Molecular mechanics of the interactions of spermine with DNA: DNA bending as a result of ligand binding.

We used energy minimization of a molecular mechanical force field to evaluate spermine interactions with B-form DNA oligomers with either alternating purine/pyrimidine or homopolymeric sequences. Four different positions for spermine docking--within, along, and bridging the minor groove and bridging the major groove--were assessed for each sequence. Interaction at the major groove of alternating purine/pyrimidine sequences appears to be the most favorable of all models assessed, and are associated with significant bending of DNA. Interactions at the major groove of homopolymers were less favorable than those of heteropolymers and showed little or no bending. Interactions with the minor groove were most favorable for spermine positioned near the base of the groove, and became less favorable as spermine was moved toward the top of the groove. Association along the phosphate backbone alone was the least favorable of the interactions.     

Kinetics of dissociation of nogalamycin from DNA: comparison with other anthracycline antibiotics

Stopped-flow spectrometry and simple mixing techniques have been employed to investigate the detergent-induced dissociation of anthracycline antibiotics from natural and synthetic DNAs. Both daunomycin and nogalamycin dissociate more slowly from poly(dG-dC) than from poly(dA-dT) but the difference is much more marked for nogalamycin. With an equimolar mixture of poly(dG-dC) and poly(dA-dT), or with poly(dA-dC).poly(dG-dT), dissociation of nogalamycin occurs very slowly. In all cases the release of antibiotic from a synthetic polynucleotide is a one-step process following a single exponential. Dissociation of daunomycin, adriamycin and iremycin from calf thymus DNA is a more complex reaction which requires a two-exponential fit, in contrast to earlier reports, but differences between the behaviour of the three antibiotics are minor. Dissociation of nogalamycin from natural DNA requires a three-exponential fit, is in general far slower, and depends upon the base composition, the level of binding and the time allowed for the complex to equilibrate. It is concluded that sequence selectivity is minimal or lacking for daunomycin, whereas nogalamycin binding is sequence dependent and probably involves migration of the antibiotic between DNA binding sites. There is an inverse correlation between dissociation rate constants and antibacterial potency in simple tests.     

Kinetics of dissociation of nogalamycin from DNA: comparison with other anthracycline antibiotics

Stopped-flow spectrometry and simple mixing techniques have been employed to investigate the detergent-induced dissociation of anthracycline antibiotics from natural and synthetic DNAs. Both daunomycin and nogalamycin dissociate more slowly poly(dG-dC) than from poly(dA-dT), but the difference is much more marked for nogalamycin. With an equimolar mixture of poly(dG-dC) and poly(dA-dT), or with poly(dA-dC)·poly(dG-dT), dissociation of nogalamycin occurs very slowly. In all cases the release of antibiotic from a synthetic polynucleotide is a one-step process following a sinigle exponential. Dissociation of daunomycin, adrianmycin and iremycin from calf thymus DNA is a more complex reaction which requires a two-exponential fit, in contrast to earlier reports, but differences between the behaviour of the three antibotics are minor. Dissociation of nogalamycin from natural DNA requires a three-exponential fit, is in general far slower, and depends upon the base composition, the level of binding and the time allowed for the complex to equilibrate. It is concluded that sequence selectivity is minimal or lacking for daunomycin, whereas nogalamycin binding is sequence dependent and probably involves migration of the antibiotic between DNA binding sites. There is an inverse correlation between dissociation rate constants and antibacterial potency in simple tests.     

The interaction of spermine and pentamines with DNA.

We studied the effects of spermine, two naturally-occurring pentamines isolated from the thermophile Thermus thermophilus and one synthetic pentamine on the aggregation and 'melting' temperature of calf-thymus DNA and on the B-to-Z transition of poly(dG-me5dC). All pentamines caused aggregation of DNA at much lower concentrations than that of spermine. Concentrations that increased the melting temperature of DNA and induced the B-to-Z transition in poly(dG-me5dC) were different for each pentamine, but were comparable with the concentration of spermine needed to cause these effects. Our results suggest that both the total charge and the distance separating the charge, which is a function of the length of the carbon chains between amino groups, are important for the induction of conformational changes in DNA. The biological role of pentamines in T. thermophilus appears to be related to their ability to promote DNA condensation at high temperature.     

