Withdrawn: Variations in Altered Global Gene Expression caused by Topoisomerase II Poisons Assessed with Reference to Differences in DNA Binding

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Characterisation of bis(4-aminoquinoline)s as α1A adrenoceptor allosteric modulators.

The development of sub-type selective α1 adrenoceptor ligands has been hampered by the high sequence similarity of the amino acids forming the orthosteric binding pocket of the three α1 adrenoceptor subtypes, along with other biogenic amine receptors. One possible approach to overcome this issue is to target allosteric sites on the α1 adrenoceptors. Previous docking studies suggested that one of the quinoline moieties of a bis(4-aminoquinoline), comprising a 9-carbon methylene linker attached via the amine groups, could interact with residues outside of the orthosteric binding site while, simultaneously, the other quinoline moiety bound within the orthosteric site. We therefore hypothesized that this compound could act in a bitopic manner, displaying both orthosteric and allosteric binding properties. To test this proposition, we investigated the allosteric activity of a series of bis(4-aminoquinoline)s with linker lengths ranging from 2 to 12 methylene units (designated C2-C12). A linear trend of increasing [3H]prazosin dissociation rate with increasing linker length between C7 and C11 was observed, confirming their action as allosteric modulators. These data suggest that the optimal linker length for the bis(4-aminoquinoline)s to occupy the allosteric site of the α1A adrenoceptor is between 7 and 11 methylene units. In addition, the ability of C9 bis(4-aminoquinoline) to modulate the activation of the α1A adrenoceptor by norepinephrine was subsequently examined, showing that C9 acts as a non-competitive antagonist. Our findings indicate that the bis(4-aminoquinolines) are acting as allosteric modulators of orthosteric ligand binding, but not efficacy, in a bitopic manner.

Molecular dynamic simulations of diacridine binding to DNA: Indications that C6 diacridine can bisintercalate spanning two base pairs.

Dimers of 9-aminoacridine linked via the 9-amino group with polymethylene chains, termed diacridines, are known to bisintercalate into DNA when the linker comprises 6 or more methylene units. There are no literature reports of crystal or NMR solution structures for bisintercalated diacridine-DNA complexes, and the issue of the structure of the C6 ([CH2 ]n linker where n = 6) diacridine complex remains unresolved. Previously, based on simple geometric considerations, it was proposed that C6 diacridine could only span a single base pair, which requires that its bifunctional reaction violates the widely-observed "neighbor exclusion principle" where bound intercalators are separated by at least 2 base pairs. Here we have explored the structure of diacridine-DNA complexes using unrestrained molecular dynamics in explicit solvent using the parmbsc0 forcefield in AMBER14. We studied the C4 to C8 dimers, intercalated via both the minor and major DNA grooves, to a variety of nucleotide sequences. We find that C6, C7, and C8 diacridine are able to form 2 base pair bisintercalated complexes from either groove, whereas the C4 and C5 homologues cannot. We conclude that C6 diacridine does have the capacity to bisintercalate without violating neighbor exclusion, and that the previous proposed binding model needs revision.

Structures and dynamics of DNA complexes of the desmethyl analog of the cytotoxin MLN944: Insights into activity when a methyl isn't futile.

Structure activity relationships for tricyclic-carboxamide topoisomerase II poisons indicate that cytotoxicity is enhanced by the presence of methyl, and other, groups in the position peri to the carboxamide. Linked dimers of phenazine-1-carboxamides are potent cytotoxins and one phenazine dimer, MLN944 (alternatively XR5944), has been in clinical trial. MLN944 is a template inhibitor of transcription, whereas corresponding monomers are not. Nevertheless, its cytotoxic potency is also diminished by removal of its peri methyl groups. Here, we describe NMR and molecular dynamic studies of the interaction of desmethyl MLN944 with d(ATGCAT)2 , d(TATGCATA)2 , and d(TACGCGTA)2 to investigate the influence of the nine-methyl group on the structure of MLN944 complexes. As with MLN944, the carboxamide group hydrogen bonds to the phenazine ring nitrogen, the ligand sandwiches the central GC base pairs in the major groove, and the protonated linker amines hydrogen bond primarily to the O6 atom of the guanines. Molecular dynamics studies reveal that the linker exists in multiple conformations, none of which produce an ideal set of hydrogen bonds. In distinction, however, the carboxamide-to-phenazine ring nitrogen hydrogen bond is weaker, the overall helix winding is less and the NMR resonances are broader in the desmethyl complexes. Exchange between free and complexed DNA, quantified using two-dimensional NOESY spectra, is faster for the desmethyl MLN944 complexes than for MLN944 complexes. Overall, the data suggest that desmethyl MLN944 DNA complexes are "looser" and more unwound at the binding site, leading to faster dissociation rates, which could account for the diminished efficacy of the desmethyl analog.

