Interaction of unfused tricyclic aromatic cations with DNA: a new class of intercalators

Unfused tricyclic aromatic ring systems 1-6 with one or two cationic side chains have been synthesized and their interactions with DNA and synthetic polymers probed with a variety of techniques. Molecular mechanics calculations indicate that the torsional angle between ring planes in the minimum energy conformation of the tricyclic molecules can range from 0 degree to as high as 50 degrees depending on the type of rings and substituents. Viscometric titrations with linear and supercoiled DNA, linear dichroism, and NMR studies indicated that all compounds with torsional angles of approximately 20 degrees or less bind to DNA by intercalation. The more highly twisted intercalators caused significant perturbation of DNA structure. Unfused intercalators with twist angles of approximately 20 degrees have reduced binding constants, suggesting that they could not form an optimum interaction with the DNA base pairs. Unfused intercalators with twist less than 20 degrees formed strong complexes with DNA. The structures of these unfused intercalators are more analogous to typical groove-binding molecules, and an analysis of their interaction with DNA provides a better understanding of the subtle differences between intercalation and groove-binding modes for aromatic cations. The results indicate that intercalation and groove-binding modes should be viewed as two potential wells on a continuous energy surface. The results also suggest design strategies for intercalators that can optimally complement DNA base pair propeller twist or that can induce bends in DNA at the intercalation site.     

Inhibition of the HIV-1 rev-RRE complex formation by unfused aromatic cations

RNA viruses cause a wide range of human diseases. Development of new agents to target such viruses is an active area of research. Towards this goal, a series of diphenylfuran cations as potential inhibitors of the Rev-RRE complex have been designed and synthesized. Analysis of the interaction of the diphenylfurans with RRE and TAR RNA model systems by gel shift assays indicates that they exhibit both sequence and structure-dependent binding modes. Our results show a strong interaction between the diphenylfuran ring system and RRE bases, while the TAR interactions are much weaker with the compounds that are the best inhibitors of Rev-RRE.     

DNA cleavage by aromatic amines.

A series of aryl amines was found to induce cleavage of DNA. Subsequent refinement led to an efficient family of dimeric derivatives capable of cleavage at low concentration. Initial investigations suggest this is an unprecedented mode of DNA cleavage, which may be ultimately applied to the development of sequence-specific agents.     

Amplification of bleomycin-induced DNA cleavage by pyrrole triamide.

We investigated the amplification of bleomycin-induced DNA cleavage by synthetic pyrrole triamide (PyPyPy) using 32P-labeled DNA fragments obtained from human genes. Peplomycin, a kind of bleomycins, plus Fe(II) caused DNA cleavage at the 5'-GC-3' and 5'-GT-3' sequences (damaged bases are underlined). The addition of PyPyPy enhanced the cleavage at cytosine and thymine residues 3' to consecutive guanines, particularly at the 5'-GGGGC-3' and 5'-GGGGT-3' sequences. These results suggest that PyPyPy binds to DNA to induce its conformational change, resulting in alteration of the site specificity and amplification of DNA cleavage. The present study on amplifiers of antitumor drugs would show a novel approach to the establishment of more effective chemotherapy.     

Amplification of C1027-induced DNA cleavage and apoptosis by a quinacrine-netropsin hybrid molecule in tumor cell lines

We examined the effect of a newly synthesized DNA-binding ligand, quinacrine-netropsin hybrid molecule (QN), on cytotoxicity, apoptosis, and DNA strand breaks induced by an enediyne antitumor antibiotic, C1027. QN significantly enhanced C1027-induced cellular DNA strand breaks, caspase-3 activation, and DNA ladder formation, characteristic of apoptosis, in human HL-60 cells. Flow cytometry revealed that C1027-induced intracellular H(2)O(2) generation was enhanced by QN, suggesting that QN enhances C1027-induced cytotoxic effect through H(2)O(2)-mediated apoptosis. QN also significantly enhanced C1027-induced apoptosis in BJAB cells, and the inhibition of apoptosis was observed in BJAB cells transfected with Bcl-2 gene. The experiment using (32)P-labeled DNA fragments showed that the addition of QN enhanced C1027-induced double-stranded DNA cleavage at the 5'-AGG-3'/3'-TCC-5' sequence (cutting sites are underlined). These results suggest that QN enhances C1027-induced antitumor effect via DNA cleavage and apoptosis. The present study shows a novel approach to the potentially effective anticancer therapy.     

