DNA Groove Binding Research Articles

Abstract 3517: Complete remission of xenograft tumor in nude mice induced by N-mustard-quinoline conjugates bearing hydrazinecarboxamide linker

Abstract Designing DNA-directed alkylating agents is a promising strategy to overcome the drawbacks of alkylating agents by enhancing DNA specific cross-linking and reducing the chemical reactivity of N-mustard. These compounds are generally synthesized by linking the alkylating warhead (such as alkyl or phenyl N-mustard pharmacophore) to the DNA-affinic carrier (such as DNA intercalating or groove binding agents). Recently, we reported that N-mustard-9-anilinoacridine conjugates via a urea, carbamate, or carbonate linker exhibited potent cytotoxicity and therapeutic efficacy against various human tumor xenografts indicating that these linkers are able to stabilize the reactive alkylating war head. Thus, these derivatives have a long half-life in plasma. Some of these conjugates are able to induce complete tumor remission (CR) in inhibiting human tumor xenograft model with low toxicity. To continue our research in finding potent DNA-directed alkylating agents, we have recently synthesized a series of N-mustard-quinoline conjugates bearing a hydrazinecarboxamide linker. These conjugates were prepared in good yield by the condensation of appropriate 4-hydrazinoquinolines with 4-bis(2-chloroethyl)- aminophenyl isocynate. The results showed that these conjugates possessed significant cytotoxicity against human lymphoblastic leukemia (CCRF-CEM) and various solid tumor xenografts (such as MX-1, HCT-116, H1299, CL 1-0, CL 1-5, PC3 and MCF-7) cell growth in vitro. Among these agents, BO-1392 and BO-1393 were able to induce CR against human breast carcinoma MX-1 xenograft and significantly suppress against human ovarian SK-OV3 xenograft in animal model with relative low toxicity. Interestingly, we found that BO-1393 (iv. injection) can significantly prolong the life span of nude mice bearing human glioma SK-NA-S xenograft (orthotropic implanted in brain). The experiment was compared with Temozolomide (oral administration). The modified comet assay and alkaline agarose gel shift assay showed that these conjugates are capable of inducing DNA interstrand cross-linking in human non-small lung cancer H1299 cells. In conclusion, the present studies demonstrated that the newly synthesized N-mustard-quinoline conjugates having a hydrazinecarboxamide linker exhibited a broad spectrum of antitumor activity and are chemically stable. The remarkable therapeutic efficacy of BO-1393 demonstrated that this agent has high potential for clinical development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3517.

Photocytotoxic Lanthanum(III) and Gadolinium(III) Complexes of Phenanthroline Bases Showing Light-Induced DNA Cleavage Activity

Lanthanide complexes of formulation [La(B)(2)(NO(3))(3)] (1-3) and [Gd(B)(2)(NO(3))(3)] (4-6), where B is a N,N-donor phenanthroline base, namely, 1,10-phenanthroline (phen in 1, 4), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq in 2, 5) and dipyrido[3,2-a:2',3'-c]phenazine (dppz in 3, 6), have been prepared, characterized from physicochemical data, and their photoinduced DNA and protein cleavage activity studied. The photocytotoxicity of the dppz complexes 3 and 6 has been studied using HeLa cancer cells. The complexes exhibit ligand centered bands in the UV region. The dppz complexes show the lowest energy band at 380 nm in N,N-dimethylformamide (DMF). The La(III) complexes are diamagnetic. The Gd(III) complexes (4-6) have magnetic moments that correspond to seven unpaired electrons. The complexes are 1:1 electrolytic in aqueous DMF. The dpq and dppz complexes in DMF show ligand-based reductions. The complexes display moderate binding propensity to calf thymus DNA giving binding constant values in the range of 5.7 x 10(4)-5.8 x 10(5) M(-1) with a relative order: 3, 6 (dppz) > 2, 5 (dpq) > 1, 4 (phen). The binding data suggest DNA surface and/or groove binding nature of the complexes. The complexes do not show any hydrolytic cleavage of plasmid supercoiled pUC19 DNA. The dpq and dppz complexes efficiently cleave SC DNA to its nicked circular form on exposure to UV-A light of 365 nm at nanomolar complex concentration. Mechanistic studies reveal the involvement of singlet oxygen ((1)O(2)) and hydroxyl radical (HO*) as the cleavage active species. The complexes show binding propensity to bovine serum albumin (BSA) protein giving K(BSA) values of approximately 10(5) M(-1). The dppz complexes 3 and 6 show BSA protein cleavage activity in UV-A light of 365 nm. The dppz complexes 3 and 6 exhibit significant photocytotoxicity in HeLa cells giving respective IC(50) values of 341 nM and 573 nM in UV-A light of 365 nm for an exposure time of 15 min (IC(50) > 100 microM in dark for both the complexes). Control experiments show significant dark and phototoxicity of the dppz base alone (IC(50) = 413 nM in light with 4 h incubation in dark and 11.6 microM in dark with 24 h incubation). A significant decrease in the dark toxicity of the dppz base is observed on binding to the lanthanide ions while retaining similar phototoxicity.

