Bleomycin interactions with DNA studies on the role of the C-terminal cationic group of bleomycin A2 in association with and degradation of DNA

The role of the cationic dimethylsulfonium group of Abeomycin A₂ in the binding of the drug to poly(dA-dT) has been investigated b proton NMR studies on the S-demethyated derivative. In contrast to the parent drug, the demethyl congener shows no intercalation of the aromatic bithiazole group which is adjacent to the former cationic group. However, chemical studies show that the demethyl derivative retains the capability to degrade DNA in the presence of iron(II), albeit at a reduced rate and to a lesser extent than the intact bleomycin A₂. Thus, the cationic group is necessary for the intercalation of the bithiazole portion of the drug molecule; however, intercalation is not essential for the degradation of DNA.     

Interaction of bleomycin A2 with poly(deoxyadenylylthymidylic acid). A proton nuclear magnetic resonance study of the influence of temperature, pH, and ionic strength

The binding of bleomycin A2 to poly(deoxyadenylylthymidylic acid) [poly(dA-dT)] has been monitored by proton nuclear magnetic resonance spectroscopy. This study includes an analysis of the effects of temperature, ionic strength, and pH. Sites of drug-nucleic acid interaction have been delineated on the basis of chemical shift perturbations of drug and nucleic acid resonances. The data indicate that the binding of the antibiotic occurs with partial intercalation of the aromatic bithiazole group and immobilization of the cationic dimethylsulfonium group. This complex dissociates as the nucleic acid is denatured to the single-stranded form. The absence of significant pH effects suggests that the N terminus of bleomycin A2, which contains the titratable groups, does not contribute to the interaction of the drug molecule with poly(dA-dT). The problems associated with assigning a specific geometry to the drug-nucleic acid complex are discussed.     

The interaction of ellipticine derivatives with nucleic acids studied by optical and 1H-nmr spectroscopy: Effect of size of the heterocyclic ring system

The DNA interaction of derivatives of ellipticine with heterocyclic ring systems with three, four, or five rings and a dimethylaminoethyl side chain was studied. Optical spectroscopy of drug complexes with calf thymus DNA, poly [(dA-dT) · (dA-dT)], or poly [(dG-dC) · (dG-dC)] showed a 10 nm bathochromic shift of the light absorption bands of the pentacyclic and tetracyclic compounds upon binding to the nucleic acids, which indicates binding by intercalation. For the tricyclic compound a smaller shift of 1–3 nm was observed upon binding to the nucleic acids. Flow linear dichroism studies show that the geometry of all complexes is consistent with intercalation of the ring system, except for the DNA and poly [(dG-dC) · (dG-dC)] complexes of the tricyclic compound, where the average angle between the drug molecular plane and the DNA helix axis was found to be 65°. One-dimensional ¹H-nmr spectroscopy was used to study complexes between d(CGCGATCGCG)₂ and the tricyclic and pentacyclic compounds. The results on the pentacyclic compound show nonselective broadening due to intermediate chemical exchange of most oligonucleotide resonances upon drug binding. The imino proton resonances are in slow chemical exchange, and new resonances with upfield shifts approaching 1 ppm appear upon drug binding, which supports intercalative binding of the pentacyclic compound. The results on the tricyclic compound show more rapid binding kinetics and very selective broadening of resonances. The data suggest that the tricyclic compound is in an equilibrium between intercalation and minor groove binding, with a preference to bind close to the AT base pairs with the side chain residing in the minor groove. © 1994 John Wiley & Sons, Inc.     

Novel DNA photocleaving agents with high DNA sequence specificity related to the antibiotic bleomycin A2.

