Dependence of Mode of DNA Binding to Benzocrown Derivatives of Actinocin on the Size and Distance from the Chromophore of Crown Fragments

Complexes of DNA with benzocrown derivatives of actinocin were studied by viscometry and dynamic birefringence. Changes in the macromolecular structure of DNA caused by complex formation were determined. Models of DNA binding to the studied compounds were suggested on the basis of data obtained. The intercalation of actinocin chromophore of benzocrown derivatives of actinocin was shown to occur only when benzocrown groups were bound to the chromophore via the glycine spacer. A change of the distance between the crown group and the chromophore prevented ligand intercalation. Increase of the ionic strength resulted in appearance of a new, nonintercalative binding mode for crown-containing compounds determined by interaction of the crown groups with DNA.     

Dependence of mode of DNA binding to benzo crown derivatives of actinocin on the size and distance from the chromophore of crown fragments

Complexes of DNA with benzocrown derivatives of actinocin were studied by viscometry and dynamic birefringence. Changes in the macromolecular structure of DNA caused by complex formation were determined. Models of DNA binding to the studied compounds were suggested on the basis of data obtained. The intercalation of actinocin chromophore of benzocrown derivatives of actinocin was shown to occur only when benzocrown groups were bound to the chromophore via glycine fragment. A change in the distance between the crown group and the chromophore prevents ligand intercalation. Increase in medium ionic strength results in appearance of new nonintercalational binding mode for crown-containing compounds with DNA caused by interaction of the crown groups with DNA.     

Interaction of DNA with benzocrown derivatives of actinocin

Complexes of DNA with actinocin derivatives containing benzocrown groups at the 1 and/or 9 positions of the chromophore were studied by spectrophotometric titration and circular dichroism. The actinocin chromophore and the crown fragments are the binding sites of the ligands with DNA. The mode of ligand-DNA binding is shown to depend on the size of the crown group, its distance to the actinocin chromophore, and the ionic strength of the medium. Selective toward Na+ ion benzocrown fragments combine with DNA phosphate groups. The simultaneous interaction of the actinocin chromophore with the DNA bases is possible only at optimal distance between both the binding sites of ligand molecule.     

Interactions of DNA with ampholytes based on actinocin

The interaction of DNA with actinocin monoamides containing a substitute with the cationoide center in one of the amide groups were studied by spectrophotometry, induced circular dichroism, viscometry and flow birefringence methods. At pH values of solution exceeding their isoelectric point, these substances, which are in nature ampholytes, occur as zwitterions. At lover pH values they occur in the cationoid form. The constants of binding of these compounds to DNA were determined, and changes in DNA macromolecular structure caused by complexation were revealed. Models for the binding of these compounds to DNA were advanced. It was shown that compounds in the zwitterion form do not intercalate into the DNA double helix. The mode of binding of compounds to DNA in the cationoid form depends on the position of the cationoid center within the chromophore actinocin. Circular dichroism spectra of actinocyn-based ampholytes that bind to DNA in a different manner were obtained.     

Interaction of DNA with Benzocrown Derivatives of Actinocin

Complexes of DNA with actinocin derivatives containing benzocrown groups at positions 1 and/or 9 of the chromophore were studied by spectrophotometric titration and circular dichroism. The actinocin chromophore and the crown fragments are the DNA-binding sites of the ligands. The mode of ligand–DNA binding is shown to depend on the size of the crown group, its distance to the actinocin chromophore, and the ionic strength of the medium. Na⁺-selective benzocrown fragments combine with DNA phosphate groups. The simultaneous interaction of the actinocin chromophore with the DNA bases is possible only at an optimal distance between the two binding sites of ligand molecule.     

