Strand invasion of DNA quadruplexes by PNA: comparison of homologous and complementary hybridization.

Molecular recognition of DNA quadruplex structures is envisioned to be a strategy for regulating gene expression at the transcriptional level and for in situ analysis of telomere structure and function. The recognition of DNA quadruplexes by peptide nucleic acid (PNA) oligomers is presented here, with a focus on comparing complementary, heteroduplex-forming and homologous, heteroquadruplex-forming PNAs. Surface plasmon resonance and optical spectroscopy experiments demonstrated that the efficacy of a recognition mode depended strongly on the target. Homologous PNA readily invades a quadruplex derived from the promoter regulatory region found upstream of the MYC proto-oncogene to form a heteroquadruplex at high potassium concentration mimicking the intracellular environment, whereas complementary PNA exhibits virtually no hybridization. In contrast, complementary PNA is superior to the homologous in hybridizing to a quadruplex modeled on the human telomere sequence. The results are discussed in terms of the different structural morphologies of the quadruplex targets and the implications for in vivo recognition of quadruplexes by PNAs.

Loop and backbone modifications of peptide nucleic acid improve g-quadruplex binding selectivity.

Targeting guanine (G) quadruplex structures is an exciting new strategy with potential for controlling gene expression and designing anticancer agents. Guanine-rich peptide nucleic acid (PNA) oligomers bind to homologous DNA and RNA to form hetero-G-quadruplexes but can also bind to complementary cytosine-rich sequences to form heteroduplexes. In this study, we incorporated backbone modifications into G-rich PNAs to improve the selectivity for quadruplex versus duplex formation. Incorporation of abasic sites as well as chiral modifications to the backbone were found to be effective strategies for improving selectivity as shown by UV-melting and surface plasmon resonance measurements. The enhanced selectivity is due primarily to decreased affinity for complementary sequences, since binding to the homologous DNA to form PNA-DNA heteroquadruplexes retains high affinity. The improved selectivity of these PNAs is an important step toward using PNAs for regulating gene expression by G-quadruplex formation.

Antitumor polycyclic acridines. 20. Search for DNA quadruplex binding selectivity in a series of 8,13-dimethylquino[4,3,2-kl]acridinium salts: telomere-targeted agents.

The growth-inhibitory activities of an extensive series of quaternized quino[4,3,2- kl]acridinium salts against tumor cell lines in vitro have been measured and their biological properties interpreted in the light of differential binding to different DNA isoforms. Selectivity for quadruplex DNA binding and stabilization by compounds were explored through an array of methods: UV absorption and fluorescence emission spectroscopy, surface plasmon resonance, and competition dialysis. Quadruplex DNA interaction was further characterized through FRET and DNA polymerase arrest assays. Telomerase inhibition, inferred from the TRAP assay, is attributed to quadruplex stabilization, supported by the strong correlation (R(2) = 0.81) across the series between quadruplex DNA binding affinity and TRAP inhibition potency. Growth inhibition potency in the NCI60 human tumor cell line panel is more marked in compounds with greater DNA duplex binding affinity (R(2) = 0.82). Quantification of relative quadruplex and duplex binding affinity constants puts some of these ligands among the most selective quadruplex DNA interactive agents reported to date.

High-affinity homologous peptide nucleic acid probes for targeting a quadruplex-forming sequence from a MYC promoter element.

Guanine-rich DNA and RNA sequences are known to fold into secondary structures known as G-quadruplexes. Recent biochemical evidence along with the discovery of an increasing number of sequences in functionally important regions of the genome capable of forming G-quadruplexes strongly indicates important biological roles for these structures. Thus, molecular probes that can selectively target quadruplex-forming sequences (QFSs) are envisioned as tools to delineate biological functions of quadruplexes as well as potential therapeutic agents. Guanine-rich peptide nucleic acids have been previously shown to hybridize to homologous DNA or RNA sequences forming PNA-DNA (or RNA) quadruplexes. For this paper we studied the hybridization of an eight-mer G-rich PNA to a quadruplex-forming sequence derived from the promoter region of the MYC proto-oncogene. UV melting analysis, fluorescence assays, and surface plasmon resonance experiments reveal that this PNA binds to the MYC QFS in a 2:1 stoichiometry and with an average binding constant Ka = (2.0 +/- 0.2) x 10(8) M(-1) or Kd = 5.0 nM. In addition, experiments carried out with short DNA targets revealed a dependence of the affinity on the sequence of bases in the loop region of the DNA. A structural model for the hybrid quadruplex is proposed, and implications for gene targeting by G-rich PNAs are discussed.

Trisubstituted acridines as G-quadruplex telomere targeting agents. Effects of extensions of the 3,6- and 9-side chains on quadruplex binding, telomerase activity, and cell proliferation.