We have designed and synthesized a series of novel DNA photocleaving agents which break DNA with high sequence specificity. These compounds contain the non-diffusible photoactive p-nitrobenzoyl group covalently linked via a dimethylene (or tetramethylene) spacer to thiazole analogues of the DNA binding portion of the antibiotic bleomycin A2. By using a variety of 5' or 3' 32P-end labeled restriction fragments from plasmid pBR322 as substrate, we have shown that photoactive bithiazole compounds bind DNA at the consensus sequence 5'-AAAT-3' and induce DNA cleavage 3' of the site. Analysis of cleavage sites on the complementary DNA strand and inhibition of DNA breakage by distamycin A indicates these bithiazole derivatives bind and attack the minor groove of DNA. A photoactive unithiazole compound was less specific inducing DNA breakage at the degenerate site 5'-(A/T)(AA/TT)TPu(A/T)-3'. DNA sequence recognition of these derivatives appears to be determined by the thiazole moiety rather than the p-nitrobenzoyl group: use of a tetramethylene group in place of a dimethylene spacer shifted the position of DNA breakage by one base pair. Moreover, much less specific DNA photocleavage was observed for a compound in which p-nitrobenzoyl was linked to the intercalator acridine via a sequence-neutral hexamethylene spacer. The 5'-AAAT-3' specificity of photoactive bithiazole derivatives contrasts with that of bleomycin A2 which cleaves DNA most frequently at 5'-GPy-3' sequences. These results suggest that the cleavage specificity exhibited by bleomycin is not simply determined by its bithiazole/sulphonium terminus, and the contributions from other features, e.g. its metal-chelating domain, must be considered. The novel thiazole-based DNA cleavage agents described here should prove useful as reagents for probing DNA structure and for elucidating the molecular basis of DNA recognition by bleomycin and other ligands.     

Interaction of ellipticine and an indolo[2,3b]-quinoxaline derivative with DNA and synthetic polynucleotides

The non-covalent DNA interaction of the anticancer drug ellipticine (Scheme I, 1a) as well as an indolo[2,3-b]-quinoxaline derivative (Scheme I, 3b) with a dimethylaminoethyl side chain has been studied by light absorption, linear dichroism (LD) and fluorescence. Compound 3b (Scheme I) has antitumorigenic as well as antiviral activity. Both compounds bind to DNA or synthetic polynucleotides such as poly(dA-dT).(dA-dT) and poly(dG-dC).(dG-dC) by intercalation. In contrast to ellipticine, compound 3b (Scheme I) exhibits a significant binding specificity for alternating AT sequences. Its fluorescence is strongly enhanced in AT sequences and quenched in GC sequences. Fluorescence titrations evaluated as Scatchard plots show that both ellipticine and compound 3b (Scheme I) bind to the nucleic acids according to a non-cooperative neighbor exclusion model.     

The effect of intercalator structure on binding strength and base-pair specificity in DNA interactions

The interaction of naphthothiophene, phenanthrene and anthracene ring systems, which have amide and ester side chains with cationic groups (synthesized from the aromatic acid chlorides and appropriate amines and alcohols), with calf thymus DNA has been investigated by using viscometric titrations, spectrophotometric binding experiments and 1H-, 31P- and 17O-NMR methods. The viscosity and NMR experiments suggest that all of these compounds bind to DNA by intercalation. These experiments and spectrophotometric binding studies, however, indicate that there is considerable variation in the interaction of these compounds with DNA. These variations can all be explained by the geometry of the ring systems, the position of protons adjacent to the side chains, and the relative sizes of the amide and ester side chains. With the naphthothiophene ester and amide, for example, the planar amide cannot rotate into the plane of the naphthothiophene ring whereas the smaller planar ester can. With this ring system the ester has a significantly higher binding constant than the amide derivative. Additional binding studies with poly[d(A-T)2] and poly[d(G-C)2] have shown that all of these compounds bind more strongly to the A-T- than the G-C-containing polymer. Since the ester compounds do not have hydrogen bond donating groups proximate to the aromatic ring, these results suggest a model for the A-T specificity of these compounds that involves a solvent-mediated hydrogen bond between the C-2 carbonyl of thymine and the carbonyl group of the intercalators.     

DNA-binding affinities and sequence specificities of protoberberine alkaloids and their demethylated derivatives: a comparative study

Berberrubine (1a), jatrorubine (2a), and palmatrubine (3a) have been chemically prepared by partial demethylation of berberine (1), jatrorrhizine (2), and palmatine (3), respectively. Their interactions with calf thymus (CT) DNA, poly(dA-dT)poly(dA-dT), poly(dG-dC)poly(dG-dC), and eight AT-rich 12-mer double-stranded DNAs have been investigated by means of competitive ethidium bromide (EB) displacement experiments. The results showed that DNA-binding affinities of these protoberberine alkaloids have been significantly improved by partial demethylation, and that all of these alkaloids have the preferable binding affinities with AT-rich DNA. Especially, the sequence specificities of DNA-binding of demethylated derivatives 1a, 2a, and 3a had changed to a certain extent when compared with the parent alkaloids 1, 2, and 3, respectively. The binding mode of these alkaloids was further confirmed by UV spectroscopic titration experiments. All the compounds bind to double-stranded DNA most probably via an intercalating mode.     

