New acridone derivatives for the electrochemical DNA-hybridisation labelling.

In the field of DNA sensing, DNA hybridisation detection is generally performed by fluorescence microscopy. However, fluorescence instrumentation is difficult to miniaturise in order to produce fully integrated DNA chips. In this context, electrochemical detection of DNA hybridisation may avoid this limitation. Therefore, the use of DNA intercalators is particularly attractive due to their selectivity toward DNA double strand enabling DNA labelling without target chemical modification and, for most of them, to their electroactivity. We have synthesized a pyridoacridone derivative dedicated to DNA hybridisation electrochemical-sensing which presents good electrochemical reversibility, electroactivity at mild potentials and specificity toward DNA double strand. The electrochemical behaviour of this molecule has been assessed using cyclic voltammetry (CV). DNA/intercalator interactions were studied by differential pulse voltammetry (DPV) before application to hybridisation detection onto DNA sensors based on polypyrrole modified electrodes.

Interest of acridine derivatives in the anticancer chemotherapy.

DNA is considered as one of the main targets for anticancer drug design. The planar structure of acridines confers to the molecules the ability to bind DNA by intercalation and therefore to interfere with metabolic processes. A large number of natural alkaloids and synthetic acridine derivatives have been tested as anticancer agents. So far, a few molecules have entered clinical trials and have been approved for chemotherapy. The mechanisms of action are not fully understood. Cytotoxicity may be related to potent enzyme inhibition. Topoisomerase and telomerase activities may be strongly affected by acridines. The affinity of acridines for DNA has also been used to design new active compounds in which a DNA modifying group is tethered to the acridine nucleus. Acridine derivatives display other pharmacological properties such as antibacterial and antimalarial activities. They are also tested for Alzheimer's disease.

Diaminopurine-acridine heterodimers for specific recognition of abasic site containing DNA. Influence on the biological activity of the position of the linker on the purine ring.

Three acridine-diaminopurine heterodimers tethered by a linker containing an N,N'-substituted guanidine were prepared. The molecules differ by the site of introduction of the linker on the 2,6-diaminopurine. The interactions of the new heterodimers with abasic site containing oligonucleotide were compared, and their cytotoxicity was measured in the presence or absence of the antitumor alkylating agent BCNU.

Potentiation of BCNU cytotoxicity by molecules targeting abasic lesions in DNA.

We describe the synthesis, DNA binding measurements and pharmacological properties of a series of new heterodimeric molecules, in which a 2,6-diaminopurine is linked to a 9-aminoacridine chromophore. The linking chain contains a central N,N'-disubstituted guanidine, connected to the two chromophores by polymethylenic units of variable length.

Enantiospecific recognition of DNA sequences by a proflavine Tröger base.

The DNA interaction of a chiral Tröger base derived from proflavine was investigated by DNA melting temperature measurements and complementary biochemical assays. DNase I footprinting experiments demonstrate that the binding of the proflavine-based Tröger base is both enantio- and sequence-specific. The (+)-isomer poorly interacts with DNA in a non-sequence-selective fashion. In sharp contrast, the corresponding (-)-isomer recognizes preferentially certain DNA sequences containing both A. T and G. C base pairs, such as the motifs 5'-GTT. AAC and 5'-ATGA. TCAT. This is the first experimental demonstration that acridine-type Tröger bases can be used for enantiospecific recognition of DNA sequences.

The abasic site as a target for generation of locally multiply damaged sites.

Abasic sites in DNA have been specifically targeted by synthetic compounds able to cleave DNA at abasic sites and to induce photodamages in the vicinity of the lesion. The synthesis and the photoactivity of the drugs on abasic sites containing DNA and oligonucleotides are reported.

Synthesis and study of a new adenine-acridine tandem, inhibitor of exonuclease III.

A new heterodimer adenine-chain-acridine containing a mixed amido-guanidinium linker chain was synthesized. To achieve the synthesis a new method of introduction of aminoalkyl chain at position 9 of adenine was designed. The heterodimer interacts specifically with the abasic sites in DNA and inhibits the major base excision repair enzyme in Escherichia coli, Exonuclease III.

Abasic site recognition in DNA as a new strategy to potentiate the action of anticancer alkylating drugs?

