Acridine dimers: influence of the intercalating ring and of the linking-chain nature on the equilibrium and kinetic DNA-binding parameters

The rigidity of the linking chain of bifunctional intercalators in the ditercalinium series was shown to be critical for antitumor activity. In order to study the influence of the rigidity of the linking chain on the DNA-binding properties of DNA bifunctional intercalators, fluorescent 9-aminoacridine and 2-methoxy-6-chloro-9-aminoacridine analogues with chains of variable rigidity were synthesized. 1H-NMR studies show that the conformation of 9-aminoacridine dimers is almost independent of the nature of the linking chain. A strong self-stacking of the aromatic rings of the 2-methoxy-6-chloro-9-aminoacridine is observed for dimers with flexible chains but not for those with rigid chains. All the dimers having a linking chain long enough to bisintercalate in DNA according to the excluded site model are indeed bisintercalators. The kinetic association constant of all monomers and dimers for poly[d(A-T)].poly[d(A-T)] are in the same range (2-4 x 10(7) M-1 s-1). The large increase of DNA binding affinity observed for the dimers is always associated with the expected decrease of the dissociation rate constant. The effect of chain rigidity and pH on the calf thymus DNA binding of 9-aminoacridine and 2-methoxy-6-chloro-9-aminoacridine dimers is quite different. In the series of 9-aminoacridine the pKa of the dimers remains high and therefore no difference of DNA-binding affinity is observed between pH 5 and 7.4. The rigidity of the linking chain does not significantly alter the DNA-binding affinity. In the 2-methoxy-6-chloro-9-aminoacridine series, the pKa of all dimers became smaller than the physiological pH and a dramatic decrease of DNA-binding affinity is observed when the pH is increased from pH 5 to 7.4. This decrease appears significantly smaller for dimers with rigid chains. A similar dramatic decrease of binding affinity at pH 7.4 is not observed for poly[d(A-T)].poly[d(A-T)]. This factor makes these dimers strongly specific for the alternating polymer at pH 7.4.     

Structural limitations on the bifunctional intercalation of diacridines into DNA

An homologous series of diacridines containing two 9-aminoacridine chromophores linked via a simple methylene chain has been studied in order to investigate the minimum interchromophore separation required to permit bifunctional intercalation. Viscometric, sedimentation, and electric dichroism experiments show that compounds having one to four methylene groups in the linker are restricted to monofunctional intercalation, whereas the interaction becomes bifunctional when the chain length is increased to six carbons or more. The results indicate that bifunctional reaction occurs with an interchromophore distance not exceeding 8.8 A, implying that intercalation by these compounds is not subject to neighbor exclusion if the mode of binding is of the classical intercalation type.     

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.     

Molecular mechanisms of chemical mutagenesis: 9-aminoacridine inhibits DNA replication in vitro by destabilizing the DNA growing point and interacting with the DNA polymerase

9-Aminoacridine was found to inhibit dNTP incorporation into DNA homopolymer duplexes by phage T4 DNA polymerase in vitro. Systematic variation of the molar ratio of 9-aminoacridine to DNA, to DNA polymerase, and to DNA precursors demonstrated that this inhibition at 9-aminoacridine concentrations below 10 microM was mainly due to interaction of 9-aminoacridine with the DNA and suggested that the basis for the preferential inhibition of incorrect precursor incorporation was destabilization of the DNA growing point. Consistent with destabilization, 9-aminoacridine stimulated the hydrolysis of correctly base paired DNA by the 3'-5' exonuclease activity of phage T4 DNA polymerase. This is the first indication to my knowledge that an intercalating dye destabilizes the DNA growing point, whereas it raises the overall Tm of the DNA. At 9-aminoacridine concentrations above 10 microM overall incorporation of dNTPs was inhibited by 9-aminoacridine interaction with the DNA polymerase. A possible explanation for the induction of both deletion and addition frameshift mutations by 9-aminoacridine during DNA biosynthesis is discussed in light of growing-point destabilization.     

9-Aminoacridine blocks NMDA and AMPA receptors by different mechanisms

Tricyclic mono- and dicationic compounds (derivatives of 9-aminoacridine) antagonize AMPA and NMDA glutamate receptors. The aim of the present study was to compare mechanisms of the 9-aminoacridine action on AMPA and NMDA receptors. Experiments were carried out by whole-cell patch-clamp technique on native receptors from rat brain neurons. An important peculiarity of the 9-aminoacridine action on NMDA receptors is the large slope of the concentration dependence, which suggests the binding of two molecules in the channel. AMPA receptors blockade also demonstrated interesting features. In contrast to the NMDA receptor channel block, inhibition of AMPA receptors is voltage-independent. 9-Aminoacridine and its dicationic analog demonstrated similar anti-AMPA activity. For classical AMPA-receptor channel blockers (derivatives of adamantane and phenylcyclohexyl) it was demonstrated that dicationic analogs are much more potent than monocationic analogs. We conclude that 9-aminoacridine binds to a specific site in AMPA receptors. This finding opens a possibility to develop a new family of non-competitive antagonists of AMPA receptors.     

