CpG methylation increases the DNA binding of 9-aminoacridine carboxamide Pt analogues

This study investigated the effect of CpG methylation on the DNA binding of cisplatin analogues with an attached aminoacridine intercalator. DNA-targeted 9-aminoacridine carboxamide Pt complexes are known to bind at 5′-CpG sequences. Their binding to methylated and non-methylated 5′-CpG sequences was determined and compared with cisplatin. The damage profiles of each platinum compound were quantified via a polymerase stop assay with fluorescently labelled primers and capillary electrophoresis. Methylation at 5′-CpG was shown to significantly increase the binding intensity for the 9-aminoacridine carboxamide compounds, whereas no significant increase was found for cisplatin. 5′-CpG methylation had the largest effect on the 9-ethanolamine-acridine carboxamide Pt complex, followed by the 9-aminoacridine carboxamide Pt complex and the 7-fluoro complex. The methylation state of a cell’s genome is important in maintaining normal gene expression, and is often aberrantly altered in cancer cells. An analogue of cisplatin which differentially targets methylated DNA may be able to improve its therapeutic activity, or alter its range of targets and evade the chemoresistance which hampers cisplatin efficacy in clinical use.     

Complexes of derivatives of 1-nitro-9-aminoacridine with DNA.

Substituted 1-nitro-9-aminoacridine derivatives were shown to inhibit RNA and to a lesser extent protein synthesis in cultured human cells. Complex formation between the compounds studied and DNA were considered to be responsible for their cytostatic action. Two types of complexes differing in their binding forces were found. The biological activity of the studied compounds seems not to be dependent on the existence of a positive charge on the acridine ring.     

Adenosine-guanosine preferential photocleavage of DNA by azido-benzoyl- and diazocyclopenta-dienylcarbonyloxy derivatives of 9-aminoacridine.

The photoreactions of 9-[6-(4-azidobenzamido)hexylamino]acridine (AHA) and 9-[6-(2-diazocyclopentadienylcarbonyloxy)hexylamino]acridine (DHA) with double stranded DNA result in formation of single strand nicks and alkali labile sites (adducts) with an efficiency of 6 x 10(-3) nicks per AHA and 3 x 10(-2) nicks per DHA molecule. The alkali dependent DNA cleavage by AHA shows a pronounced A+G preference whereas that by DHA is practically sequence independent. In the presence of diacridines, however, DHA exhibits a preference for cleavage at guanosines. These DNA photocleaving reagents could be useful for DNA photofootprinting and photosequencing.     

The sequence selectivity of DNA-targeted 9-aminoacridine cisplatin analogues in a telomere-containing DNA sequence

In this study, the detailed DNA sequence specificity of four acridine Pt complexes was examined and compared with that of cisplatin. The DNA sequence specificity was determined in a telomere-containing DNA sequence using a polymerase stop assay, with a fluorescent primer and an automated capillary DNA sequencer. The Pt compounds included an acridine intercalating moiety that was modified to give a 9-aminoacridine derivative, a 7-methoxy-9-aminoacridine derivative, a 7-fluoro-9-aminoacridine derivative and a 9-ethanolamine-acridine derivative. Compared with cisplatin, the DNA sequence specificity was most altered for the 7-methoxy-9-aminoacridine compound, followed by the 9-aminoacridine derivative, the 7-fluoro-9-aminoacridine compound and the 9-ethanolamine-acridine derivative. The DNA sequence selectivity for the four acridine Pt complexes was shifted away from runs of consecutive guanines towards single guanine bases, especially 5′-GA dinucleotides and sequences that contained 5′-CG. The sequence specificity was examined in telomeric and non-telomeric DNA sequences. Although it was found that telomeric DNA sequences were extensively damaged by the four acridine Pt complexes, there was no extra preference for telomeric sequences.     

A theoretical investigation on the sequence selective binding of mitoxantrone to double-stranded tetranucleotides.

Theoretical computations are performed on the comparative binding energetics of mitoxantrone (MX), a newly synthesized intercalating anthraquinone antitumor drug, to six representative double-stranded tetranucleotides: d(GCGC)2, d(CGCG)2, d(ATAT)2, d(TATA)2, d(GTGT), d(ACAC), and d(CCGG)2. The computations are performed with the SIBFA procedure, which uses empirical formulas based on ab initio SCF computations. The best binding configuration of mitoxantrone locates its two side chains in the major groove. A considerable preference is elicited for intercalation of the chromophore ring in a pyrimidine (3'-5') purine sequence rather than the isomeric purine (3'-5') pyrimidine sequence. Contrary to the situation encountered with "simple" intercalators, in which this preference is generally attributed solely to differences in the energies of unstacking necessary to generate the intercalation sites, the preference is dictated in MX to a large extent by the intermolecular interaction energy term. This result is imposed by the interactions of the side chains of MX with the oligonucleotide.     