Binuclear copper and zinc complexes possessing bio-potential ligands: synthesis, characterization, antimicrobial, SOD mimetic, DNA binding, and cleavage studies

Schiff bases (L) viz, N,N′,N′′,N′′′-tetra-3,4-dimethoxybenzalidene-3,3′-diaminobenzidine (TDBD), N,N′,N′′,N′′′-tetra-4-hydroxy-3-methoxybenzalidene-3,3′-diaminobenzidine (THMBD), and N,N′,N′′,N′′′-tetra-3-hydroxy-4-nitrobenzalidene-3,3′-diaminobenzidine (THNBD) afford binuclear [M2LCl4] complexes where M = Cu(II) or Zn(II). These Schiff bases and their binuclear complexes have been characterized by analytical and spectral data showing square-planar geometry on metalation with Cu2+. Intercalative binding of these complexes with DNA has been investigated by electronic absorption spectroscopy, viscosity measurements, cyclic voltammetry, and differential pulse voltammetry. Control DNA cleavage experiments using pUC19 supercoiled (SC) DNA and minor groove binder distamycin suggest that these synthesized complexes bind to the major groove. In the presence of a reducing agent like 3-mercaptopropionic acid (MPA), they show chemical nuclease activity. They also show an efficient photo-induced DNA cleavage on irradiation with a monochromatic UV light of 360 nm in the presence of inhibitors. Control experiments indicate the inhibition of cleavage in the presence of singlet oxygen quencher like sodium azide and the enhancement of cleavage in D2O show the formation of singlet oxygen as reactive species. The superoxide dismutase (SOD)-mimetic activity of the synthesized complexes has been assessed for their ability to inhibit the reduction of nitroblue tetrazolium chloride (NBT). The complexes have promising SOD-mimetic activity. The antimicrobial results indicate that the complexes inhibit the growth of bacteria and fungi more than free ligands.

Anaerobic DNA cleavage activity in red light and photocytotoxicity of (pyridine-2-thiol)cobalt(iii) complexes of phenanthroline bases

Cobalt(iii) complexes [Co(pnt)(B)(2)](NO(3))(2) (1-3) of pyridine-2-thiol (pnt) and phenanthroline bases (B), viz. 1,10-phenanthroline (phen in 1), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq in ) and dipyrido[3,2-a:2',3'-c]phenazine (dppz in 3), have been prepared, characterized and their photo-induced anaerobic DNA cleavage activity studied. The crystal structure of 1a as mixed ClO(4)(-) and PF(6)(-) salt of 1 shows a Co(III)N(5)S coordination geometry in which the pnt and phen showed N,S- and N,N-donor binding modes, respectively. The complexes exhibit Co(iii)/Co(ii) redox couple near -0.3 V (vs. SCE) in 20% DMF-Tris-HCl buffer having 0.1 M TBAP. The complexes show binding propensity to calf thymus DNA giving K(b) values within 2.2 x 10(4)-7.3 x 10(5) M(-1). Thermal melting and viscosity data suggest DNA surface and/or groove binding of the complexes. The complexes show significant anaerobic DNA cleavage activity in red light under argon atmosphere possibly involving sulfide anion radical or thiyl radical species. The DNA cleavage reaction under aerobic medium in red light is found to involve both singlet oxygen and hydroxyl radical pathways. The dppz complex shows non-specific BSA and lysozyme protein cleavage activity in UV-A light of 365 nm via both hydroxyl and singlet oxygen pathways. The dppz complex exhibits photocytotoxicity in HeLa cervical cancer cells giving IC(50) values of 767 nM and 19.38 microM in UV-A light of 365 nm and in the dark, respectively. A significant reduction of the dark toxicity of the dppz base (IC(50) = 8.34 microM in dark) is observed on binding to the cobalt(iii) center.