The interaction with DNA of unfused aromatic systems containing terminal piperazino substituents. Intercalation and groove-binding

A number of unfused tricyclic aromatic intercalators have shown excellent activity as amplifiers of the anticancer activity of the bleomycins and the 4',6-diphenylpyrimidines, 2a and 2b, with terminal basic functions (4-methylpiperazino groups) have been synthesized to test the structural requirements for amplifier-DNA interactions. The terminal piperazine rings are bulky, have limited flexibility, and are twisted out of the phenyl ring plane in both 2a and 2b. With 2a the pyrimidine is unsubstituted at position 5 and the conformation predicted by molecular mechanics calculations has a 25-30 degrees twist between the phenyl and pyrimidine ring planes. With 2b the 5-position is substituted with a methyl group and this causes a larger twist angle (50-60 degrees) between the phenyl and pyrimidine planes. These conformational variations lead to markedly different DNA interactions for 2a and 2b. Absorption, CD and NMR spectral, viscometric, flow dichroism and kinetics results indicate that 2a binds strongly to DNA by intercalation while 2b binds more weakly in a groove complex. The general structure and conformation of 2a, a slightly twisted, unfused-aromatic system with terminal piperazino groups is more similar to groove-binding agents such as Hoechst 33258 than to intercalators. The fact that 2a forms a strong intercalation complex with DNA is unusual but in agreement with studies on other amplifiers of anticancer drug action. Molecular modeling studies provide a second unusual feature of the 2a intercalation complex. While most well-characterized intercalators bind with their bulky and/or cationic substitutents in the DNA minor groove, the cationic piperazino groups of 2a are too large to bind in the minor groove in an intercalation complex but can form strong interactions with DNA in the major groove. The tricyclic aromatic ring system of 2a stacks well with adjacent base-pairs in the major-groove complex and the piperazino groups have good electrostatic and van der Waals interactions with the DNA backbone.     

Recognition of ATGA sequences by the unfused aromatic dication DB293 forming stacked dimers in the DNA minor groove

Furamidine and related diamidines represent a promising series of drugs active against widespread parasites, in particular the Pneumocystic carinii pathogen. In this series, the phenylfuranbenzimidazole diamidine derivative DB293 was recently identified as the first unfused aromatic dication capable of forming stacked dimers in the DNA minor groove of GC-containing sequences. Here we present a detailed biochemical and biophysical characterization of the DNA sequence recognition properties of DB293. Three complementary footprinting techniques using DNase I, Fe(II)-EDTA, and an anthraquinone photonuclease were employed to locate binding sites for DB293 in different DNA restriction fragments. Two categories of sites were identified by DNase I footprinting: (i) 4/5 bp sequences containing contiguous A.T pairs, such as 5'-AAAA and 5'-ATTA; and (ii) sequences including the motif 5'-ATGA.5'-TCAT. In particular, a 13-bp sequence including two contiguous ATGA motifs provided a highly preferential recognition site for DB293. Quantitative footprinting analysis revealed better occupancy of the 5'-ATGA site compared to the AT-rich sites. Preferential binding of DB293 to ATGA sites was also observed with other DNA fragments and was confirmed independently by means of hydroxyl radical footprinting generated by the Fe(II)-EDTA system, as well as by a photofootprinting approach using the probe anthraquinone-2-sulfonate (AQS). In addition, this photosensitive reagent revealed the presence of sites of enhanced cutting specific to DB293. This molecule, but not other minor groove binders such as netropsin, induces specific local structural changes in DNA near certain binding sites, as independently shown by DNase I and the AQS probe. Recognition of the ATGA sequence by DB293 was investigated further using melting temperature experiments and surface plasmon resonance (SPR). The use of different hairpin oligonucleotides showed that DB293 can interact with AT sites via the formation of 1:1 drug-DNA complexes but binds much more strongly, and cooperatively, to ATGA-containing sequences to form 2:1 drug-DNA complexes. DB293 binds strongly to ATGA sequences with no significant context dependence but is highly sensitive to the orientation of the target sequence. The formation of 2:1 DB293/DNA complexes is abolished by reversing the sequence 5'-ATGA-->3'-ATGA, indicating that directionality plays an important role in the drug-DNA recognition process. Similarly, a single mutation in the A[T-->G]GA sequence is very detrimental to the dimer interactions of DB293. From the complementary footprinting and SPR data, the 5'-ATGA sequence is identified as being a highly favored dimer binding site for DB293. The data provide clues for delineating a recognition code for diamidine-type minor groove binding agents, and ultimately to guide the rational design of gene regulatory molecules targeted to specific sites of the genetic material.     