Non stacking binding of 7-amino-actinomycin D and actinomycin D to DNA and model nucleotide systems in solutions

Mechanism of actinomycin D (AMD) and 7-aminoactinomycin D (7AAMD) interaction with DNA and model nucleotide compounds was studied by absorption and fluorescence spectroscopy (steady-state, phase-modulation, and polarization). It was shown that complex formation does not result in energy transfer from photoexcited nucleotides to phenoxazone chromophore of 7AAMD that indicates the absence of stacking-like intercalation. This fact is fundamentally important to explain the biological effect of actinomycin on cells. It was revealed a fundamental difference in the complex-forming properties of AMD and 7AAMD. Thus AMD is capable of binding to guanine micelles to destroy them. 7AAMD forms complexes neither guanine micelles nor polyguanilic acid. 7AAMD binding sites on DNA can differ substantially from AMD binding sites. However, a strong competition is observed between AMD and 7AAMD for binding site in oligonucleotide HP1 used as DNA hairpin model. The efficient diameters of 7AAMD-HP1 complex and free 7AAMD were determined using the Levshin-Perren equation.     

Single-strand DNA binding of actinomycin D with a chromophore 2-amino to 2-hydroxyl substitution

A modified actinomycin D was prepared with a hydroxyl group that replaced the amino group at the chromophore 2-position, a substitution known to strongly reduce affinity for double-stranded DNA. Interactions of the modified drug on single-stranded DNAs of the defined sequence were investigated. Competition assays showed that 2-hydroxyactinomycin D has low affinity for two oligonucleotides that have high affinities (K(a) = 5-10 x 10(6) M(-1) oligomer) for 7-aminoactinomycin D and actinomycin D. Primer extension inhibition assays performed on several single-stranded DNA templates totaling around 1000 nt in length detected a single high affinity site for 2-hydroxyactinomycin D, while many high affinity binding sites of unmodified actinomycin D were found on the same templates. The sequence selectivity of 2-hydroxyactinomycin D binding is unusually high and approximates the selectivity of restriction endonucleases. Binding appears to require a complex structure, including residues well removed from the polymerase pause site.     

Interaction of DNA with actinocin amides containing cationoid centers in the amide groups

Complexes of DNA with actinocin derivatives containing ω-dialkylaminoalkyl groups in the1 and/or 9 positions of the chromophore were studied by spectrophotometric titration, circular dichroism, and viscometry. Induced circular dichroism (ICD) spectra of the DNA-ligand complexes were compared for the cases of the complexes of known structure established by other methods. It was shown that the presence of an isoelliptic point in the long-wavelength absorption band of the ICD spectra of the ligand under monomeric binding conditions could indicate intercalation of the actinocin chromophore into DNA. The separation of the cationoid center and the chromosphore upon elongation of the methylene chain increases the aggregability of the ligand pn the surface of the DNA double helix, which prevents the intercalation of the chromophore.     

Hydration effects accompanying the formation of DNA complexes with some ligands

In the range of millimeter wavelengths the dielectric properties of aqueous solutions of some biologically active ligands (potential anticarcinogen chlorophyllin; pharmacological drug caffeine; polyamine putrescine; mutagens proflavine and ethidium bromide; actinocin derivative, an analogue of antitumor antibiotic actinomycin D) and DNA complexes with these substances were studied. It was shown that complex formation is accompanied by the change in dielectric properties of the solution. These changes during interaction of DNA with the first three compounds correspond to a decrease in hydration (compared with the total hydration of free components), and in other cases they cause an increase in hydration. The number of water molecules bound with both the ligand and DNA nucleotide in the complex was estimated. The results were compared with existing models of DNA interaction with the studied substances.     

Nonstacking Binding of 7-Aminoactinomycin D and Actinomycin D to DNA and Model Nucleotide Systems in Solutions

The mechanism of actinomycin D (AMD) and 7-aminoactinomycin D (7AAMD) interaction with DNA and model nucleotide compounds was studied by absorption and fluorescence spectroscopy (steady-state, phase-modulation, and polarization). It was shown that complex formation does not result in energy transfer from photoexcited nucleotides to the phenoxazone chromophore of 7AAMD, which indicates the absence of stacking-like intercalation. This fact is fundamentally important to explain the biological effect of actinomycin on cells. A basic difference was revealed in the complex-forming properties of AMD and 7AAMD. Thus AMD is capable of binding to guanine micelles to destroy them; 7AAMD forms no complexes with either guanine micelles or polyguanylic acid. 7AAMD binding sites on DNA can differ substantially from AMD binding sites. However, strong competition is observed between AMD and 7AAMD for the binding site in oligonucleotide HP1 used as a DNA hairpin model. The effective diameters of 7AAMD–HP1 complex and free 7AAMD were determined using the Levshin–Perren equation.     