Oxovanadium(iv) complexes of phenanthroline bases: the dipyridophenazine complex as a near-IR photocytotoxic agent

Oxovanadium(iv) complexes [VOCl(B)(2)]Cl (1-3) of phenanthroline bases (B), viz. 1,10-phenanthroline (phen in ), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq in ) and dipyrido[3,2-a:2',3'-c]phenazine (dppz in ), have been prepared, characterized and their DNA and protein binding, photo-induced DNA and protein cleavage activity and photocytotoxicity have been studied. Complex , structurally characterized by X-ray crystallography, shows the presence of a vanadyl group in VOClN(4) coordination geometry. The dpq ligand displays a chelating mode of binding with a N-donor site trans to the oxo-group. The chloride ligand is cis to the oxo-group. The one-electron paramagnetic complexes show a d-d band near 715 nm in 15% DMF-Tris-HCl buffer. The complexes are redox active exhibiting a V(iv)/V(iii) redox couple within -0.5 to -0.7 V vs. SCE in 20% DMF-Tris-HCl/0.1 M KCl. The complexes bind to calf thymus (CT) DNA in the order: (dppz) > (dpq) > (phen). The binding data reveal the groove and/or partial intercalative DNA binding nature of the complexes. The complexes show "chemical nuclease" activity in the dark in the presence of 3-mercaptopropionic acid or hydrogen peroxide via a hydroxyl radical pathway. The dpq and dppz complexes are efficient photocleavers of DNA in UV-A light of 365 nm forming reactive singlet oxygen ((1)O(2)) and hydroxyl radical ( OH) species. Complexes and also show DNA cleavage activity in red light (>750 nm) by an exclusive OH pathway. The complexes display a binding propensity to bovine serum albumin (BSA) protein giving K(BSA) values in the range of 7.1 x 10(4)-1.8 x 10(5) M(-1). The dppz complex shows BSA and lysozyme protein cleavage activity in UV-A light of 365 nm via OH pathway. The dppz complex exhibits significant PDT effect in human cervical cancer HeLa cells giving IC(50) values of 1.0 microM and 12.0 microM in UV-A and visible light, respectively (IC(50) = >100 microM in the dark).

Photocytotoxic Oxovanadium(IV) Complexes Showing Light-Induced DNA and Protein Cleavage Activity

Oxovanadium(IV) complexes [VO(L)(B)]Cl(2) (1-3), where L is bis(2-benzimidazolylmethyl)amine and B is 1,10-phenanthroline (phen), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq) or dipyrido[3,2-a:2',3'-c]phenazine (dppz), have been prepared, characterized, and their photo-induced DNA and protein cleavage activity studied. The photocytotoxicity of complex 3 has been studied using adenocarcinoma A549 cells. The phen complex 1, structurally characterized by single-crystal X-ray crystallography, shows the presence of a vanadyl group in six-coordinate VON(5) coordination geometry. The ligands L and phen display tridentate and bidentate N-donor chelating binding modes, respectively. The complexes exhibit a d-d band near 740 nm in 15% DMF-Tris-HCl buffer (pH 7.2). The phen and dpq complexes display an irreversible cathodic cyclic voltammetric response near -0.8 V in 20% DMF-Tris-HCl buffer having 0.1 M KCl as supporting electrolyte. The dppz complex 3 exhibits a quasi-reversible voltammogram near -0.6 V (vs SCE) that is assignable to the V(IV)-V(III) couple. The complexes bind to calf thymus DNA giving binding constant values in the range of 6.6 x 10(4)-2.9 x 10(5) M(-1). The binding site size, thermal melting and viscosity binding data suggest DNA surface and/or groove binding nature of the complexes. The complexes show poor "chemical nuclease" activity in dark in the presence of 3-mercaptopropionic acid or hydrogen peroxide. The dpq and dppz complexes are efficient photocleavers of plasmid DNA in UV-A light of 365 nm via a mechanistic pathway that involves formation of both singlet oxygen and hydroxyl radicals. The complexes show significant photocleavage of DNA in near-IR light (>750 nm) via hydroxyl radical pathway. Among the three complexes, the dppz complex 3 shows significant BSA and lysozyme protein cleavage activity in UV-A light of 365 nm via hydroxyl radical pathway. The dppz complex 3 also exhibits photocytotoxicity in non-small cell lung carcinoma/human lung adenocarcinoma A549 cells giving IC(50) value of 17 microM in visible light (IC(50) = 175 microM in dark).