Homobivalent Conjugation Increases the Allosteric Effect of 9-aminoacridine at the α1-Adrenergic Receptors.

The α1-adrenergic receptors are targets for a number of cardiovascular and central nervous system conditions, but the current drugs for these receptors lack specificity to be of optimal clinical value. Allosteric modulators offer an alternative mechanism of action to traditional α1-adrenergic ligands, yet there is little information describing this drug class at the α1-adrenergic receptors. We have identified a series of 9-aminoacridine compounds that demonstrate allosteric modulation of the α1A- and α1B-adrenergic receptors. The 9-aminoacridines increase the rate of [3H]prazosin dissociation from the α1A- and α1B-adrenergic receptors and noncompetitively inhibit receptor activation by the endogenous agonist norepinephrine. The structurally similar compound, tacrine, which is a known allosteric modulator of the muscarinic receptors, is also shown to be a modulator of the α1-adrenergic receptors, which suggests a general lack of selectivity for allosteric binding sites across aminergic G protein-coupled receptor. Conjugation of two 9-aminoacridine pharmacophores, using linkers of varying length, increases the potency and efficacy of the allosteric effects of this ligand, likely through optimization of bitopic engagement of the allosteric and orthosteric binding sites of the receptor. Such a bivalent approach may provide a mechanism for fine tuning the efficacy of allosteric compounds in future drug design efforts.

Novel DNA topoisomerase IIα inhibitors from combined ligand- and structure-based virtual screening.

DNA topoisomerases are enzymes responsible for the relaxation of DNA torsional strain, as well as for the untangling of DNA duplexes after replication, and are important cancer drug targets. One class of topoisomerase inhibitors, "poisons", binds to the transient enzyme-DNA complex which occurs during the mechanism of action, and inhibits the religation of DNA. This ultimately leads to the accumulation of DNA double strand breaks and cell death. Different types of topoisomerases occur in human cells and several poisons of topoisomerase I and II are widely used clinically. However, their use is compromised by a variety of side effects. Recent studies confirm that the inhibition of the α-isoform of topoisomerase II is responsible for the cytotoxic effect, whereas the inhibition of the ß-isoform leads to development of adverse drug reactions. Thus, the discovery of agents selective for topoisomerase IIα is an important strategy for the development of topoisomerase II poisons with improved clinical profiles. Here, we present a computer-aided drug design study leading to the identification of structurally novel topoisomerase IIα poisons. The study combines ligand- and structure-based drug design methods including pharmacophore models, homology modelling, docking, and virtual screening of the National Cancer Institute compound database. From the 8 compounds identified from the computational work, 6 were tested for their capacity to poison topoisomerase II in vitro: 4 showed selective inhibitory activity for the α- over the ß-isoform and 3 of these exhibited cytotoxic activity. Thus, our study confirms the applicability of computer-aided methods for the discovery of novel topoisomerase II poisons, and presents compounds which could be investigated further as selective topoisomerase IIα inhibitors.

Human α1-adrenoceptor subtype selectivity of substituted homobivalent 4-aminoquinolines.