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

Theoretical computations are performed on the structural and energetical factors involved in the sequence selective binding of adriamycin (ADM) to five self-complementary double-stranded hexanucleotides. Among the two regularly alternating hexanucleotides d (TATATA)2 and d (CGCGCG)2, a stronger binding is predicted for the former. The strongest complex is computed, however, for the mixed hexanucleotide d (CGTACG)2, containing the intercalation site between two CG base pairs and an adjacent TA base pair. The overall sequence preference is the result of an intricate interplay of sequence preferences of the constituents in particular of daunosamine and the 9-OH substituent. Altogether, the selective base pair recognition by adriamycin cannot be defined in terms of the two base pairs implicated in the intercalation site alone but must be expressed in terms of a triplet of base pairs.     

Inhibition of RNA synthesis in vitro by 9-aminoacridine carboxamide antitumor agents. Effects on overall RNA synthesis and synthesis of the initiating dinucleotide.

A series of 9-aminoacridine carboxamide derivatives of systematically varied structure was assayed in an RNA synthesis in vitro system. Escherichia coli DNA-dependent RNA polymerase and DNA derived from phage T7 or calf thymus were used to measure the effect of the drugs on overall RNA and the initiating dinucleotide (pppApU) syntheses. By means of multiple linear regression analysis it was shown that the inhibition of these reactions depends both on the drug equilibrium binding constant and kinetic parameters of dissociation of drug-DNA complexes.     

Photofootprinting of drug-binding sites on DNA using diazo- and azido-9-aminoacridine derivatives

It is demonstrated that DNA photofootprinting analysis of the intercalating depsipeptide echinomycin, and the minor groove-binders distamicyn, 4',6-diamidino-2-phenylindole (DAPI) and Hoechst 33258 can be performed using 9-[6-(2-diazocyclopentadienylcarbonyloxy)hexylamino]acridine (DHA) [Nielsen et al. (1988) Nucleic Acids Res. 16, 3877-3888] or 2-methoxy-6-azido-9-aminoacridine (MAA) [Jeppesen et al. (1988) Nucleic Acids Res. 16, 5755-5770]. Both the extent of the drug-binding sites and their relative strength can be determined with either reagent. DNA has the advantage of giving virtually sequence-uniform DNA photocleavage. On the other hand, structural changes in the DNA are detected by MAA. Using the 232-base-pair EcoRI-PvuII pUC19 restriction fragment, it is found that cleavage protection by distamycin, DAPI and Hoechst 33258 all require an (A.T)4 sequence, whereas protection by echinomycin was confined to a G + C-rich 8-base-pair region.     

Binding of 9-aminoacridine to deoxydinucleoside phosphates of defined sequence: Preferences and stereochemistry

The effect of sequence on the binding of 9-aminoacridine to DNA has been investigated by studying its interaction with deoxydinucleoside phosphates of different sequences using proton nuclear magnetic resonance. Quantitative binding information can be obtained by comparison of the proton chemical shift behavior of 9-aminoacridine upon addition of dinucleoside phosphate to various models for the interaction using least-squares computer fitting procedures. The simplest model that fits the data includes (1) dimerization of 9-aminoacridine and (2) a mixture of 1:1 and 2:1 (dinucleoside phosphate/9-aminoacridine) complexes. The computed parameters allow comparison of binding constants and stereochemistry for different sequences. The 1:1 complexes seem to involve interaction of the ring nitrogen with the backbone phosphate and stacking of one or both chromophores on the acridine; preference in binding is observed for alternating (purine-pyrimidine or pyrimidine-purine) over non-alternating (purine-purine) dinucleoside phosphates. The 2:1 complexes involve intercalation of the acridine between two complementary dinucleoside phosphate strands with weak sequence preferences in binding. The stereochemistry of intercalation differs between non-alternating purine-purine sequences and the alternating pyrimidine-purine or purine-pyrimidine sequences in having the 9-aminoacridine stacked with the purines of one strand rather than straddling the purines on opposite strands. The difference in stereochemistry could possibly be a determining factor in frameshift sequence 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)     

Interactions of some nitro-derivatives of substituted 9-aminoacridine with DNA.

The mechanism of the biological activity of the 1-nitro and 2-nitro aminoacridine derivatives containing the dimethylaminopropyl side chain was studied. RNA synthesis in the isolated rat liver nuclei was only slightly influenced by both compounds. They do not differ in their ability to form an intercalative complex with DNA. Only the 1-nitro derivative exhibited strong inhibitory effect on RNA biosynthesis and caused distinct ultrastructural changes (nucleolar segregation, chromatine margination etc.) in a living cell. The 1-nitro derivative binds covalently to DNA in vivo resulting in crosslink formation. It is concluded that the biological activity of 1-nitro acridine derivatives depends more on their crosslinking activity than on their ability to intercalate into DNA.     