Ternary copper(II) complexes of levofloxacin and phenanthroline derivatives: In-vitro antibacterial,DNA interactions,and SOD-like activity

A series of ternary copper(II) complexes have been derived using levofloxacin and five phenanthroline derivatives. Complexes were characterized using infrared spectroscopy, Thermogravimetric (TG)-analysis, fast atom bombardment mass spectroscopy and reflectance spectra. Synthesized complexes exhibit the only d-d band at ～ 666?nm points toward a distorted square pyramidal geometry at metal centre with one unpaired electron responsible for paramagnetic behaviour of whole moiety. Binding behaviour of the complexes toward Herring Sperm DNA were determined using ultraviolet-Vis (UV-Vis) absorption titration and viscometric titration experiment, where as the cleavage efficacy of the complexes toward pUC19 DNA was determined by electrophoresis in presence of ethidium bromide. Complexes exhibit superoxide dismutase–like activity with their IC₅₀ values ranging from 0.7917 to 1.7432 µM.     

Ternary copper(II) complexes of levofloxacin and phenanthroline derivatives: in-vitro antibacterial, DNA interactions, and SOD-like activity

A series of ternary copper(II) complexes have been derived using levofloxacin and five phenanthroline derivatives. Complexes were characterized using infrared spectroscopy, Thermogravimetric (TG)-analysis, fast atom bombardment mass spectroscopy and reflectance spectra. Synthesized complexes exhibit the only d-d band at ～ 666?nm points toward a distorted square pyramidal geometry at metal centre with one unpaired electron responsible for paramagnetic behaviour of whole moiety. Binding behaviour of the complexes toward Herring Sperm DNA were determined using ultraviolet-Vis (UV-Vis) absorption titration and viscometric titration experiment, where as the cleavage efficacy of the complexes toward pUC19 DNA was determined by electrophoresis in presence of ethidium bromide. Complexes exhibit superoxide dismutase-like activity with their IC(50) values ranging from 0.7917 to 1.7432 μM.     

A scanning calorimetric study of natural DNA and antitumoral anthracycline antibiotic-DNA complexes

Thermal denaturation of natural DNA in the absence and presence of antitumor anthracycline antibiotics has been studied by adiabatic differential scanning calorimetry. The helix-coil transition is operationally irreversible as measured by DSC. Both the melting temperature and the overall molar transition enthalpy of the DNA samples was dependent on the percentage of GC base pairs. Calorimetric traces of anthracycline-DNA complexes have qualitatively similar features and the significance of this characteristic is discussed. The unsaturated drug-DNA complex melts through complex thermal transitions with one broad endotherm in the same temperature region as free DNA and the other at a higher temperature which is rf (mol ligand per mol DNA in base pairs) value dependent. Antibiotic binding at concentrations close to saturating conditions (rf = 0.2) reverts the melting range to a value near to its original one and increases the thermal stability of the duplex structure by around 30 degrees C. In addition, the calorimetric enthalpy is increased by between 64% and 150%, depending on which ligand was used.     

Noncovalent interactions in ternary complexes of spermine and copper(II) with adenosine 5'-triphosphate and tripolyphosphate

Thermodynamic characteristics pertinent to the formation equilibria of two ternary systems: 1) Copper(II), 4,9-diazadodecane-1,12-diamine (spermine, Spe), and adenosine 5'-triphosphate (ATP) and 2) Copper(II), Spe, and tripolyphosphate (TPP) have been determined by means of potentiometric and calorimetric techniques, together with the parent binary complex characteristics. Ternary complexes involving ATP can give information useful in the interpretation of bioenergetic reactions and of biological interactions between nucleic acids and polyamines. As a model system, the TPP-containing ternary complexes have been studied, together with the parent binary complexes. The thermodynamic study of these systems is very important because it can give information about the structural environment of the complexes; moreover, it can help in outlining different noncovalent interactions such as coulombic forces and hydrogen bonds.     