A series of ring-substituted ethyl- and heptyl-linked 4-aminoquinoline dimers were synthesized and evaluated for their affinities at the 3 human α(1)-adrenoceptor (α(1)-AR) subtypes and the human serotonin 5-HT(1A)-receptor (5-HT(1A)-R). We find that the structure-specificity profiles are different for the two series at the α(1)-AR subtypes, which suggests that homobivalent 4-aminoquinolines can be developed with α(1)-AR subtype selectivity. The 8-methyl (8-Me) ethyl-linked analogue has the highest affinity for the α(1A)-AR, 7 nM, and the greatest capacity for discriminating between α(1A)-AR and α(1B)-AR (6-fold), α(1D)-AR (68-fold), and the 5-HT(1A)-R (168-fold). α(1B)-AR selectivity was observed with the 6-methyl (6-Me) derivative of the ethyl- and heptyl-linked 4-aminoquinoline dimers and the 7-methoxy (7-OMe) derivative of the heptyl-linked analogue. These substitutions result in 4- to 80-fold selectivity for α(1B)-AR over α(1A)-AR, α(1D)-AR, and 5-HT(1A)-R. In contrast, 4-aminoquinoline dimers with selectivity for α(1D)-AR are more elusive, since none studied to date has greater affinity for the α(1D)-AR over the other two α(1)-ARs. The selectivity of the 8-Me ethyl-linked 4-aminoquinoline dimer for the α(1A)-AR, and 6-Me ethyl-linked, and the 6-Me and 7-OMe heptyl-linked 4-aminoquinoline dimers for the α(1B)-AR, makes them promising leads for drug development of α(1A)-AR or α(1B)-AR subtype selective ligands with reduced 5-HT(1A)-R affinity.

The solution structure of bis(phenazine-1-carboxamide)-DNA complexes: MLN 944 binding corrected and extended.

MLN 944 is a bisintercalating DNA-binding antitumor agent known to be a template inhibitor of transcription. Previous (1) H NMR studies of its d(ATGCAT)2 complex concluded that its phenazine chromophores are protonated. However, we find that this is not so, which has important consequences for the charged state of the ligand, for the orientation of its 1-carboxamide group in the complex, and for the details of the interaction of its protonated interchromophore linker with the DNA base pairs. Here, we report a corrected solution structure of the MLN 944-d(ATGCAT)2 complex, and extend the study to complexes with d(TATGCATA)2 , and d(TACGCGTA)2 , using a variety of (1) H and (31) P NMR methods and molecular dynamics simulations employing the AMBER 12 force field. We find that for all three complexes MLN 944 binds as a dication, in which the chromophores are uncharged, in the DNA major groove spanning the central 2 GC base pairs in a manner that maintains the dyad symmetry of the DNA. The carboxamide group lies in the plane of the chromophore, its NH making hydrogen bonding interactions with the phenazine N10 nitrogen, and the protonated linkers form hydrogen bonds with the O6 atom of guanine. The dynamics simulations reveal extensive solvent interactions involving the linker amines, the carboxamide group, and the DNA bases.

α1-Adrenoceptor and serotonin 5-HT(1A) receptor affinity of homobivalent 4-aminoquinoline compounds: an investigation of the effect of linker length.

α1-adrenoceptor (α1-AR) subtype-selective ligands lacking off-target affinity for the 5-HT(1A) receptor (5-HT(1A)-R) will provide therapeutic benefits in the treatment of urogenital conditions such as benign prostatic hyperplasia. In this study we determined the affinity of 4-aminoquinoline and eleven homobivalent 4-aminoquinoline ligands (diquinolines) with alkane linkers of 2-12 atoms (C2-C12) for α(1A), α(1B) and α(1D)-ARs and the 5-HT(1A)-R. These ligands are α(1A)-AR antagonists with nanomolar affinity for α(1A) and α(1B)-ARs. They display linker-length dependent selectivity for α(1A/B)-ARs over α(1D)-AR and the 5-HT(1A)-R. The C2 diquinoline has the highest affinity for α1A-AR (pKi 7.60±0.26) and greater than 30-fold and 600-fold selectivity for α(1A)-AR over α(1D)-AR and 5-HT(1A)-R respectively. A decrease in affinity for α1-ARs is observed as the linker length increases, reaching a nadir at 5 (α(1A/1B)-ARs) or 6 (α(1D)-AR) atoms; after which affinity increases as the linker is lengthened, peaking at 9 (α(1A/1B/1D)-ARs) or 8 (5-HT(1A)-R) atoms. Docking studies suggest that 4-aminoquinoline and C2 bind within the orthosteric binding site, while for C9 one end is situated within the orthosteric binding pocket, while the other 4-aminoquinoline moiety interacts with the extracellular surface. The limited α(1D)-AR and 5-HT(1A)-R affinity of these compounds makes them promising leads for future drug development of α(1A)-AR selective ligands without α(1D)-AR and the 5-HT(1A)-R off-target activity.