A theoretical investigation of the sequence specificity in the binding of the antitumor drug anthramycin to DNA

A theoretical study is presented concerning DNA-anthramycin adducts. By explicit energy minimisations using a semi-empirical energy formula and an advanced algorithm the structural properties and the energetics of this system are analysed. The results obtained demonstrate that the formation of a covalently bound adduct in which anthramycin is attached to the N2 site of a guanine within a DNA fragment is accompanied by a considerable change in the nucleic acid conformation as confirmed by recent experimental evidence. With the use of the "SIR" methodology for treating DNA flexibility the general features of this change are characterised. The sequence specificity of anthramycin binding is investigated and the important role of sequence dependent nucleic acid flexibility brought to light. This theoretical treatment thus provides new elements for the interpretation of the origins of ligand binding specificities.     

Local base sequence preferences for DNA cleavage by mammalian topoisomerase II in the presence of amsacrine or teniposide.

Several classes of antitumor drugs are known to stabilize topoisomerase complexes in which the enzyme is covalently bound to a terminus of a DNA strand break. The DNA cleavage sites generally are different for each class of drugs. We have determined the DNA sequence locations of a large number of drug-stimulated cleavage sites of topoisomerase II, and find that the results provide a clue to the possible structure of the complexes and the origin of the drug-specific differences. Cleavage enhancements by VM-26 and amsacrine (m-AMSA), which are representative of different classes of topoisomerase II inhibitors, have strong dependence on bases directly at the sites of cleavage. The preferred bases were C at the 3' terminus for VM-26 and A at the 5' terminus for m-AMSA. Also, a region of dyad symmetry of 12 to 16 base pairs was detected about the enzyme cleavage positions. These results are consistent with those obtained with doxorubicin, although in the case of doxorubicin, cleavage requires the presence of an A at the 3' terminus of at least one the pair of breaks that constitute a double-strand cleavage (Capranico et al., Nucleic Acids Res., 1990, 18: 6611). These findings suggest that topoisomerase II inhibitors may stack with one or the other base pair flanking the enzyme cleavage sites.     

9-aminoacridine inhibits the B-Z transition of poly(dA-dT).

9-Aminoacridine is the parent compound of a family of pharmacologically active model substances that bind to DNA through intercalation between base pairs. In the present study we show that 9-aminoacridine inhibits the B-to-Z isomerization of poly(dA-dT) in conditions that otherwise cause it to occur (5 M NaCl and 123 mM Ni(ClO4)2). Higher concentrations of Ni(ClO4)2 (155 mM) are able to induce the Z-form due to the disruption of the drug-polynucleotide interaction by the metal ion. Additionally, the dye reverses the Z-form in certain conditions. Thus, the data from this study indicate that 9-aminoacridine binds preferentially to the B-form of poly(dA-dT).     

Acridin-9-yl exchange: a proposal for the action of some 9-aminoacridine drugs

The finding that several derivatives of 9-aminoacridine were deacridinylated in the presence of primary aliphatic amines during the solid phase synthesis of acridine-peptide conjugates prompted us to investigate the acridin-9-yl moiety transfer from a relatively low-molecular acridine source to a high-molecular carrier. The hydrophobic polymer was used as a model of hydrophobic core of biologically active proteins. While the alpha-amino group in the peptide was found to play the role of weak acridine acceptor, the epsilon-amino group of lysine appeared to serve as a moderate acceptor of acridine moiety. The covalent modification of the lysine residues side chain in the hydrophobic core of prion protein aggregates could thus explain the discrepancy between the ability of the acridine drug quinacrine to reduce efficiently the incidence of prion protein in cell culture and its weak prion binding affinity.     

Induction of deletion and insertion mutations by 9-aminoacridine. An in vitro model

The ability of 9-aminoacridine to induce mutagenic lesions during DNA replication in vitro was investigated. The ampicillinase gene of pBR322 was replicated in vitro in the presence of 9-aminoacridine. Transfection of the replicated DNA into Escherichia coli gave Amps mutants. Determination of the base changes in 76 of these mutants indicated that the spectrum of mutations induced by 9-aminoacridine was consistent with its action in vivo. Both large (407-base) and small (1- and 2-base) deletions were induced at repetitive sequences. The frequency of deletion mutations depended on the identity of the base deleted and sequences surrounding the deletions. The characteristics of the frameshift mutations induced were consistent with the interactions of 9-aminoacridine with DNA. These results establish that 9-aminoacridine can induce frameshift mutations during the replication process and provide an in vitro model of frameshift induction for mechanistic studies.     

A theoretical method to predict DNA permutation gel electrophoresis from the sequence.

The gel electrophoretic permutation assays of DNA fragments experimentally investigated by different authors were theoretically reproduced using our theoretical model of sequence-dependent curvature. The general pattern of agreement obtained suggests that our method can be usefully adopted as an alternative to the experimental assay, in particular where the lack of a sufficient number of unique restriction sites in the fragment prevents the correct localization of the main bend site.