Sequence Specific Binding of Beta Carboline Alkaloid Harmalol with Deoxyribonucleotides: Binding Heterogeneity,Conformational, Thermodynamic and Cytotoxic Aspects

Background

Base dependent binding of the cytotoxic alkaloid harmalol to four synthetic polynucleotides, poly(dA).poly(dT), poly(dA-dT).poly(dA-dT), poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) was examined by various photophysical and calorimetric studies, and molecular docking.

Methodology/Principal Findings

Binding data obtained from absorbance according to neighbor exclusion model indicated that the binding constant decreased in the order poly(dG-dC).poly(dG-dC)>poly(dA-dT).poly(dA-dT)>poly(dA).poly(dT)>poly(dG).poly(dC). The same trend was shown by the competition dialysis, change in fluorescence steady state intensity, stabilization against thermal denaturation, increase in the specific viscosity and perturbations in circular dichroism spectra. Among the polynucleotides, poly(dA).poly(dT) and poly(dG).poly(dC) showed positive cooperativity where as poly(dG-dC).poly(dG-dC) and poly(dA-dT).poly(dA-dT) showed non cooperative binding. Isothermal calorimetric data on the other hand showed enthalpy driven exothermic binding with a hydrophobic contribution to the binding Gibbs energy with poly(dG-dC).poly(dG-dC), and poly(dA-dT).poly(dA-dT) where as harmalol with poly(dA).poly(dT) showed entropy driven endothermic binding and with poly(dG).poly(dC) it was reported to be entropy driven exothermic binding. The study also tested the in vitro chemotherapeutic potential of harmalol in HeLa, MDA-MB-231, A549, and HepG2 cell line by MTT assay.

Conclusions/Significance

Studies unequivocally established that harmalol binds strongly with hetero GC polymer by mechanism of intercalation where the alkaloid resists complete overlap to the DNA base pairs inside the intercalation cavity and showed maximum cytotoxicity on HepG2 with IC₅₀ value of 14 µM. The results contribute to the understanding of binding, specificity, energetic, cytotoxicity and docking of harmalol-DNA complexation that will guide synthetic efforts of medicinal chemists for developing better therapeutic agents.     

A theoretical study of anthracene and phenanthrene derivatives acting as A-T specific intercalators.

Theoretical computations are performed on the comparative A-T versus G-C binding selectivities of two DNA intercalating molecules recently synthesized by Wilson et al. These are derivatives of phenanthrene and anthracene with side chains containing an hydroxy group bound to its C alpha carbon and a cationic amino group bound to its C beta carbon. We have optimized the binding energies of these phenanthrene and anthracene derivatives (1 and 2, respectively) to the double-stranded tetramers d(ATAT)2 and d(GCGC)2, the intercalation occurring in the central pyrimidine (3'-5') purine sequence. The sum of the intercalator-oligonucleotide intermolecular interaction energy plus the conformational energy variation of the intercalator upon binding were computed by the SIBFA procedures, which use empirical formulas based on ab initio SCF computations. Both compounds are found to bind more favourably to the AT sequence than to the GC one. Moreover, the affinity of 1 for the AT oligomer is computed to be larger than that of 2, whereas conversely that of 2 is larger than that of 1 for the GC oligomer. The AT versus GC binding selectivity of 1 is significantly larger than that of 2. These results are in excellent agreement with the experimental findings of Wilson et al. However, contrary to the suggestion of these authors the alpha-hydroxy group of the side chain of the intercalators does not seem to play a decisive role in determining the A-T specificity.     