Exploring DNA topoisomerase I ligand space in search of novel anticancer agents.

DNA topoisomerase I (Top1) is over-expressed in tumour cells and is an important target in cancer chemotherapy. It relaxes DNA torsional strain generated during DNA processing by introducing transient single-strand breaks and allowing the broken strand to rotate around the intermediate Top1-DNA covalent complex. This complex can be trapped by a group of anticancer agents interacting with the DNA bases and the enzyme at the cleavage site, preventing further topoisomerase activity. Here we have identified novel Top1 inhibitors as potential anticancer agents by using a combination of structure- and ligand-based molecular modelling methods. Pharmacophore models have been developed based on the molecular characteristics of derivatives of the alkaloid camptothecin (CPT), which represent potent antitumour agents and the main group of Top1 inhibitors. The models generated were used for in silico screening of the National Cancer Institute (NCI, USA) compound database, leading to the identification of a set of structurally diverse molecules. The strategy is validated by the observation that amongst these molecules are several known Top1 inhibitors and agents cytotoxic against human tumour cell lines. The potential of the untested hits to inhibit Top1 activity was further evaluated by docking into the binding site of a Top1-DNA complex, resulting in a selection of 10 compounds for biological testing. Limited by the compound availability, 7 compounds have been tested in vitro for their Top1 inhibitory activity, 5 of which display mild to moderate Top1 inhibition. A further compound, found by similarity search to the active compounds, also shows mild activity. Although the tested compounds display only low in vitro antitumour activity, our approach has been successful in the identification of structurally novel Top1 inhibitors worthy of further investigation as potential anticancer agents.

Intracellular trafficking as a determinant of AS-DACA cytotoxicity in rhabdomyosarcoma cells.

BACKGROUND: Rhabdomyosarcoma (RMS) is a malignant soft tissue sarcoma derived from skeletal muscle precursor cells, which accounts for 5-8% of all childhood malignancies. Disseminated RMS represents a major clinical obstacle, and the need for better treatment strategies for the clinically aggressive alveolar RMS subtype is particularly apparent. Previously, we have shown that the acridine-4-carboxamide derivative AS-DACA, a known topoisomerase II poison, is potently cytotoxic in the alveolar RMS cell line RH30, but is 190-fold less active in the embryonal RMS cell line RD. Here, we investigate the basis for this selectivity, and demonstrate in these RMS lines, and in an AS-DACA- resistant subclone of RH30, that AS-DACA-induced cytotoxicity correlates with the induction of DNA double strand breaks. RESULTS: We show that inhibition of the multidrug-resistance associated protein (MRP1) has no effect on AS-DACA sensitivity. By exploiting the pH-dependent fluorescence properties of AS-DACA, we have characterized its intracellular distribution, and show that it concentrates in the cell nucleus, as well as in acidic vesicles of the membrane trafficking system. We show that fluorescence microscopy can be used to determine the localization of AS-DACA to the nuclear and cytoplasmic compartments of RMS cells grown as spheroids, penetrance being much greater in RH30 than RD spheroids, and that the vesicular signal leads the way into the spheroid mass. EEA1 and Rab5 proteins, molecular markers expressed on early-endosomal vesicles, are reduced by >50% in the sensitive cell lines. CONCLUSION: Taking the evidence as a whole, suggests that endosomal vesicle trafficking influences the toxicity of AS-DACA in RMS cells.

Effects of DNA minor groove binding agents on global gene expression.