The synthesis is reported of a group of 3,6,9-trisubstituted acridine compounds as telomeric quadruplex-stabilizing ligands with systematic variations at the 3-, 6-, and 9-positions. A new microwave-assisted methodology has been developed for trisubstituted acridine synthesis. Structure-activity relationships are reported using surface plasmon resonance and a fluorescence melting assay to examine quadruplex binding, together with a telomerase inhibition assay. These reveal relationships between G-quadruplex stabilization and telomerase inhibition and optimal 3,6- and 9-substituent side-chain lengths for maximal activity. Qualitative molecular modeling using molecular dynamics simulations has been undertaken on four quadruplex-DNA complexes. Long-term exposure of MCF7 cancer cells to a subset of the most active compounds, at doses lower than the IC(50) values, showed that one compound produced a marked decrease in population growth, accompanied by senescence, which is consistent with telomere targeting by this agent.

Dications that target the DNA minor groove: compound design and preparation, DNA interactions, cellular distribution and biological activity.

Fluorescence microscopy of trypanosomes from drug treated mice shows that biologically active heterocyclic diamidines that target the DNA minor groove bind rapidly and specifically to parasite kinetoplast DNA (k-DNA). The observation that the kinetoplast is destroyed, generally within 24 hours, after drug treatment is very important for understanding the biological mechanism, and suggests that the diamidines may be inhibiting some critical opening/closing step of circular k-DNA. Given the uncertainties in the biological mechanism, we have taken an empirical approach to generating a variety of synthetic compounds and DNA minor groove interactions for development of improved and new biological activities. Furamidine, DB75, is a diphenyl-diamidine that has the curvature to match the DNA minor groove as expected in the classical groove interaction model. Surprisingly, a linear diamidine with a nitrogen rich linker has significantly stronger binding than furamidine due to favorable linker and water-mediated DNA interactions. The water interaction is very dependant on compound structure since other linear compounds do not have similar interactions. Change of one phenyl of furamidine to a benzimidazole does not significantly enhance DNA binding but additional conversion of the furan to a thiophene (DB818) yields a compound with ten times stronger binding. Structural analysis shows that DB818 has a very favorable curvature for optimizing minor groove interactions. It is clear that there are many ways for compounds to bind to k-DNA and exert specific effects on kinetoplast replication and/or transcription that are required to obtain an active compound.

Targeting DNA with novel diphenylcarbazoles.

Double-stranded DNA is a therapeutic target for a variety of anticancer and antimicrobial drugs. Noncovalent interactions of small molecules with DNA usually occur via intercalation of planar compounds between adjacent base pairs or minor-groove recognition by extended crescent-shaped ligands. However, the dynamic and flexibility of the DNA platform provide a variety of conformations that can be targeted by structurally diverse compounds. Here, we propose a novel DNA-binding template for construction of new therapeutic candidates. Four bisphenylcarbazole derivatives, derived from the combined molecular architectures of known antitumor bisphenylbenzimidazoles and anti-infectious dicationic carbazoles, have been designed, and their interaction with DNA has been studied by a combination of biochemical and biophysical methods. The substitutions of the bisphenylcarbazole core with two terminal dimethylaminoalkoxy side chains strongly promote the interaction with DNA, to prevent the heat denaturation of the double helix. The deletion or the replacement of the dimethylamino-terminal groups with hydroxyl groups strongly decreased DNA interaction, and the addition of a third cationic side chain on the carbazole nitrogen reinforced the affinity of the compound for DNA. Although the bi- and tridentate molecules both derive from well-characterized DNA minor-groove binders, the analysis of their binding mode by means of circular and linear dichroism methods suggests that these compounds form intercalation complexes with DNA. Negative-reduced dichroism signals were recorded in the presence of natural DNA and synthetic AT and GC polynucleotides. The intercalation hypothesis was validated by unwinding experiments using topoisomerase I. Prominent gel shifts were observed with the di- and trisubstituted bisphenylcarbazoles but not with the uncharged analogues. These observations, together with the documented stacking properties of such molecules (components for liquid crystals), prompted us to investigate their binding to the human telomeric DNA sequence by means of biosensor surface plasmon resonance. Under conditions favorable to G4 formation, the title compounds showed only a modest interaction with the telomeric quadruplex sequence, comparable to that measured with a double-stranded oligonucleotide. Their sequence preference was explored by DNase I footprinting experiments from which we identified a composite set of binding sequences comprising short AT stretches and a few other mixed AT/GC blocks with no special AT character. The variety of the binding sequences possibly reflects the coexistence of distinct positioning of the chromophore in the intercalation sites. The bisphenylcarbazole unit represents an original pharmacophore for DNA recognition. Its branched structure, with two or three arms suitable to introduce a structural diversity, provides an interesting scaffold to built molecules susceptible to discriminate between the different conformations of nucleic acids.

Trisubstituted acridine derivatives as potent and selective telomerase inhibitors.