Synthesis, characterization, DNA binding, photo-induced DNA cleavage, and antimicrobial activity of metal complexes of a Schiff base derived from bis(3-aminophenyl)malonamide

The peptide linkage Schiff base (H2L) and its complexes have been synthesized and fully characterized by elemental analysis, UV–Vis, FTIR, 1H-NMR, 13C-NMR, EPR, and FAB-mass spectra. The stoichiometry of the complexes is [ML] (where M = Cu(II), Co(II), Ni(II), Zn(II), and VO(IV)). All the complexes exhibit square-planar geometry except the vanadyl complex which has square-pyramidal geometry. Interactions of the complexes and free ligand with double-stranded calf thymus DNA (CT-DNA) are studied by UV-spectrophotometric, electrochemical, and viscosity measurements. The data suggest that all the complexes form adducts with DNA and distort the double helix by changing the base stacking. Vanadyl complex forms a weaker adduct to CT-DNA than other complexes, probably due to the square-pyramidal geometry. CT-DNA induces extensive distortion in the planarity of vanadyl complex as EPR spectral calculations reveal. The intrinsic binding constants (K b) of [ZnL], [CuL], [CoL], and [NiL] are 1.1 × 105, 1.4 × 105, 0.8 × 105, and 0.6 × 105 M−1, respectively. Photo-induced DNA cleavage indicates that all complexes cleave DNA effectively. Control DNA cleavage experiments using pUC19 supercoiled (SC) DNA and minor groove binder distamycin suggest major groove binding for the synthesized complexes. The antimicrobial results indicate that the complexes inhibit the growth of bacteria and fungi more than the free ligand.

Novel metal-based pharmacologically dynamic agents of transition metal(II) complexes: Designing, synthesis, structural elucidation, DNA binding and photo-induced DNA cleavage activity

Novel Schiff base Cu(II), Ni(II), Co(II) and Zn(II) complexes have been designed and synthesized using the macrocyclic ligand derived from the condensation of diethylphthalate with Schiff base, obtained from benzene-1,2-diamine and 3-benzylidene-pentane-2,4-dione. The ligand and its complexes have been characterized by analytical and spectral techniques. DNA binding properties of these complexes have been investigated by UV-vis, viscosity measurements, cyclic voltammetric and differential pulse voltammogram studies. The intrinsic binding constants for Co(II), Ni(II), Cu(II) and Zn(II) complexes are 1.6x10(6), 1.8x10(6), 2.0x10(6) and 1.5x10(6) M(-1) respectively which are obtained from electronic absorption experiment. Control DNA cleavage experiments using pUC19 supercoiled (SC) DNA and minor groove binder (distamycin) suggest the major groove binding tendency for the synthesized complexes. In the presence of a reducing agent like 3-mercaptopropionic acid (MPA), the synthesized complexes show chemical nuclease activity under dark reaction condition. The complexes also show efficient photo-induced DNA cleavage activity on irradiation with a monochromatic UV light of 360 nm in the presence of inhibitors. Control experiments show inhibition of cleavage in the presence of singlet oxygen quencher like sodium azide and enhancement of cleavage in D(2)O, suggesting the formation of singlet oxygen as a reactive species in a type-II process.

In Vitro Study of Damaging Effects of 2,4-Dichlorophenoxyacetic Acid on DNA Structure by Spectroscopic and Voltammetric Techniques

2,4-Dichlorophenoxyacetic acid (2,4-D) is a genotoxic organochlorinated herbicide, and its interaction with DNA was studied by UV/Vis, fluorescence, circular dichroism viscosity measurements, and alternative current voltammetry techniques. Using these analyses, the binding constant of 2,4-D to DNA has been calculated by two different techniques. The binding constant of 2,4-D to DNA calculated by fluorescence and circular dichroism spectra was found to be 3.5 x 10(3) M(-1) and 5.02 x 10(3) M(-1), respectively. Analyses of fluorescence spectra, viscosity measurements, and alternative current voltammetry interactions indicated that 2,4-D is a groove binder of DNA. Ethidium bromide displacement studies revealed that 2,4-D does not have any effect on ethidium bromide-bound DNA, which is indicative of groove binding.