Influence of cyanine dye structure on self-aggregation and interaction with nucleic acids: a kinetic approach to TO and BO binding

Kinetics and equilibria of cyanine dyes thiazole orange (TO) and benzothiazole orange (BO) self-aggregation and binding to CT-DNA are investigated in aqueous solution at 25 degrees C and pH 7. Absorbance spectra and T-jump experiments reveal that BO forms J-aggregates while TO forms more stable H-aggregates. Fluorescence and absorbance titrations show that TO binds to DNA more tightly than BO. TO stacks externally to DNA for low polymer-to-dye concentration ratios (C(P)/C(D)) while dye intercalation occurs for high values of C(P)/C(D). T-jump and stopped-flow experiments performed at high C(P)/C(D) agree with reaction scheme D+S <=> D,S <=> DS(I) <=> DS(II) where the precursor complex D,S evolves to a partially intercalated complex DS(I) which converts to the more stable intercalate DS(II). Non-electrostatic forces play a major role in D,S stabilization. Last step is similar for both dyes suggesting accommodation of the common benzothiazole residue between base pairs. Experiments using poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC) confirm base pair preference for TO.     

Binding of pyrene to DNA, base sequence specificity and its implication.

Solubilization as well as spectral studies of pyrene in natural DNA and synthetic deoxypolynucleotide solutions at neutral pH reveal at least two binding modes. Sites I are predominant in native DNA and in poly(dA-dT): poly(dA-dT) whereas sites II are found with denatured DNA and other polynucleotides such as poly(dA):poly(dT) and three different types of guanine containing copolymers which solubilize pyrene to a lesser extent. Spectral comparison with the covalent adducts of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10- tetrahydro-benzo(a)pyrene (anti-BPDE) and the physical complexes of its tetraols lead to the suggestion of a base sequence specific binding model for this carcinogenic metabolite to account for the puzzling fact that although its physical binding is predominantly intercalative, the covalent adducts appear not to be intercalated. It is speculated that in neutral solutions, intercalation may have little, if any, to do with the chemical lesion of this metabolite to the guanine base of the DNA and may, on the contrary, provide an efficient pathway for detoxification.     

Spectrophotometric and NMR analysis of the interaction of fluorinated mono- and bifunctional intercalators with DNA, poly(dA-dT), and poly(dG-dC)

We have synthesized and investigated the DNA binding properties of three fluorinated acridine derivatives—a monomer (I), a short dimer (II) and a long dimer (III). Only III has a sufficiently long chain bridging the two acridine nuclei to permit binding by bisintercalation. Analysis of the equilibrium and kinetic binding properties of these compounds to poly(dA-dT) demonstrates that they behave very similarly to their unfluorinated parent compounds. Helix extension, as determined by viscosity measurements, shows that both compounds I and II bind by monointercalation while III binds by bisintercalation. These results are confirmed by ¹⁹F-nmr analysis, which indicates, in particular, that the two chromophores of III share the same molecular environment as that of I in the presence of either calf thymus DNA or poly(dA-dT). Negative nuclear Overhauser effects in the presence of DNA indicate tight binding such that the motion of the ligands is governed by the polynucleotide dynamics. Optical titrations establish that in 4M NaCl, both I and III bind to calf thymus DNA, but no binding was observed with poly(dG-dC). This result is in contrast to those for dimers of ethidium, which show substantial binding to polynucleotides under high salt conditions. Nuclear magnetic resonance experiments, however, carried out at considerably higher concentrations, show that compound I does indeed bind to poly(dG-dC) under these high salt conditions, albeit weakly, and leads to a conversion of the polynucleotide from a left-handed to a right-handed conformation.     

Interaction of synthetic analogues of distamycin with poly(dA-dT): role of the conjugated N-methylpyrrole system