Inhibition of abasic site repair in the cell seems an attractive strategy to potentiate the action of antitumor DNA alkylating drugs. Molecules that bind specifically and strongly to the abasic site are possible candidates to achieve such inhibition. We explored this strategy by preparing molecule 4 that incorporates (1) an aminoacridine intercalator for DNA binding, (2) an adenine moiety for abasic site recognition, and (3) a linker containing two guanidinium functions to increase binding to DNA without inducing cleavage at the base-sensitive abasic site. Compound 4 was compared to analogues containing secondary amines, i.e., 1. We report on synthesis of the new heterodimer 4. We show by physicochemical studies-including determination of association constants with calf-thymus DNA, T(m) measurements, and high-field NMR examination of the complexes formed with abasic DNA duplexes-that 4 binds specifically and more strongly to the abasic site than the analogues. Compound 4 does not cleave abasic plasmid DNA. Compound 4 shows apparent synergy with the anticancer bischloroethylnitrosourea (BCNU) in L1210 and A549 cell lines in vitro. It potentiates BCNU in the in vivo tests. The results favor the pertinence of the strategy.

Synthesis of an imidazo[1,2-e]purine-acridine heterodimer for targeting abasic sites in DNA.

Cyclization of 8-bromo-9-alkylaminoethyl-adenine quantitatively affords a substituted imidazo[1,2-e]purine. The corresponding heterodimer, imidazo[1,2-e]purine-acridine, was prepared and its interaction with abasic site containing oligonucleotides was studied.

Abasic DNA structure, reactivity, and recognition.

Loss of a base in DNA, i.e., creation of an abasic site leaving a deoxyribose residue in the strand, is a frequent lesion that may occur spontaneously, or under the action of radiations and alkylating agents, or enzymatically as an intermediate in the repair of modified or abnormal bases. The abasic site lesion is mutagenic or lethal if not repaired. From a chemical point of view,the abasic site is an alkali-labile residue that leads to strand breakage through beta- and delta- elimination. Progress in the understanding of the chemistry and enzymology of abasic DNA largely relies upon the study of synthetic abasic duplexes. Several efficient synthetic methods have thus been developed to introduce the lesion (or a stable analogue) at defined position in the sequence. Physicochemical and spectroscopic examination of such duplexes, including calorimetry, melting temperature, high-field nmr and molecular modeling indicate that the lesion strongly destabilizes the duplex, although remaining in the canonical B-form with structural modifications strictly located at the site of the lesion. Probes have been developed to titrate the damage in DNA in vitro. Series of molecules have been devised to recognize specifically the abasic site, exhibiting a cleavage activity and mimicking the AP nucleases. Others have been prepared that bind strongly to the abasic site and show promise in potentiating the cytotoxic and antitumor activity of the clinically used nitrosourea (bis-chloroethylnitrosurea).

Introduction of a nitroxide group on position 2 of 9-phenoxyacridine: easy access to spin labelled DNA-binding conjugates.

In the search for spin labelled intercalators of general use to construct DNA-binding conjugates, 6-chloro-2-[(1-oxyl-2,2,5,5-tetramethyl- pyrrolin-3-yl)methyloxy]-9-phenoxy-acridine 5, has been prepared. This key-intermediate reacts with amines to give the corresponding labelled 9-amino substituted acridines. Comparative EPR and fluorescence measurements show that the label causes only little modification of the binding properties of acridine.

Search for DNA repair inhibitors: selective binding of nucleic bases-acridine conjugates to a DNA duplex containing an abasic site.

The abasic site is one of the most frequent DNA lesions generated by spontaneous or enzymatic cleavage of the N-glycosidic bond. The abasic site is also an intermediate in the nucleotide and base excision DNA repair. We examined molecules which recognize and cleave DNA at the abasic site with high efficiency. These molecules incorporate in their structure a nucleic base for abasic site recognition, an intercalator for DNA binding, and a polyamino linker for ionic interaction and DNA cleavage. Such compounds, by interfering with abasic sites in DNA, are also inhibitors of DNA repair. In order to better understand the parameters of the interaction, we carried out a UV thermal denaturation study of synthetic oligonucleotides containing the lesion both in the absence and in the presence of the drugs. A similar study was also carried out using the corresponding nonmodified oligonucleotide. The results indicate selective binding of the base-chain-intercalator conjugates to the abasic site containing oligonucleotides.

NMR and molecular modeling studies of the interaction of artificial AP lyases with a DNA duplex containing an apurinic abasic site model.