Amplification of apoptosis through sequential caspase cleavage of the MET tyrosine kinase receptor

Activation of the MET tyrosine kinase receptor by hepatocyte growth factor/scatter factor is classically associated with cell survival. Nonetheless, stress stimuli can lead to a caspase-dependent cleavage of MET within its juxtamembrane region, which generate a proapoptotic 40 kDa fragment (p40 MET). We report here that p40 MET is in fact generated through an additional caspase cleavage of MET within its extreme C-terminal region, which removes only few amino acids. We evidenced a hierarchical organization of these cleavages, with the C-terminal cleavage favoring the juxtamembrane one. As a functional consequence, the removal of the last amino acids of p40 MET increases its apoptotic capacity. Finally, cells expressing a MET receptor mutated at the C-terminal caspase site are unable to generate p40 MET and are resistant to apoptosis, indicating that generation of p40 MET amplifies apoptosis. These results revealed a two-step caspase cleavage of MET resulting in the reshaping of this survival receptor to a proapoptotic factor.     

Proteolytic cleavage of retinoblastoma protein upon DNA damage and Fas-mediated apoptosis

Proteolytic cleavage of key cellular proteins by caspases (ICE, CPP32, and Ich-1/Nedd2) may be crucial to the apoptotic process. The retinoblastoma tumor suppressor gene is a negative regulator of cell growth and the retinoblastoma protein (pRb) exhibits anti-apoptotic function. We show that pRb is cleaved during apoptosis induced by either UV irradiation or anti-Fas antibody. Our studies implicate CPP32-like activity in the proteolytic cleavage of pRb. The kinetics of proteolytic cleavage of pRb during apoptosis differ from that observed for other cellular proteins, suggesting that the specific cleavage of pRb during apoptosis may be an important event.     

Association between DNA cleavage during apoptosis and regions of chromatin replication

We have addressed the association between the site of DNA cleavage during apoptosis and DNA replication. DNA double strand breaks were introduced into chromatin containing pulse labeled nascent DNA by the induction of apoptosis or autocleavage of isolated nuclei. The location of these breaks in relation to nascent DNA were revealed by Bal 31 exonuclease digestion at the cut sites. Our data show that Bal31 accessible cut sites are directly linked to regions enriched in nascent DNA. We suggest that these regions coincide with the termini of replication domains, possibly linked by strong DNA-matrix interactions with biophysically defined topological structures of 0.5 - 1.3 Mbp in size. The 50 kbp fragments that are commonly observed as products of apoptosis are also enriched in nascent DNA within internal regions but not at their termini. It is proposed that these fragments contain a subset of replicon DNA that is excised during apoptosis through recognition of their weak attachment to the nuclear matrix within the replication domain.J. Cell. Biochem. 70:604-615, 1998. © 1998 Wiley-Liss, Inc. © 1998 Wiley-Liss, Inc.     

