicate that all these antibiotics effectively bind copper(ll) at physiological pH. Cyclic voltammetry investigations and kinetic studies of H(2)o(2) disproportionation and hydroxyl radicals detection made it possible to support the mechanism of oxidative reactivity of cupric complexes of aminoglycosides, which involves Cu(1) and Cu(lll) redox states and metal-bound, rather than free radical species. The mechanism of this process appears to be complicated, and may have deleterious side-effects by leaking radical intermediates. The presence of these reactive oxygen species may be responsible for modulating the biological activity of these drugs.The interactions of copper(ll) complexes of aminoglycosides with oxidation-susceptible biomolecules: 2'-deoxyguanosine, plasmid DNA and yeast tRNA(phe) in both the presence and absence of hydrogen peroxide showed that the complexes with H(2)o(2) are the most efficient oxidants, converting dG to its 8-oxo derivative, generating strand breaks in plasmid DNA and multiple cleavages in tRNA(phe). Some of these reactions may play a role in aminoglycoside-induced ototoxicity and nephrotoxicity; moreover, they may suggest that Cu(ll)-aminoglycosides are potentially dangerous genotoxic agents. These complexes were also screened for their antibacterial activity and bioassays were engaged to find out the possibility of Cu(ll)-kanamycin A complexes to induce tumor necrosis factor (TNF), interferon (IFN) and interleukin-10 (IL-10) in human peripheral blood leukocytes. The aim of these studies was to compare the biological action of antibiotic alone and complexed with copper(ll) ions in both neutral and oxidative environment.

Correlations between complexation modes and redox activities of Ni(II)-GSH complexes.

The formation of Ni(II) complexes of GSH in conditions of 4-fold GSH excess over Ni(II) was studied by potentiometric titrations, UV-vis and CD spectroscopies, and magnetic susceptibility measurements. The following set of complexes was obtained in the pH range of 6-12: NiHL, Ni(2)L(2)(2)(-), NiHL(2)(3)(-), NiL(2)(4)(-), and NiH(-)(1)L(2)(5)(-). The first of these is an octahedral species, coordinated through the donors of the Glu moiety of GSH, while the remaining ones are largely square-planar, with participation of the thiol in Ni(II) coordination. Magnetic moments indicate the presence of a spin equilibrium for Ni(2)L(2)(2)(-), NiHL(2)(3)(-), and NiL(2)(4)(-) complexes. Phosphate ions apparently decompose the Ni(2)L(2)(2)(-) complex, converting it into a monomeric, high spin, ternary species. Among the molecular forms of GSH, HL(2)(-) is the one most susceptible to air oxidation, due to a presence of ionic interactions between its protonated amine and deprotonated thiol moieties. The complexation of Ni(II) accelerates air oxidation of GSH in alkaline solutions by a factor of 4, but this effect is absent at neutral pH. The damage to plasmid DNA by H(2)O(2) is facilitated by Ni(II) ions and inhibited by excess of GSH. However, the analysis of the concentration profile of this process indicates that octahedral Ni(II) complexes with GSH are involved in the formation of double strand breaks. Finally, numerical simulations of intracellular Ni(II) distribution, made possible by the determination of stability constants of Ni(II) complexes of GSH, indicate that histidine and ATP, rather than GSH, may act as ligands for Ni(II) in vivo. Altogether, our results suggest that the direct impact of GSH on Ni(II) toxicity may be of a limited character.

In vitro oxidative activity of cupric complexes of kanamycin A in comparison to in vivo bactericidal efficacy.

The interactions of copper(II) complexes of kanamycin A with oxidation-susceptible biomolecules: 2'-deoxyguanosine, plasmid DNA and yeast tRNA(Phe) were studied in both the presence and absence of hydrogen peroxide. The mixture of complex with H(2)O(2) was found to be an efficient oxidant, converting dG to its 8-oxo derivative, generating strand breaks in plasmid DNA and multiple cleavages in tRNA(Phe). Some of these reactions may play a role in toxic effects of aminoglycoside antibiotics. These complexes were screened for their antibacterial activity. The microbiological studies undertaken to compare the bactericidal action of kanamycin A alone and complexed with copper(II) ions in both neutral and oxidative environment revealed that the enhancement of bactericidal action by Cu(II) was not statistically significant.