The interaction of 7-substituted actinomycin D analogs with DNA

The interaction of actinomycin D and three new 7-substituted analogs with calf thymus DNA has been studied by a number of physical techniques. The methods utilized in this investigation include visible absorption spectrometry and ultrafiltration methodology for the determination of equilibrium binding constants; viscometry; and circular dichroism. The studies show that the 7-substituted actinomycin D analogs retain the G . C base pair specific DNA binding demonstrated by actinomycin D. The mode of binding to native DNA, despite substitution at position 7, is practically unaltered. The retention of this binding specificity by these analogs seems to be unaffected by changes in the electon properties of the chromophore.     

Phenoxazinone biosynthesis: accumulation of a precursor, 4-methyl-3-hydroxyanthranilic acid, by mutants of Streptomyces parvulus.

Mutants of Streptomyces parvulus that are blocked in the synthesis of the phenoxazinone-containing antibiotic, actinomycin, were isolated by the 'agar piece' method (after ultraviolet irradiation or treatment with 8-methoxypsoralen plus near-ultraviolet light). Radiolabelling experiments in conjunction with paper, thin-layer and column chromatography revealed that 4-methyl-3-hydroxyanthranilic acid (MHA) is a major metabolite accumulated by these mutants. Studies in vitro and in vivo provided evidence that MHA is a precursor of the phenoxazinone chromophore, actinocin. Normally MHA does not accumulate during growth or antibiotic synthesis by the parental strains. Protoplasts derived from the mutant strain AM5 synthesized MHA in significant amounts. A scheme is proposed for the biosynthesis of actinomycin D that accounts for the accumulation of MHA by the mutants.     

Flexible docking of DNA fragments and actinocin derivatives

We have applied molecular docking methods to systems containing nucleic acids as targets and biologically active substances as ligands. The complexes of DNA fragments and actinocin derivatives with different lengths of aminoalkyl side chains were obtained by molecular docking. It was observed that actinocin derivatives could form energetically favourable complexes with DNA both as intercalators and minor groove binders. It was shown that small changes in the binding energy (～1?kcal/mol) could result in complexes with substantially different structure. The complexes of actinocin derivatives and DNA fragments were stabilized by hydrogen bonding upon intercalation and minor groove binding. It was found that the change of solvent-accessible surface area upon binding of the actinocin derivative to DNA linear increased with the growth of methylene groups' number in ligand side chains. The solvation energy change upon binding of actinocin derivatives to DNA calculated by the WSAS method was favourable in the case of small uncharged ligands and unfavourable for positively charged ligands.     

Stereochemistry of actinomycin binding to DNA : II. Detailed molecular model of actinomycin-DNA complex and its implications

The three-dimensional structure of a crystalline complex containing actinomycin D and deoxyguanosine (described in the previous paper) has shed light on the stereochemistry of actinomycin binding to DNA. The phenoxazone ring system on actinomycin intercalates between the base-paired dinucleotide sequence, GpC, while the peptide subunits lie in the narrow groove of the DNA helix and interact with deoxyguanosine residues on opposite chains through specific hydrogen bonds. The binding of actinomycin to DNA demonstrates a general principle which several classes of proteins may utilize in recognizing symmetrically arranged nucleotide sequences on the DNA helix.     

Formation of DNA Complexes with Actinomycins in Aqueous Solutions and Films

The mechanisms of DNA interaction with actinomycin D (AMD), 7-amino-actinomycin D (7-AAMD), and ethidium bromide (EtBr) were studied in aqueous solutions and in the condensed state (films coating plates). The use of the methods of absorption (UV, IR, and visible spectral ranges) and fluorescence (steady-state, polarization, and phase-modulation) spectroscopy revealed that (1) the formation of DNA complexes with 7-AAMD in solution was not accompanied by energy transfer from photoexcited nucleotides to phenoxazone chromophore and (2) the mechanism of ligand incorporation was distinct from stacking. In the film of the DNA–7-AAMD complex, which simulated the native state in a biological cell, the energy transfer efficiency was high. This indicates that a stacking-type mechanism underlies actinomycin intercalation into DNA. In the presence of high concentrations of 7-AAMD in the film, DNA denatured and its double-helical structure degraded. In the DNA–AMD complex, the native B-form of DNA molecule was conserved both in films and in solution.     