Role for caspase-mediated cleavage of Rad51 in induction of apoptosis by DNA damage

We report here that the Rad51 recombinase is cleaved in mammalian cells during the induction of apoptosis by ionizing radiation (IR) exposure. The results demonstrate that IR induces Rad51 cleavage by a caspase-dependent mechanism. Further support for involvement of caspases is provided by the finding that IR-induced proteolysis of Rad51 is inhibited by Ac-DEVD-CHO. In vitro studies show that Rad51 is cleaved by caspase 3 at a DVLD/N site. Stable expression of a Rad51 mutant in which the aspartic acid residues were mutated to alanines (AVLA/N) confirmed that the DVLD/N site is responsible for the cleavage of Rad51 in IR-induced apoptosis. The functional significance of Rad51 proteolysis is supported by the finding that, unlike intact Rad51, the N- and C-terminal cleavage products fail to exhibit recombinase activity. In cells, overexpression of the Rad51(D-A) mutant had no effect on activation of caspase 3 but did abrogate in part the apoptotic response to IR exposure. We conclude that proteolytic inactivation of Rad51 by a caspase-mediated mechanism contributes to the cell death response induced by DNA damage.     

XIAP regulates DNA damage-induced apoptosis downstream of caspase-9 cleavage

The IAP (inhibitor of apoptosis) family of anti-apoptotic proteins regulates programmed cell death. Of the six known human IAP-related proteins, XIAP is the most potent inhibitor. To study the mechanistic effects of XIAP on DNA damage-induced apoptosis, we prepared U-937 cells that stably overexpress XIAP. The results demonstrate that XIAP inhibits apoptosis induced by 1-[beta-d-arabinofuranosyl]cytosine (ara-C) and other genotoxic agents. XIAP had no detectable effect on ara-C-induced release of mitochondrial cytochrome c and attenuated cleavage of procaspase-9. In addition, we show that ara-C induces the association of XIAP with the cleaved fragments of caspase-9 and thereby inhibition of caspase-9 activity. The results also demonstrate that ara-C induces cleavage of procaspase-3 by a caspase-8-dependent mechanism and that XIAP inhibits caspase-3 activity. These results demonstrate that XIAP functions downstream of procaspase-9 cleavage as an inhibitor of both proteolytically processed caspase-9 and -3 in the cellular response to genotoxic stress.     

Detection of apoptosis induced DNA cleavage in scrapie-infected sheep brain

Abstract The pathogenesis and molecular basis of nerve cell death which accompanies scrapie infections in sheep are not well understood. Degeneration of neurons in culture caused by prion fragments has been reported to be consistent with mechanisms of cell death by apoptosis or programmed cell death. Apoptosis activation during prion-related encephalopathies has not yet been established in vivo. We report here the detection of DNA damage consistent with apoptosis in the brain cells of sheep infected with scrapie using laser scanning microscopic analysis of the single cell gel assay. We suggest that this DNA fragmentation is the result of the activation of the mechanisms characteristic of apoptotic cell death.     

DNA condensation by multivalent cations

In the presence of multivalent cations, high molecular weight DNA undergoes a dramatic condensation to a compact, usually highly ordered toroidal structure. This review begins with an overview of DNA condensation : condensing agents, morphology, kinetics, and reversibility, and the minimum size required to form orderly condensates. It then summarizes the statistical mechanics of the collapse of stiff polymers, which shows why DNA condensation is abrupt and why toroids are favored structures. Various ways to estimate or measure intermolecular forces in DNA condensation are discussed, all of them agreeing that the free energy change per base pair is very small, on the order of 1% of thermal energy. Experimental evidence is surveyed showing that DNA condensation occurs when about 90% of its charge is neutralized by counterions. The various intermolecular forces whose interplay gives rise to DNA condensation are then reviewed. The entropy loss upon collapse of the expanded wormlike coil costs free energy, and stiffness sets limits on tight curvature. However, the dominant contributions seem to come from ions and water. Electrostatic repulsions must be overcome by high salt concentrations or by the correlated fluctuations of territorially bound multivalent cations. Hydration must be adjusted to allow a cooperative accommodation of the water structure surrounding surface groups on the DNA helices as they approach. Undulations of the DNA in its confined surroundings extend the range of the electrostatic forces. The condensing ions may also subtly modify the local structure of the double helix. © 1998 John Wiley & Sons, Inc. Biopoly 44: 269–282, 1997     