The capacity of two minor groove binding agents that differ in their DNA sequence selectivity to modulate gene expression in human leukaemia cells was investigated. The chosen compounds were the chromomycin A3, a GC selective minor groove binder, and alkamin, an AT selective minor groove binder. As revealed by DNA microarray analysis of 6000 genes, at equitoxic doses, 5×IC(50) values for growth inhibition, the two drugs disturbed transcription, resulting in both up- and down-regulation of many hundreds of genes, 24 h after drug exposure. Direct comparisons between the most affected genes and also the cluster analysis indicated a relatively low degree of similarity between the tow expression profiles. Moreover, the ontological and the pathway responses also indicated a distinguished biological responses. Chromomycin treatment was characterized by many negative impacts on the important cellular functions and by the activation for those functions that usually take the cells towards apoptosis. In the second biological profile, the domination of many positive functions might indicate that the cells were attempting to overcome and repair the alkamin assault. Examples of these functions are positive regulation of gene expression, positive regulation of macromolecule biosynthetic processes, the cell cycle pathway and DNA repair.

[Co(III)(cyclen)Cl2]Cl is selectively cytotoxic to human leukaemia cells.

Co(III)-cyclen complexes are known to cause DNA strand breaks by hydrolytically cleaving the phosphodiester backbone via a mechanism that does not require oxidation. Here, we report the first cytotoxicity study of [Co(III)(cyclen)Cl(2)]Cl (2), the parental example of this class of agent, which reveals that (2) is selectively toxic towards CCRF-CEM (IC(50)=32+/-10 microM), THP-1 (IC(50)=110+/-40 microM), and HL-60 (IC(50)=70+/-35 microM) human leukaemia cells, compared to human skin and lung fibroblasts (IC(50)>10 mM). Investigations of its effect on CCRF-CEM cells show it kills by apoptosis which was characterised by microscopy, flow cytometry, and in vitro NMR experiments. The latter involved measurement of the ratio of methylene and methyl (1)H resonances at 1.3 and 0.9 ppm, respectively, associated with the externalisation of membrane bound phosphatidyl serine. The NMR data indicate increasing lactate production during apoptosis, which implies involvement of the intrinsic mitochondrial pathway, a notion supported by down-regulation of Bcl-2 and up-regulation of Bax levels as detected by Western blotting.

In vitro assessment of novel transcription inhibitors and topoisomerase poisons in rhabdomyosarcoma cell lines.

PURPOSE: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Current chemotherapy regimes include the topoisomerase II poison etoposide and the transcription inhibitor actinomycin D. Poor clinical response necessitate identification of new agents to improve patient outcomes. METHODS: We assessed the in vitro cytotoxicity (MTT assay) of DNA intercalating agents in five established human RMS cell lines. These include novel classes of transcription inhibitors and topoisomerase poisons, previously shown to have potential as anti-cancer agents. RESULTS: Amongst the former agents, bisintercalating bis(9-aminoacridine-4-carboxamides) linked through the 9-position, and bis(phenazine-1-carboxamides) linked via their side chains, are compared with established transcription inhibitors. Amongst the latter, monofunctional acridine-4-carboxamides related to N-[2-(dimethylamino)ethyl]acridine-4-carboxamide, DACA, are compared with established topoisomerase poisons. CONCLUSIONS: Our findings specifically highlight the topoisomerase poison 9-amino-DACA, its 5-methylsulphone derivative, AS-DACA, and the bis(phenazine-1-carboxamide) transcription inhibitor MLN944/XR5944, currently in phase I trial, as candidates for further research into new agents for the treatment of RMS.

Mass spectrometry studies of the binding of the minor groove-directed alkylating agent alkamin to AT-tract oligonucleotides.