Tailor-made molecules, DTAc and ATAc, that incorporate a nucleic base (adenine or 2,6-diaminopurine) linked by a diamino chain to an intercalator (9-amino-6-chloro-2-methoxyacridine) selectively recognize and efficiently cleave abasic sites in DNA via a beta-elimination reaction. The three-dimensional structure of the complexes of DTAc and ATAc bound to a DNA undecamer, the 5'd(C1G2C3A4C5X6C7A8C9G10C11)3' x 3'd(G22C21G20T19G18T17G16T15G14C13G12)5' duplex in which the X residue is a stable abasic site [3-hydroxy-2-(hydroxymethyl)tetrahydrofuran], has been studied by combined NMR-energy minimization methods. Analysis of the NMR spectra reveals that DTAc and ATAc interact with a very similar fashion and form two different complexes with DNA, present in a ratio of 70/30 (+/-10). In both complexes, the acridine ring intercalates exclusively between the C3 x G20 and A4 x T19 base pairs, the linker is located in the minor groove, and the base moiety docks in the abasic site. The principal difference between the major and the minor complexes consists of a 180 degrees rotation of the acridine ring around the Acr-C-N bond within the same intercalation site. Molecular modeling studies with few intermolecular ligand-DNA restraints were used to investigate the geometry of the base pair formed between the diaminopurine of DTAc and the T17 ring. The most energetically favored complex has the 2,6-diaminopurine of DTAc base paired with the T17 ring in a Hoogsteen conformation. The models DTAc and ATAc are also discussed as nuclease mimics and cleaving agents at abasic sites.

Design of molecules that specifically recognize and cleave apurinic sites in DNA.

We have prepared a series of tailor-made molecules that recognize and cleave DNA at apurinic sites in vitro. These molecules incorporate in their structure different units designed for specific function: an intercalator for DNA binding, a nucleic base for abasic site recognition and a linking chain of variable length and nature (including amino and/or amido functions). The cleavage efficiency of the molecules can be modulated by varying successively the nature of the intercalating agent, the nucleic base and the chain. All molecules bind to native calf thymus DNA with binding constants ranging from 10(4) to 10(6) M-1. Their cleavage activity was determined on plasmid DNA (pBR 322) containing 1.8 AP-sites per DNA-molecule. The minimum requirements for cleavage are the presence of the three units, the intercalator, the nucleic base and at least one amino function in the chain. The most efficient molecules cleave plasmid DNA at nanomolar concentrations. Enzymatic experiments on the termini generated after cleavage of AP-DNA suggest a strand break induced by a beta-elimination reaction. In order to get insight into the mode of action (efficiency, selectivity, interaction), we have used synthetic oligonucleotides containing either a true abasic site at a determined position to analyse the cleavage parameters of the synthetic molecules by HPLC or a chemically stable analog (tetrahydrofuran) of the abasic site for high field 1H NMR spectrometry and footprinting experiments. All results are consistent with a beta-elimination mechanism in which each constituent of the molecule exerts a specific function as indicated in the scheme: DNA targeting, abasic site recognition, phosphate binding and beta-elimination catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Formation of a cyclic adenine adduct with a 4-nitroquinoline N-oxide metabolite model.

Pentacyclic adducts are obtained in the reaction of adenine derivatives with the diacetyl ester of 4-hydroxyamino quinoline oxide, the postulated metabolite of the potent carcinogen, 4-nitroquinoline N-oxide (4-NQO).

Identification of 4-acetoxyaminoquinoline from the hydrolysis of 1-acetoxy-4-acetoxyimino-1,4-dihydroquinoline, in vitro and in vivo properties.

4-Acetoxyaminoquinoline (Ac-4-HAQ) (1) was identified as a hydrolysis product of 1-acetoxy-4-acetoxyimino-1,4-dihydroquinoline (diAc-4-HAQO). The reaction allowing the obtention of (1) obeys to a reduction mechanism implying the N1-O cleavage. The carcinogenic properties of (1) observed by Sato et al. (Japan J. Exp. Med., 40 (1970) 475) in mice were studied in rats with the in vivo system we used previously with 4-nitroquinoline-1-oxide (4-NQO) and 4-hydroxyaminoquinoline-1-oxide (4-HAQO). In rats (1) does not covalently bind DNA. It was, therefore, possible to propose an interpretation of the results obtained by Enomoto et al. (Proc. Soc. Exp. Biol. Med., 136 (1971) 1206) who injected diAc-4-HAQO s.c. to mice and rats. Compound 1 could be responsible for the carcinogenic effects observed through the following pathway: (1) should be formed by hydrolysis of diAc-4-HAQO and reactivated by an enzymatic system to N-oxide derivative, the 4-acetoxyaminoquinoline-1-oxide (Ac-4-HAQO), which constitutes an ultimate carcinogen model of 4-NQO.