Comparison of the hammerhead cleavage reactions stimulated by monovalent and divalent cations

Although the hammerhead reaction proceeds most efficiently in divalent cations, cleavage in 4 M LiCl is only approximately 10-fold slower than under standard conditions of 10 mM MgCl2 (Murray et al., Chem Biol, 1998, 5:587-595; Curtis & Bartel, RNA, 2001, this issue, pp. 546-552). To determine if the catalytic mechanism with high concentrations of monovalent cations is similar to that with divalent cations, we compared the activities of a series of modified hammerhead ribozymes in the two ionic conditions. Nearly all of the modifications have similar deleterious effects under both reaction conditions, suggesting that the hammerhead adopts the same general catalytic structure with both monovalent and divalent cations. However, modification of three ligands previously implicated in the binding of a functional divalent metal ion have substantially smaller effects on the cleavage rate in Li+ than in Mg2+. This result suggests that an interaction analogous to the interaction made by this divalent metal ion is absent in the monovalent reaction. Although the contribution of this divalent metal ion to the overall reaction rate is relatively modest, its presence is needed to achieve the full catalytic rate. The role of this ion appears to be in facilitating formation of the active structure, and any direct chemical role of metal ions in hammerhead catalysis is small.     

Copper-dependent cleavage of DNA by bleomycin

DNA strand scission by bleomycin in the presence of Cu and Fe was further characterized. It was found that DNA degradation occurred readily upon admixture of Cu(I) or Cu(II) + dithiothreitol + bleomycin, but only where the order of addition precluded initial formation of Cu(II)--bleomycin or where sufficient time was permitted for reduction of the formed Cu(II)--bleomycin to Cu(I)--bleomycin. DNA strand scission mediated by Cu + dithiothreitol + bleomycin was inhibited by the copper-selective agent bathocuproine when the experiment was carried out under conditions consistent with Cu chelation by bathocuproine on the time scale of the experiment. Remarkably, it was found that the extent of DNA degradation obtained with bleomycin in the presence of Fe and Cu was greater than that obtained with either metal ion alone. A comparison of the sequence selectivity of bleomycin in the presence of Cu and Fe using 32P-end-labeled DNA duplexes as substrates revealed significant differences in sites of DNA cleavage and in the extent of cleavage at sites shared in common. For deglycoblemycin and decarbamoylbleomycin, whose metal ligation is believed to differ from that of bleomycin itself, it was found that the relative extents of DNA cleavage in the presence of Cu were not in the same order as those obtained in the presence of Fe. The bleomycin-mediated oxygenation products derived from cis-stilbene were found to differ in type and amount in the presence of added Cu vs. added Fe. Interestingly, while product formation from cis-stilbene was decreased when excess Fe was added to a reaction mixture containing 1:1 Fe(III) and bleomycin, the extent of product formation was enhanced almost 4-fold in reactions that contained 5:1, as compared to 1:1, Cu and bleomycin. The results of these experiments are entirely consistent with the work of Sugiura [Sugiura, Y. (1979) Biochem. Biophys. Res. Commun. 90, 375-383], who first demonstrated the generation of reactive oxygen species upon admixture of O2 and Cu(I)--bleomycin.     

Specificity of DNA cleavage by ultrasound

Cleavage of double-stranded DNA fragments with known nucleotide sequences upon sonication at 22 and 44 kHz was studied by PAGE. The cleavage rate was shown to depend on the fragment size, pH, ionic strength, and temperature. Double-strand breaks occurred preferentially in 5′-CpG-3′ dinucleotides. The strand was broken between C and G so that the phosphate group was at the 5′ side of G in the products. The cleavage rate proved to depend on the sequences flanking the cleavage site. The character of cleavage changed in the presence of Pt-bis-netropsin, a sequence-specific ligand that alters the local conformation of DNA.     