Minor groove binding alkylating agents, which have potential as cancer drugs, generate cytotoxic DNA adducts that are relatively resistant to repair as a consequence of locating covalent attachment at purine N3 nitrogen atoms. Recently, we used electrospray and matrix-assisted laser desorption ionization mass spectrometry to study the binding of the minor groove-directed polybenzamide bis-half-mustard alkamin, and its monofunctional analogue alkamini, to the oligonucleotide d(CGCGAATTCGCG)(2), identifying a number of inter- and intrastrand alkamin cross-links involving the GAATTC sequence [ Abdul Majid , A. M. S. , Smythe , G. , Denny , W. A. , and Wakelin , L. P. G. ( 2007 ) Mol. Pharmacol. 71 , 1165 - 1178 ]. Here, we extend these studies to d(CGCAAATTTGCG)(2), A3T3, and d(CGCAAAAAAGCG).d(CGCTTTTTTGCG), A6/T6, in which the opportunity for both inter- and intrastrand cross-linking is enhanced. We find that both ligands alkylate all adenines in the longer AT-tracts, as well as the abutting guanines, whether they are in the same strand as the adenines or not, in a manner consistent with covalent attack on purine N3 atoms from the minor groove. Alkamin forms intrastrand cross-links involving A4 and A6 and A6 and G10 in A3T3 and all of the purines in the A6/T6 purine tract, including G10. In addition, it forms interstrand cross-links between A4, A5, A6 and A4', A5', A6', between G10 and the latter adenines in A3T3, and between G22 and adenines A5 and A6 in A6/T6. The reactivity of the abutting guanines provides unexpected opportunities for both inter- and intrastrand cross-linking by alkamin, such as the interstrand cross-link in the CAAAAAAG sequence. We conclude that positioning monofunctional mustard groups on either end of a minor groove-directed polybenzamide has the capacity to enhance interstrand cross-links at all manner of AT-tracts, including most in which the adenines are all in one strand.

Effects of DNA threading bis(9-aminoacridine-4-carboxamides) on global gene expression.

The capacity of series of DNA-threading bis(9-aminoacridine-4-carboxamides) comprising ethylmorpholino, ethylpiperidine and N-methylpiperidin-4-yl sidechains joined by different linkers, to modulate gene expression in human leukaemia cells was investigated. The chosen compounds provided the opportunity for probing the relationships between the structure ligand structure and the drug effects on transcription, information that might lead to a greater understanding of their potential as antitumour agents. As revealed by DNA microarray analysis of 6000 genes, at equitoxic doses, 5xIC(50) values for growth inhibition, all of the drugs perturb transcription, resulting in both up- and down-regulation of many hundreds of genes, 24 h after drug exposure. Under these conditions, the capacity to inhibit transcription decreases in the order C3NC3 morpholino > C2pipC2 morpholino > C8 piperidine > C8NMP > C2pipC2 piperidine. Cluster analysis segregated the examined agents into two groups: the first included C2pipC2 morpholino and C3NC3 morpholino and the second C2pipC2 piperidine, C8 piperidine and C8NMP. This classification agreed with the ontological analysis for the markedly up-regulated genes that showed a relatively specific profile for each group. Interestingly, the general up-regulation responses for the first group (C3NC3 morpholino and C2pipC2 morpholino) indicated marked up-regulation amongst the transcription gene set, which suggests that the transcription machinery is the main target for the members of this group. While in the second group (C2pipC2 piperidine, C8 piperidine, C8NMP), the general up-regulation responses for the three agents are dominated by the protein modification process ontological class, implying at least involvement of topoisomerase poisoning in their mode of action.

DNA threading bis(9-aminoacridine-4-carboxamides): effects of piperidine sidechains on DNA binding, cytotoxicity and cell cycle arrest.

We describe the synthesis of a series of DNA-threading bis(9-aminoacridine-4-carboxamides) comprising ethylpiperidino and N-methylpiperidin-4-yl sidechains, joined via neutral flexible alkyl chains, charged flexible polyamine chains and a semi-rigid charged piperazine linker. Their cytotoxicity towards human leukaemic cells gives IC(50) values ranging from 99 to 1100 nM, with the ethylpiperidino series generally being more cytotoxic than the N-methylpiperidin-4-yl series. Measurements with supercoiled DNA indicate that they bisintercalate.

Structure of the d(CGCGAATTCGCG)2 complex of the minor groove binding alkylating agent alkamin studied by mass spectrometry.