Formation of DNA complexes with actinomycins in aqueous solutions and films

The mechanisms of DNA interaction with actinomycin D (AMD), 7-amino-actinomycin D (7-AAMD), and ethidium bromide (EtBr) were studied in aqueous solutions and in condensed state (films coating plates). The use of the methods of absorption (UV, IR, and visible spectral ranges) and fluorescence (steady-state, polarization, and phase-modulation) spectroscopy revealed that (1) the formation of DNA complexes with 7-AAMD in solution was not accompanied by energy transfer from photoexcited nucleotides to phenoxazone chromophore and (2) the mechanism of ligand incorporation was distinct from stacking. In the film of the DNA-7-AAMD complex, which simulated the native state in a biological cell, the efficiency of the energy transfer was high. This indicates that a stacking-type mechanism underlies actinomycin intercalation into DNA. In the presence of high concentrations of 7-AAMD in the film, DNA denatured and its double-helical structure, degraded. In the DNA-AMD complex, the native B-form of DNA molecule was conserved both in films and in solution.     

Solution structure of the ActD-5'-CCGTT3GTGG-3' complex: drug interaction with tandem G.T mismatches and hairpin loop backbone

Binding of actinomycin D (ActD) to the seemingly single-stranded DNA (ssDNA) oligomer 5'-CCGTT3 GTGG-3' has been studied in solution using high-resolution nuclear magnetic resonance (NMR) techniques. A strong binding constant (8 x 10(6) M(-1)) and high quality NMR spectra have allowed us to determine the initial DNA structure using distance geometry as well as the final ActD-5'-CCGTT3 GTGG-3' complex structure using constrained molecular dynamics calculations. The DNA oligomer 5'-CCGTT3GTGG-3' in the complex forms a hairpin structure with tandem G.T mismatches at the stem region next to a loop of three stacked thymine bases pointing toward the major groove. Bipartite T2O-GH1 and T2O-G2NH2 hydrogen bonds were detected for the G.T mismatches that further stabilize this unusual DNA hairpin. The phenoxazone chromophore of ActD intercalates nicely between the tandem G.T mismatches in essentially one major orientation. Additional hydrophobic interactions between the ActD quinoid amino acid residues with the loop T5-T6-T7 backbone protons were also observed. The hydrophobic G-phenoxazone-G interaction in the ActD-5'-CCGTT3GTGG-3' complex is more robust than that of the classical ActD- 5'-CCGCT3GCGG-3' complex, consistent with the roughly 2-fold stronger binding of ActD to the 5'-CCGTT3GTGG-3' sequence than to its 5'-CCG CT3GCGG-3' counterpart. Stabilization by ActD of a hairpin containing non-canonical stem base pairs further strengthens the notion that ActD or other related compounds may serve as a sequence- specific ssDNA-binding agent that inhibits human immunodeficiency virus (HIV) and other retroviruses replicating through ssDNA intermediates.     

Study of effect of water on the interaction of DNA and actinocin derivatives with various aminoalkyl chain length by infrared spectrophotometry and computer modeling

A comparative study of the effect of water on the interaction of DNA with actinocin derivatives having different numbers of methylene groups in side chains was performed by IR spectroscopy. It was found that, as relative humidity increases, water molecules simultaneously bind to hydrate-active sites of DNA and ligands. The absorption band at v = 1137 cm-1, caused by oscillations of the C-O and P-O groups of atoms in the DNA-ligand complex having two methylene groups, is due to the interactions between the cationic groups of the ligand and the sugar-phosphate backbone of DNA, which may be one of the reasons for the high stability of this complex. Using computer simulation of interaction of DNA fragments and actinocin derivatives in water environment, molecular models of the formation of their complexes for two ways of binding were constructed.     

Stereochemistry of Actinomycin–DNA Binding

The three dimensional structure of a crystalline complex containing actino-mycin D and deoxyguanosine has shed light on the stereochemistry of actino-mycin binding to DNA. The phenoxa-zone ring system of actinomycin intercalates into the DNA helix, while deoxyguanosine residues interact with both cyclic peptides through specific hydrogen bonds.