Copper-catalyzed cleavage of DNA by arenes

DNA was found to be cleaved in neutral solutions containing arenes and copper (II) salts. The reaction is comparable in efficiency with the DNA cleavage by such systems as Cu(II)-phenanthroline and Cu(II)-ascorbic acid, but, in contrast to the latter, the system Cu(2+)-arene does not require the presence of an exogenous reducing agent or hydrogen peroxide. The system Cu(2+)-arene does not cleave DNA under anaerobic conditions. Catalase, sodium azide, and bathocuproine, which is a specific chelator of Cu(I), completely inhibit the reaction. The data obtained allow one to suppose that Cu(I) ions, superoxide radical, and singlet oxygen participate in the reaction. It has been shown by the EPR method using spin traps that the reaction proceeds with formation of alkoxyl radicals, which can insert breaks in the DNA molecule. For effective cleavage of DNA in the Cu(II)-o-bromobenzoic acid system, the radicals have to be generated by a specific copper-DNA-o-bromobenzoic acid complex, in which copper ions are most probably coordinated with oxygen atoms of the DNA phosphate groups. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.     

Light-induced DNA cleavage by esperamicin and neocarzinostatin

Ultraviolet radiation of the enediyne drugs is effective in causing nicks in supercoiled DNA. Of special interest is the fact that the observed nucleotide cleaving specificity for the UV light- and thiol-activated antibiotics was the same with esperamicin A1, but was different with neocarzinostatin. In addition to the preferred cutting of T and A bases, the light-activated neocarzinostatin attacked certain G bases which were rarely cleaved by the thiol-activated neocarzinostatin. It should be noted that these enediyne antibiotics lose the DNA breakage activity after light-exposure for 30 min.     

Influence of compound structure on affinity, sequence selectivity, and mode of binding to DNA for unfused aromatic dications related to furamidine

In the course of a program aimed at developing sequence-specific gene-regulatory small organic molecules, we have investigated the DNA interactions of a new series of nine diphenylfuran dications related to the antiparasitic drug furamidine (DB75). Two types of structural modifications were tested: the terminal amidine groups of DB75 were shifted from the para to the meta position, and the amidines were replaced with imidazoline or dimethyl-imidazoline groups, to test the importance of both the position and nature of positively charged groups on DNA interactions. The interactions of these compounds with DNA and oligonucleotides were studied by a combination of biochemical and biophysical techniques. Absorption and CD measurements suggested that the drugs bind differently to AT and GC sequences in DNA. The para-para dications, like DB75, bind into the minor groove of poly(dAT)(2) and intercalate between the base pairs of poly(dGC)(2), as revealed by electric linear dichroism experiments. In contrast, the meta-meta compounds exhibit a high tendency to intercalate into DNA whatever the target sequence. The lack of sequence selectivity of the meta-meta compounds containing amidines or dimethyl-imidazoline groups was also evident from DNase I footprinting and surface plasmon resonance (SPR) experiments. Accurate binding measurements using the BIAcore SPR method revealed that all nine compounds bind with similar affinity to an immobilized GC sequence DNA hairpin but exhibit very distinct affinities for the corresponding AT hairpin oligonucleotide. The minor groove-binding para-para compounds have a high specificity for AT sequences. The biophysical data clearly indicate that shifting the cationic substituents from the para to the meta position results in a loss of specificity and change in binding mode. The strong AT selectivity of the para-para compounds was independently confirmed by DNase I footprinting experiments performed with a range of DNA restrictions fragments. In terms of AT selectivity, the compounds rank in the order para-para > para-meta > meta-meta. The para dications bind preferentially to sequences containing four contiguous AT base pairs. Additional footprinting experiments with substrates containing the 16 possible [A.T](4) blocks indicated that the presence of a TpA step within an [A.T] (4) block generally reduces the extent of binding. The diverse methods, from footprinting to SPR to dichroism, provide a consistent model for the interactions of the diphenylfuran dications with DNA of different sequences. Altogether, the results attest unequivocally that the binding mode for unfused aromatic cations can change completely depending on substituent position and DNA sequence. These data provide a rationale to explain the relationships between sequence selectivity and mode of binding to DNA for unfused aromatic dications related to furamidine.