The synthesis and evaluation for telomerase-inhibitory and quadruplex DNA binding properties of three related series of rationally designed trisubstituted acridine derivatives are described. These are substituted on the acridine ring at the 2,6,9; 2,7,9; and 3,6,9 positions. The ability of several of the most potent compounds to interact with and stabilize an intramolecular G-quadruplex DNA was evaluated by surface plasmon resonance methods, and affinities were found to correlate with potency in a telomerase assay. The interactions of a number of compounds with a parallel quadruplex DNA structure were simulated by molecular modeling methods. The calculated interaction energies were compared with telomerase activity and showed generally consistent correlations between quadruplex affinity and telomerase inhibition. These data support a model for the action of these compounds that involves the stabilization of intermediate quadruplex structures that inhibit the elongation of telomeric DNA by telomerase in tumor cells.

The activity of diguanidino and 'reversed' diamidino 2,5-diarylfurans versus Trypanosoma cruzi and Leishmania donovani.

The in vitro activity of 20 dicationic molecules containing either diguanidino or reversed amidine cationic groups were evaluated versus Trypanosoma cruzi and Leishmania donovani. The most active compounds were in the reversed amidine series and six exhibited IC(50) values of less than 1 micro mol versus T. cruzi and five gave similar values versus L. donovani.

Distribution of furamidine analogues in tumor cells: targeting of the nucleus or mitochondria depending on the amidine substitution.

Diphenylfuran diamidines represent an important class of DNA minor groove binders of high therapeutic interest as antiparasitic or antitumor agents depending on the compounds structures. To exert their cytotoxic action, the compounds must first get into the cell and reach the nuclear compartment where the main target, DNA, is located. The forces that drive the drugs into cell nuclei, as well as the influence of the molecular structures on the cell distribution, are not known. To address these issues, we took advantage of the fluorescence of the molecules to analyze their intracellular distribution profiles in tumor cells of different origins (B16 melanoma, MCF7 mammary adenocarcinoma, A549 lung carcinoma, HT29 colon carcinoma, LNCaP, and PC3 prostatic carcinoma) by epifluorescence and confocal microscopy. A homogeneous series of synthetic bis-substituted alkyl or phenyl amidine and reverse amidine derivatives of furamidine was used to dissect the molecular mechanisms that control the distribution of the drugs into the cytoplasm or the nucleus of the cells. The amidine (DB75) and the various N-alkyl derivatives were found to accumulate selectively in the cell nuclei. This is also the case for a guanidine derivative but not for the phenyl-substituted compound DB569, which essentially localizes in cytoplasmic granules. Similar cytoplasmic patterns were observed with a reverse amidine analogue and a pyridine-substituted compound indicating that the presence of aromatic rings on the terminal side chain is the limiting factor that restricts the uptake of the compounds in the nuclear compartment. The use of different organelle-selective fluorescent probes, such as JC-1 and chloromethyl-X-rosamine, both specific to mitochondria and neutral red considered as a lysosome-selective probe, suggests that DB569 preferentially accumulates in mitochondria. Competition experiments with the antitumor drug daunomycin reveal that the diphenylfurans are attracted into the nuclei by the DNA. The DNA minor groove-drug interactions provide the driving force that permits massive accumulation of the fluorescent molecules in the nuclei. The DNA binding properties of the diphenylfuran derivatives were investigated by DNase I footprinting and surface plasmon resonance biosensor experiments to measure sequence selectivity and binding affinities, respectively. Furamidine and its phenyl-substituted analogue that accumulate in the cell nuclei and mitochondria, respectively, share a common selectivity for AT sites and bind equally tightly to these sites. Therefore, it is possible to modulate the intracellular distribution of the furamidine derivatives without affecting their DNA binding and sequence recognition properties. The introduction of aromatic substituents on diphenylfuran diamidines represents a novel strategy to control the intracellular compartmentalization of these DNA binding agents and directs them to mitochondria. This drug design strategy may prove useful to trigger drug-induced apoptosis.

Distribution of furamidine analogues in tumor cells: influence of the number of positive charges.

Fluorescence microscopy has been used to study the cellular distribution properties of a series of DNA binding cationic compounds related to the potent antiparasitic drug furamidine (DB75). The compounds tested bear a diphenylfuran or a phenylfuranbenzimidazole unfused aromatic core substituted with one or two amidine or imidazoline groups. The synthesis of five new compounds is reported. The B16 melanoma cell line was used to compare the capacities of mono-, bis-, and tetracations to enter the cell and nuclei. The high-resolution fluorescence pictures show that in the furamidine series, the compounds with two or four positive charges selectively accumulate in the cell nuclei whereas, in most cases, those bearing only one positive charge show reduced cell uptake capacities. One of the monocationic compounds, DB607, distributes in the cytoplasm, possibly in mitochondria, with no distinct nuclear accumulation. In sharp contrast, furamidine and benzimidazole analogues, including the drug DB293 that forms DNA minor groove dimers, efficiently accumulate in the cell nuclei and the intranuclear distribution of these DNA minor groove binders is significantly different from that seen with the DNA intercalating drug propidium iodide. The results suggest that the presence of two amidine terminal groups plays a role in facilitating nuclear accumulation into cells, probably as a result of nucleic acid binding. The determination of DNA melting temperature increases on addition of these compounds supports the importance of DNA binding in nuclear uptake.