Location of spermine and other polyamines on DNA as revealed by photoaffinity cleavage with polyaminobenzenediazonium salts

Although polyamines interact strongly with nucleic acids, X-ray and NMR studies have not revealed much structural information about spermine-DNA complexes. Therefore, it was of interest to look at the binding of polyamines to 32P-labeled DNA restriction fragments by sequencing gel electrophoresis of the photoaffinity cleavage products induced by polyaminobenzendiazonium salts. The shift of cleavage patterns observed on opposite strands as well as competition experiments with distamycin shows polyamines to be located in the minor groove of B-DNA and to depend on the nucleic acid polymorphism, jumping to the major groove in the A-form. The sequence selectivities of various polycations (spermine, putrescine, and cobalt (III) hexaammine) are similar and slightly favor A,T-rich regions. Taken together, these results show that polycations which are not point charges are guided by the electronegative potential along the nucleic acid and suggest fast crawling of the polyamine within the minor groove, due to individual NH2+ jumping between multiple equidistant and isoenergetic bidentate hydrogen-bonding sites. Such a picture could be the clue to the unexpected NMR and to the frequently silent X-ray behavior of polyamines when bound to DNA.     

Protein-protein interactions of hCsl4p with other human exosome subunits.

The exosome is a complex of 3'-->5' exoribonucleases, which functions in a variety of cellular processes, all requiring the processing or degradation of RNA. We demonstrate that the two human proteins hCsl4p and hRrp42p, which have been identified on the basis of their sequence homology with Saccharomyces cerevisiae proteins, are associated with the human exosome. By mammalian two-hybrid and GST pull-down assays, we show that the hCsl4p protein interacts directly with two other exosome proteins, hRrp42p and hRrp46p. Mutants of hCsl4p that fail to interact with either hRrp42p or hRrp46p are also not able to associate with exosome complexes in vivo. These results indicate that the association of hCsl4p with the exosome is mediated by protein-protein interactions with hRrp42p and hRrp46p.     

Interaction of spermine and DNA

The effect of spermine upon the denaturation temperature (T_m) of DNA's of various base compositions has been found to depend upon both the base composition of the DNA and the pH of the solution. Measurement of the hydrogen ion titration curve of spermine as a function of temperature reveals that the net charge of the spermine molecule is undergoing a rapid change with temperature in the range of temperatures at which DNA denatures. Since the value of T_m depends upon base composition, the correlation of the effect of spermine upon T_m with the base composition of the DNA used may be explainable in terms of the changing degree of ionization of spermine. The binding of spermine to native DNA has also been studied by dialysis equilibrium. There is no significant variation either in the number of strongly binding sites or strength of binding with base composition. It is concluded that there is no evidence of correlation between the binding of spermine and the base composition of DNA.     

Nimodipine's interactions with other drugs: I. Ethanol

Adult mice (Binghamton Heterogeneous stock) received different doses of ethanol (0.5, 1.0, or 2.0 g/kg) administered alone or in combination with the voltage-sensitive calcium channel antagonist, nimodipine (Bay e 9736). Both 20 and 60 minutes later, sensitivity to ethanol was assessed in terms of rotorod activity and changes in rectal temperatures. Nimodipine (5 mg/kg) alone did not alter rectal temperature or motor coordination, but at both observation periods nimodipine potentiated the hypothermia induced by the highest dose of alcohol (2.0 g/kg) and exaggerated alcohol-induced motor incoordination at all doses. The present set of results indicates that the inhibition of voltage-dependent calcium channels can exaggerate ethanol-induced effects.     

Nimodipine's interactions with other drugs: II. Diazepam

Adult Binghamton Heterogeneous (HET) stock mice were administered one of three doses of diazepam (0.1, 2.5, or 5.0 mg/kg) immediately followed by a second injection of either the slow calcium channel blocker, nimodipine (Bay e 9736), or its vehicle. Hypothermic responses and muscular incoordination were measured twenty and sixty minutes later as assessed by changes in rectal temperature and motoric activity on a rotating rod. Nimodipine (5 mg/kg) alone did not significantly affect body temperature or motor coordination. However, when administered in combination with the two highest doses of diazepam, nimodipine significantly potentiated the hypothermic response produced by these doses both twenty minutes and sixty minutes post-injection. Administration of high doses of diazepam (2.5 and 5.0 mg/kg) resulted in significant motor incoordination at both observation periods, but this effect was not potentiated by nimodipine.     