Two synthetic analogues of distamycin (Dst), PPA and PAP, containing a saturated beta-alanine moiety substituting for an N-methylpyrrole chromophore were studied for their interactions with the double-stranded alternating copolymer poly(dA-dT).poly(dA-dt) [abbreviated as poly(dA-dT)], with UV absorption and circular dichroism (CD) spectroscopy. The distinctive feature of these analogues is the difference in the extents of extended conjugation due to contiguous pyrrole rings: it decreases in the order Dst greater than PPA greater than PAP. Both these analogues bind to poly(dA-dT) in a way similar to Dst, as suggested from the observed red shift in the UV spectra of the ligands upon complexation and the appearance of induced Cotton effects (in the 290-350-nm region) in the CD spectra of the complexes. A comparative study of (i) the spectral features of the complexes between these ligands, Dst and netrospin (Nt) and poly(dA-dT), and (ii) the binding parameters for the association with the polynucleotide suggests that the number and relative positions of the pyrrole moieties influence the spectral features and thermodynamic stabilities of the complexes, and the latter show a progressive decrease in the order Dst greater than Nt greater than PPA greater than PAP. Implications of these results vis-à-vis the molecular basis of Dst-DNA interaction are discussed.     

Influence of some chromophore substituents on the intercalation of anthracycline antibiotics into DNA

The interaction between some chromopore-modified daunorubicin derivatives and calf thymus DNA was studied using a number of physical techniques in order to investigate the effect substituents on the aromatic ring system have on the capacity to intercalate into DNA and on the DNA binding affinity. The modifications examined include methylation of the hydroxyl groups at the 6 and 11 positions of the B ring and removal of the 11-hydroxyl group. The studies showed that only 11-deoxydaunorubicin retains the ability to bind to DNA by the intercalation mechanism typical of the parent compound, although the structural modification leads to an appreciably weaker binding. In contrast, methylation of any hydroxyl group dramatically reduces the affinity of the drug for DNA. At physiological ionic strength both methyl ether derivatives showed no evidence of intercalation. Structure activity correlations for the intercalation reaction deduced from these studies are in agreement with earlier findings and hypotheses relating to antitumour activity.     

Peptide conformations--49(1): synthesis and structure-activity relationships of side chain modified peptides of cyclo(-D-Pro-Phe-Thr-Lys-Trp-Phe.)

Cyclic hexapeptide analogues representing the modified retro sequence of the amino acid residues 7-11 of natural somatostatin are known to protect liver cells from phalloidin poisoning. To determine the influence of steric, lipophilic, and charge effects on (a) the conformation of the backbone and the aromatic side chains and (b) the biological response, the side chains of Phe2, Lys4, and Phe6 of cyclo(-D-Pro1-Phe2-Thr3-Lys(Z)4-Trp5-Phe6-), 1a, one of the most active peptides found so far, were modified by various residues. The discussion of conformationally relevant parameters proves that neither backbone conformations nor populations of aromatic side chain rotamers were altered by these substitutions. The potency of these derivatives in a cytoprotection assay varies by at most one order of magnitude (more or less active than the parent peptide 1a). A qualitative evaluation of lipophilic, steric, and charge effects reveals the dominance of lipophilic effects of aromatic residues; the most potent compounds contain aromatic substructures in the side chain of Lys4.     

The role of side chains in the interaction of new antitumor pyrimidoacridinetriones with DNA: molecular dynamics simulations

Pyrimidoacridinetriones (PATs) are a new group of highly active antitumor compounds. It seems reasonable to assume that, like for some other acridine derivatives, intercalation into DNA is a necessary, however not a sufficient condition for antitumor activity of these compounds. Rational design of new compounds of this chemotype requires knowledge about the structure of the intercalation complex, as well as about interactions responsible for its stability. Computer simulation techniques such as molecular dynamics (MD) may provide valuable information about these problems. The results of MD simulations performed for three rationally selected PATs are presented in this paper. The compounds differ in the number and position of side chains. Each of the compounds was simulated in two systems: i) in water, and ii) in the intercalation complex with the dodecamer duplex d(GCGCGCGCGCGC)2. The orientation of the side chain in relation to the ring system is determined by the position of its attachment. Orientation of the ring system inside the intercalation cavity depends on the number and position of side chain(s). The conformations of the side chain(s) of all PATs studied in the intercalation complex were found to be very similar to those observed in water.     