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.     

DNA binding, cytotoxicity and inhibitory effect on RNA synthesis of two new 1-nitro-9-aminoacridine dimers

Two 1-nitro-9-aminoacridine dimers were prepared: one bearing a spermine flexible linking chain, compound 4, the other a rigid dipiperidine-type linker, compound 7. Both dimers elicited a higher affinity constant for DNA than the parent monomeric drug nitracrine 2. This affinity was several orders lower than what was found for other dimeric compounds having the same linkers and no nitro group on the acridine ring (3, 5, 6 and 8). Bisintercalation was evidenced for compound 4 by viscosimetric measurements. In the absence of dithiothreitol, an inhibitory effect of RNA synthesis in vitro was observed for all the tested compounds except 2 and 7. In the presence of dithiothreitol, 4 and 7 formed irreversible complexes with DNA of decreased template properties. The level of the dimers binding was lower than that of the parent compound 2. Cross-links were detected by means of hydroxylapatite chromatography in a complex of the dimer bearing a flexible linking chain, compound 4 with DNA, while the compound 7-DNA complex eluted in the single-stranded DNA region. The extent of cytotoxicity of the two 1-nitro-9-aminoacridine dimers against L1210 cultured cells was different.     

Characteristics of the Quenching of 9-Aminoacridine Fluorescence by Liposomes Made from Plant Lipids

Several laboratories have determined the surface charge density of membranes utilizing methods based on vesicle-induced quenching of the fluorescence of 9-aminoacridine and its relief by other cations. However, the computational methods by which surface charge density were calculated have not been verified in a model system. In this study, the quenching of 9-aminoacridine fluorescence by liposomes made from varying amounts of digalactosyldiacylglyceride and phosphatidic acid and relief of quenching by salts was examined. Quenching of 9-aminoacridine fluorescence increased with increasing amounts of phosphatidic acid added, independent of the composition of the added liposomes. In certain instances, the computational methods did not yield the surface charge density of the liposomes expected from their composition. However, when the effects of background ionic strength on surface potential were considered, there was a positive correlation between expected and calculated values. Therefore, the data support the contention that changes in the fluorescence of 9-aminoacridine can be used to calculate surface charge density of membranes. Received: 29 November 1999/Revised: 31 July 2000     

Sodium-23 and lithium-7 NMR spin-lattice relaxation measurements in the study intercalation in DNA.

Sodium-23 spin-lattice relaxation rate (the reciprocal relaxation time) measurements have been used to study the intercalation of 9-aminoacridine in calf thymus DNA. The results are analyzed by a two state model based on the counterion condensation theory and a theory for the quadrupolar relaxation of counterions in polyelectrolyte solutions. It is shown that change of the solvent from H2O to D2O has a negligible effect on the intercalation process. Furthermore, an attempt is made to analyze the dependence of the 7Li spin-lattice relation rate on intercalation of 9-aminoacridine in LiDNA. It is shown that both quadrupolar and dipolar mechanisms contribute to the bound 7Li relation rate, and that both these contributions are reduced upon intercalation of 9-aminoacridine.     

Azido analogs of acridine: Photoaffinity probes for frameshift mutagenesis in Salmonella typhimurium

In order to identify a photoaffinity probe for 9-aminoacridine frameshift mutagenesis, 20 azido analogs of acridine were synthesized and tested in Ames' Salmonella tester strains, TA1535, TA1537, TA1538 and their corresponding excision-repair-proficient strains TA1975, TA1977, and TA1978, to determine their mutagenicity and toxicity relative to 9-aminoacridine. The substituent-mutagenicity patterns observed for these compounds agree very well with those obtained previously for non-azidoacridines. The results presented here show that the 2-azido-analog of 9-aminoacridine demonstrates biological activity similar to 9-aminoacridine prior to photolytic activation. With light activation, however, the 9-amino-2-azido derivative becomes more effective at producing frameshift mutations characteristics of 9-aminoacridine. Furthemore, this photolytic enhancement of mutagenesis appears to be due to the repairable lesion suggesting that covalent attachment of the drug occurs.     

9-Aminoacridine as a fluorescent probe of the electrical diffuse layer associated with the membranes of plant mitochondria.