Nitrogen mustard alkylating agents are important cancer drugs. Much interest has been focused on redirecting their covalent adducts from the N7 atoms of guanine in the major groove of DNA to the N3 atoms of adenine in the minor groove by attaching mustard groups to AT-selective minor groove binding ligands. Here we describe the use of electrospray ionization and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry to study the structure of the DNA complexes of two minor groove binding polybenzamide mustards, alkamin and alkamini; the former is a bis-half-mustard in which reactive groups are disposed at each end of the ligand, and the latter is its monofunctional analog. Alkamin is potently cytotoxic and active in experimental mouse tumor models, whereas alkamini is not. We have studied their interaction with the DNA dodecamer d(CGCGAATTCGCG)(2), designated A2T2, and we provide a detailed analysis of the observed DNA-ligand adduct ions and their fragmentation products. We find that alkamini alkylates A2T2 at guanine G4 and adenines A5 and A6 in a manner consistent with covalent attack on purine N3 atoms from the minor groove of the AT tract. Alkamin also forms monofunctional adducts at G4 and both adenines in which the second mustard arm is hydrolyzed but, in addition, forms a variety of interstrand cross-links between adenines A5/A6 and A5'/A6', an interstrand cross-link between G4 and A6', and an intrastrand cross-link between G4 and A6. We conclude that the marked cytotoxicity of alkamin and its experimental antitumor activity could be the consequence of its ability to cross-link cellular DNA at AT tract sequences.

Structure of 9-amino-[N-(2-dimethylamino)propyl]acridine-4-carboxamide bound to d(CGTACG)(2): a comparison of structures of d(CGTACG)(2) complexed with intercalatorsin the presence of cobalt.

The structure of the complex formed between 9-amino-[N-(2-dimethylamino)propyl]acridine-4-carboxamide and d(CGTACG)(2) has been refined to a resolution of 1.55 A. The complex crystallized in space group C222. An asymmetric unit comprises two strands of DNA, one disordered drug molecule, two cobalt(II) ions, two magnesium ions and 32 water molecules. The DNA helices stack in continuous columns, with their four central base pairs adopting a B-like motif. The terminal G.C base pairs engage in different interactions. At one end of the duplex there is a CpG dinucleotide overlap modified by ligand intercalation and terminal cytosine exchange between symmetry-related duplexes. An intercalation complex is formed involving four DNA duplexes, four disordered ligand molecules and two pairs of base tetrads. The other end of the DNA is frayed, with the terminal guanine lying in the minor groove of the next duplex in the column. The structure is stabilized by guanine N7-cobalt(II) coordination. The structure is compared with previously published isomorphous structures of d(CGTACG)(2) complexed with intercalators in the presence of cobalt and it is concluded that the formation of this crystal form is primarily determined by DNA-DNA interactions and packing forces, rather than by special interactions between the ligand and the DNA. Given the nature of the ligands found in these complexes, the relevance of the quadruplex structure to the biological activity of those agents, known to be topoisomerase poisons, is questioned.

Bisintercalating threading diacridines: relationships between DNA binding, cytotoxicity, and cell cycle arrest.

We have synthesized a series of bis(9-aminoacridine-4-carboxamides) linked via the 9-position with neutral flexible alkyl chains, charged flexible polyamine chains, and a semirigid charged piperazine-containing chain. The carboxamide side chains comprise N,N-dimethylaminoethyl and ethylmorpholino groups. The compounds are designed to bisintercalate into DNA by a threading mode, in which the side chains are intended to form hydrogen-bonding contacts with the O6/N7 atoms of guanine in the major groove, and the linkers are intended to lie in the minor groove. By this means, we anticipate that they will dissociate slowly from DNA, and be cytotoxic as a consequence of template inhibition of transcription. The dimers remove and reverse the supercoiling of closed circular DNA with helix unwinding angles ranging from 26 degrees to 46 degrees, confirming bifunctional intercalation in all cases, and the DNA complexes of representative members dissociate many orders of magnitude more slowly than simple aminoacridines. Cytotoxicity for human leukemic CCRF-CEM cells was determined, the most active agents having IC(50) values of 35-50 nM in a range extending over 20-fold, with neither the dimethylaminoethyl nor the ethylmorpholino series being intrinsically more toxic. In common with established transcription inhibitors, the morpholino series, with one exception, have no effect on cell cycle distribution in randomly dividing CCRF-CEM populations. By contrast, the dimethylaminoethyl series, with two exceptions, cause G2/M arrest in the manner of topoisomerase poisons, consistent with possible involvement of topoisomerases in their mode of action. Thus, the cellular response to these bisintercalating threading agents is complex and appears to be determined by both their side chain and linker structures. There are no simple relationships between structure, cytotoxicity, and cell cycle arrest, and the origins of this complexity are unclear given that the compounds bind to DNA by a common mechanism.