Molecular structure, stereochemistry and interactions of steffimycin B, and DNA binding anthracycline antibiotic

The crystal and molecular structure of anthracycline antibiotic steffimycin B(C29H32O13) has been determined by X-ray diffraction and the stereochemistry revealed. The orthorhombic crystals belong to space group P2(1)2(1)2(1), with the dimensions; a = 8.253 (2), b = 8.198 (2), c = 40.850 (8) A and Z = 4. Intensity data were collected for 2518 independent reflections. The structure was solved by direct methods and refined to an R value of 0.066 for 1410 reflections. The configuration in ring A is 7R,8S,9S. Ring A adopts half chair conformation, while the sugar ring has the regular chair conformation. The molecule most probably binds to double helical DNA through intercalation and hydrogen bonding.     

Photoaffinity polyamines: sequence-specific interactions with DNA.

ANB-spermine is a photoaffinity analog of the naturally-occurring polyamine, acetylspermine. ANB-spermine was used to determine its binding sites on naked double stranded DNA, at the nucleotide level, using a modification of the primer extension technique. A total of 1,275 nucleotides was examined in 5 sequences of DNA from Saccharomyces cerevisiae. Binding sites were non-random. The primary determinant of binding was the presence of a thymidine residue. Secondary determinants appeared to depend on the secondary structure of the DNA, with runs of thymidines providing unusually poor binding sites while TA and, especially, TATA providing the strongest binding sites. The 'TATA element' upstream of the URA3 gene from S. cerevisiae was the strongest binding site. The data indicate that ANB-spermine binding to DNA is a probe for DNA secondary structure and suggest a role for polyamines in regulating the structure of chromatin in vivo.     

Interaction of spermine with different forms of DNA. A conformational study

The energy of interaction of a spermine molecule with the A - and B -forms of DNA has been calculated, assuming that the molecule of spermine is fixed in the narrow groove of the DNA helix with the formation of hydrogen bonds between the amino groups of spermine and the phosphate groups of DNA. The atom–atom potentials method was used. Optimal structures for the A-DNA–spermine and B-DNA–spermine complexes are suggested. It is shown that, in agreement with the experimental data, the interaction of the spermine molecule with the A -DNA is energetically more favorable than that with the B -DNA. Two main factors are responsible for this: (1) the distance between neighboring phosphates of the chain in A -DNA (which is about 1 Å less than that in B -DNA) corresponds better to the distance between the amino groups of the propyl part of spermine; and (2) the orientation of phosphate groups in A -DNA inside the groove is preferable for complex formation with spermine to the outside groove arrangement of the phosphates in B -DNA. These conclusions are further confirmed by the calculations for DNA–propane diamine complexes.     

Adriamycin interactions with T4 DNA polymerase. Two modes of template-mediated inhibition.

We examined the effect of adriamycin on kinetics of DNA synthesis catalyzed by DNA polymerase purified from bacteriophage T4-infected Escherichia coli. Two distinct modes of enzyme inhibition occur: uncompetitive and competitive at "low" and "high" drug:DNA nucleotide molar ratios, respectively. Competitive inhibition is not observed unless an unblocked amino group is present on the sugar (daunosamine) moiety. A model is proposed to relate the enzyme inhibition kinetics to intercalative and ionic binding of adriamycin to DNA.     

Selectivity of spermine homologs on triplex DNA stabilization.

We synthesized seven homologs of spermine (H2N(CH2)3NH(CH2)nNH(CH2)3NH2, where n = 2-9; n = 4 for spermine) and studied their effects on melting temperature (Tm), conformation, and precipitation of poly(dA).2poly(dT). The triplex DNA melting temperature, Tm1 was 34.4 degrees C in the presence of 150 mM KCl. Addition of spermine homologs increased Tm1 in a concentration-dependent and structure-dependent manner, with 3-6-3 (n = 6) exerting optimal stabilization. The dTm1/dlog[polyamine] values were 9-24 for these compounds. The duplex melting temperature, Tm2 was insensitive to homolog concentration and structure, suggesting their ability to stabilize triplex DNA without altering the stability of the underlying duplex. Circular dichroism spectral studies revealed psi-DNA formation in a concentration-dependent and structure-dependent manner. Phase diagrams were constructed showing the critical ionic/polyamine concentrations stabilizing different structures. These compounds also exerted structural specificity effects on precipitating triplex DNA. These data provide new insights into the ionic/structural determinants affecting triplex DNA stability and indicate that 3-6-3 is an excellent ligand to stabilize poly(dA).2poly(dT) triplex DNA under physiologic ionic conditions for antigene therapeutics.