1. Mitochondria from Jerusalem artichoke (Helianthus tuberosus) tubers and Arum maculatum spadices caused a quenching of the fluorescence of 9-aminoacridine when mixed in a low-cation medium (approximately 1 mM-K+) and addition of chelators further decreased the fluorescence. Salts released the quenching of the 9-aminoacridine fluorescence and the efficiency of the release appeared to be mainly dependent on the valency of the cation (C3+ greater than C2+ greater than C+). 2. The results are consistent with the theory of charge screening and demonstrate that 9-aminoacridine is a convenient probe of the behaviour of cations on the membranes of mitochondria and in the diffuse layer associated with these membranes. 3. The concentration of salt required to achieve half-maximal release of quenching of 9-aminoacridine fluorescence was proportional to the concentration of mitochondria in the solution and theoretical considerations show this effect to be inherent in the Gouy-Chapman theory. 4. 9-Aminoacridine was removed from the bulk of the solution by the mitochondria to a far greater extent than was Na+ or K+, which is suggested to be due to the formation of bi- and poly-valent cations by aggregation of 9-aminoacridine molecules in the diffuse layer. This would have implications for the use of 9-aminoacridine to determine delta pH across membranes. 5. Jerusalem-artichoke mitochondria removed from 9-aminoacridine and Ca2+ from the bulk of the solution and required more ions to screen the membranes than did an equal concentration (mg of protein/ml) of Arum mitochondria, indicating that Jerusalem-artichoke mitochondria contain more negative charges per mg of protein.     

delta pH-induced fluorescence quenching of 9-aminoacridine in lipid vesicles is due to excimer formation at the membrane.

The fluorescence of 9-aminoacridine (9-AA) is quenched in vesicular suspensions containing negatively charged lipid headgroups (e.g., phosphatidylserine) upon imposition of a transmembrane (inside acidic) pH-gradient. It is shown that this fluorescence loss is accompanied by the formation of 9-AA dimers that undergo a transition in the dimer excited state to a dimer-excimer state. This result has been obtained on the basis of the specific dimer fluorescence excitation and hypochromic absorbance spectra that are redshifted by maximally 275 cm-1 (4.4 nm) with respect to the corresponding monomer spectra, as well as by the detection of the characteristic broad excimer emission band, centered at 560 nm. The existence of the spectrally distinct dimer-excimer is further corroborated by fluorescence life-time measurements that indicate an increased lifetime of up to 24 ns for this complex as compared with the normal monomer fluorescence lifetime of 16 ns. The formation of this dimer-excimer complex from the monomers can be reversed completely and the original monomeric spectral properties restored after the abolishment of the electrochemical proton gradient. In addition to the delta pH-induced dimer redshift in absorbance and fluorescence excitation, a further small redshift in monomer absorbance, fluorescence excitation, and emission spectra is observed due solely to the presence of the negatively charged phospholipid headgroups.     

Mechanisms of blockade of glutamate receptor ionic channels: Paradox of 9-aminoacridine

9-Aminoacridine and tacrine differ from other channel blockers of NMDA receptors in that their binding prevents the closing of blocked channels and subsequent dissociation of the agonist. Structural determinants of aminoacridine derivatives underlying the blocking mechanism are still unknown. The aim of this study was to elucidate the effects of a dicationic 9-aminoacridine derivative and some other tricyclic compounds on NMDA receptors of rat hippocampal pyramidal neurons. All the compounds under study are voltage-dependent blockers of NMDA channels; their IC₅₀ values recorded at −80 mV vary from 1 to 50 μM. The dicationic derivatives demonstrate the same voltage dependence of the block as the monocationic derivatives. The monoand dicationic tricyclic compounds under study are weak blockers of AMPA receptor channels and differ from adamantane, phenylcyclohexyl and other dicationic derivatives that exhibit greater voltage dependence of the NMDA channel block and are able to induce effective suppression of AMPA channels. We conclude that the mechanisms of action of the tricyclic and dicationic 9-aminoacridine derivatives are different from that of 9-aminoacridine, since these compounds do not prevent closing of the blocked channels. This suggests that the binding site for 9-aminoacridine has specific properties and high selectivity with respect to ligand structure. Original Russian Text ? K.H. Kim, V.E. Gmiro, D.B. Tikhonov, L.G. Magazanik, 2007, published in Biologicheskie Membrany, 2007, Vol. 24, No. 1, pp. 96–104.     