The interaction of multiply charged intercalating heterocycles with DNA

The interaction with DNA of two aromatic nitrogen heterocycles, 1 and 2 , which at pH 6 have two positive charges on their ring systems and two cationic side chains, have been determined. A third similar compound, 3 , with a single side chain and reduced ring charge, was analyzed as a control. Viscometric titrations with sonicated DNA indicated that all three compounds bind to DNA by intercalation. Spectrophotometric binding studies as a function of ionic strength indicated that both 1 and 2 bind to DNA as tetracations at pH 6. These are the first examples of intercalators with two charges directly on the intercalating ring system. Dissociation kinetics experiments as a function of ionic strength confirmed that 1 and 2 bind to DNA as tetracations. Compound 1 has a G · C base-pair binding preference, 2 seems to prefer binding to alternating pyrimidine–purine sequences regardless of the composition, and 3 has no significant binding specificity.     

A theoretical investigation on the sequence selective binding of daunomycin to double-stranded polynucleotides

Theoretical computations are performed on the structural and energetical factors involved in the sequence selective binding of daunomycin (DNM) to six representative self-complementary double-stranded hexanucleotides: d(CGTACG)2,d(CGATCG)2,d(CITACI)2, d(TATATA)2, d(CGCGCG)2 and d(TACGTA)2. The conformational angles of the hexanucleotides are fixed in values found in the representative crystal structure of the d(CGTACG)2-DNM complex. The intermolecular DNM-hexanucleotide interaction energies and the conformational energy changes of DNM upon binding are computed and optimized in the framework of the SIBFA procedure, which uses empirical formulas based on ab initio SCF computations. Among the two regularly alternating hexanucleotides, d(TATATA)2 and d(CGCGCG)2, a stronger binding is predicted for the former, in agreement with experimental results obtained with poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC). Altogether, however, among the six investigated sequences, the strongest complexes are computed for the mixed hexanucleotides d(CGATCG)2 and d(CGTACG)2, containing the intercalation site between two CG base pairs and an adjacent TA base pair. This situation may be related to the increased affinity of DNM for GC rich DNA's and to the situation in the crystal structure of the DNM-d(CGTACG)2 complex. Analysis of the intrinsic base sequence preferences expressed by the individual constituents of DNM, namely the daunosamine side chain, the chromophore ring and its two 9-hydroxyl and 9-acetoxy substituents, reveals that the overall sequence preference found is the result of a rather intricate interplay of intrinsic sequence preferences, in particular at the level of daunosamine and the 9-hydroxyl substituent.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA-binding affinities and sequence selectivity of quaternary benzophenanthridine alkaloids sanguinarine, chelerythrine, and nitidine

A comparative study on the intercalating binding of sanguinarine, chelerythrine, and nitidine with CT DNA, poly(dG-dC).poly(dG-dC), poly(dA-dT).poly(dA-dT), and seven sequence-designed double-stranded oligodeoxynucleotides has been performed using fluorometric and spectrophotometric techniques, aiming at providing insights into their sequence selectivity for DNA-binding. The results show that both sanguinarine and nitidine bind preferentially to DNA containing alternating GC base pairs [d(TGCGCA)(2)], while chelerythrine exhibits quite distinct sequence selectivity from sanguinarine, which shows a high specificity for DNA containing contiguous GC base pairs [5'-TGGGGA-3'/3'-ACCCCT-5'].     

Puromycin inhibition of eucaryotic ribosomes. Differences in sensitivity between polypeptide synthesis directed by endogenous mRNA and synthetic templates including poly(U).

In a protein synthesis sytem derived from porcine uteri we have made the following observations: 1. Synthesis directed by the endogenous mRNA of the system is more sensitive to inhibition by puromycin than poly(U) directed synthesis. 2. Synthesis directed by the synthetic templates poly(G,U) and poly(C,U) is more sensitive to inhibition by puromycin than poly(U) directed synthesis and frequently shows a sensitivity to puromycin similar to that directed by endogenous mRNA. 3. Protein synthesis was inhibited by three aminoacyloligonucleotides (C-A-Phe, C-A-Asp, and C-A-Pro) which are analogs of the 3' terminus of aminoacyl tRNAs. Of the three, C-A-Phe was the most active and C-A-Asp the least active but, unlike puromycin, inhibition by these compounds was the same for endogenous and poly(U) directed synthesis. These results are interpreted as supporting the proposal that the acceptor site of ribosomes contains an hydrophobic binding region which interacts with the side chain of aliphatic amino acids, and particularly with the aromatic side chain of phenylalanine.