The inhibition of ribonucleic acid polymerase by acridines

1. The aminoacridines, proflavine (3,6-diaminoacridine) and 9-aminoacridine, and a hydrogenated derivative, 9-amino-1,2,3,4-tetrahydroacridine, were shown to inhibit in vitro the DNA-primed RNA polymerase of Escherichia coli. The inhibition is strong with both proflavine and 9-aminoacridine, but weak with 9-amino-1,2,3,4-tetrahydroacridine. 2. The extent to which the three acridines bind to calf-thymus DNA in the enzyme medium was studied spectrophotometrically. The extent of binding decreases in the order: proflavine, 9-aminoacridine, 9-amino-1,2,3,4-tetrahydroacridine. Some evidence was also obtained for interaction between the nucleoside triphosphate substrates and proflavine or 9-aminoacridine; no such interaction was detectable with 9-amino-1,2,3,4-tetrahydroacridine. 3. Although the amount of acridine bound to DNA increases with increasing inhibition, a stage is reached where an increase in acridine concentration still causes an increase in inhibition, with practically no increase in the amount bound to DNA. 4. Plots of reciprocal rates against the reciprocal of DNA concentration were linear and had a common intercept when proflavine or 9-aminoacridine was present. Similar relations were obtained when the reciprocal concentration of nucleoside triphosphates was plotted. The observations are interpreted kinetically in terms of a competitive inhibition of the enzyme by proflavine or 9-aminoacridine and of a kinetic role for the DNA analogous to ;activation'. 5. This suggests that inhibitory acridine molecules can occupy the sites on the RNA polymerase that are specific for binding the nucleoside triphosphate substrate or the bases of the DNA, when these become accessible during the copying process.     

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.     

DNA polyintercalation: comparison of DNA binding properties of an acridine dimer and trimer

The DNA binding characteristics of a mono-, di- and trimeric derivative of 9-aminoacridine were studied. The length of the linking carboxamidoalkyl chains was selected to allow bis- or tris-intercalation according to the excluded-site model. Measurements of DNA unwinding angle using closed circular DNA showed that the trimeric derivative behaves as a tris-intercalating agent. Nevertheless the increase of DNA binding affinity on going from dimer to trimer was found to be relatively small. This is probably related to the large structural constraint for DNA binding of the trimeric derivative. The nature of the linking chain for the design of high-affinity DNA poly-intercalating agents appears therefore critical.     

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.     

Allosteric activation by dimerization of the PknD receptor Ser/Thr protein kinase from Mycobacterium tuberculosis

To define how extracellular signals activate bacterial receptor Ser/Thr protein kinases, we characterized the regulatory functions of a weak dimer interface identified in the Mycobacterium tuberculosis PknB and PknE receptor kinases. Sequence comparisons revealed that the analogous interface is conserved in PknD orthologs from diverse bacterial species. To analyze the roles of dimerization, we constructed M. tuberculosis PknD kinase domain (KD) fusion proteins that formed dimers upon addition of rapamycin. Dimerization of unphosphorylated M. tuberculosis PknD KD fusions stimulated phosphorylation activity. Mutations in the dimer interface reduced this activation, limited autophosphorylation, and altered substrate specificity. In contrast, an inactive catalytic site mutant retained the ability to stimulate the wild-type KD by dimerization. These results support the idea that dimer formation allosterically activates unphosphorylated PknD. The phosphorylated PknD KD was fully active even in the absence of dimerization, suggesting that phosphorylation provides an additional regulatory mechanism. The conservation of analogous dimers in diverse prokaryotic and eukaryotic Ser/Thr protein kinases implies that this mechanism of protein kinase regulation is ancient and broadly distributed.     

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).     

Auto‐activation mechanism of the Mycobacterium tuberculosis PknB receptor Ser/Thr kinase

Many Ser/Thr protein kinases are activated by autophosphorylation, but the mechanism of this process has not been defined. We determined the crystal structure of a mutant of the Ser/Thr kinase domain (KD) of the mycobacterial sensor kinase PknB in complex with an ATP competitive inhibitor and discovered features consistent with an activation complex. The complex formed an asymmetric dimer, with the G helix and the ordered activation loop of one KD in contact with the G helix of the other. The activation loop of this putative ‘substrate’ KD was disordered, with the ends positioned at the entrance to the partner KD active site. Single amino‐acid substitutions in the G‐helix interface reduced activation‐loop phosphorylation, and multiple replacements abolished KD phosphorylation and kinase activation. Phosphorylation of an inactive mutant KD was reduced by G‐helix substitutions in both active and inactive KDs, as predicted by the idea that the asymmetric dimer mimics a trans‐autophosphorylation complex. These results support a model in which a structurally and functionally asymmetric, ‘front‐to‐front’ association mediates autophosphorylation of PknB and homologous kinases.