Phase I Study of Sequence-Selective Minor Groove DNA Binding Agent SJG-136 in Patients with Advanced Solid Tumors

This phase I dose-escalation study was undertaken to establish the maximum tolerated dose of the sequence-selective minor groove DNA binding agent SJG-136 in patients with advanced solid tumors. The study also investigated antitumor activity and provided pharmacokinetic and pharmacodynamic data. Sixteen patients were assigned sequentially to escalating doses of SJG-136 (15-240 microg/m(2)) given as a 10-minute i.v. infusion every 21 days. The dose was subsequently reduced in incremental steps to 45 microg/m(2) due to unexpected toxicity. The maximum tolerated dose of SJG-136 was 45 microg/m(2). The main drug-related adverse event was vascular leak syndrome (VLS) characterized by hypoalbuminemia, pleural effusions, ascites, and peripheral edema. Other unexpected adverse events included elevated liver function tests and fatigue. The VLS and liver toxicity had delayed onset and increased in severity with subsequent cycles. Disease stabilization was achieved for >6 weeks in 10 patients; in 2 patients this was maintained for >12 weeks. There was no evidence of DNA interstrand cross-linking in human blood lymphocytes with the use of the comet assay. Evidence of DNA interaction in lymphocytes and tumor cells was shown through a sensitive gamma-H2AX assay. SJG-136 had linear pharmacokinetics across the dose range tested. SJG-136 was associated with dose-limiting VLS and hepatotoxicity when administered by short injection every 21 days. DNA damage was noted, at all dose levels studied, in circulating lymphocytes. The etiology of the observed toxicities is unclear and is the subject of further preclinical research. Alternative clinical dosing strategies are being evaluated.

Molecular Aspects of Small Molecules-Poly(A) Interaction: An Approach to RNA Based Drug Design

The use of small molecules to specifically control important cellular functions is an area of major current interest at the interface of chemical biology and medicinal chemistry. Recognition of ribonucleic acids (RNA) has emerged more recently as a critical event in many biological pathways of eukaryotic cells and consequently the opportunity of drugs targeting to diverse structures of RNA is abundant. Such RNA targeting molecules must be able to specifically bind to unique structural organizations in RNA to regulate the gene expression. One particular example in this context is the modulation of the mRNA through its polyadenylic acid [poly(A)] tail. All mRNAs in eukaryotic cells have a poly(A) tail at the 3'-end This tail of about 200-250 or so adenine residues is an important determinant in maturation, stability of poly(A) and in initiation of translation process. Small molecules that could bind to this poly(A) tail could influence and possibly inhibit mRNA function and subsequent protein production in the cell leading to the development of new type of therapeutic agents. Recent discovery of the over expression of neo polyadenylic polymerase, the enzyme that catalyses polyadenylation, in human cancer cells compared to normal cells further signifies the importance of poly(A) in cellular events in cancer progression. The structural transition in poly(A) from single strand to double strand form induced by a narrow pH, salt and temperature variations also makes it a potential target for the better understanding of structure-function relationship in nucleic acids. Over the last forty years attempts have been made for the structural elucidation of this polyribonucleotide as well as the complex formed by the interactions with various small molecules like DNA intercalators, partial intercalators and groove binders using various physico-chemical and technique. These studies have led to progress in the understanding of specificity of binding, correlation between structural and thermodynamic aspects, description of drug-RNA binding modes and influence of substitutents on drug-RNA complexes and ultimately the discovery of new novel compounds that can be used as modulators of poly(A) structure. This review focuses on the structural and biological significance of poly(A), the use of small molecules to control the structure of this RNA and the futuristic development of new small molecules targeted to poly(A) structures.

Sequence‐Specific Positions of Water Molecules at the Interface between DNA and Minor Groove Binders

Ligands able to specifically recognize DNA sequences are of fundamental interest as transcription-controlling drugs. Herein, we analyze the positions of water molecules relative to B-DNA base pairs in the minor groove of X-ray and NMR protein data bank (PDB) structures. The patterns observed for water molecules at the interface between DNA and a ligand are compared with those obtained for structures without a ligand. Although the ligand end groups are often charged, and therefore highly hydrated, they do not alter the water patterns, which show considerable differences for the AT and CG base pairs. For AT they are much more precise than for CG in both ligand-containing and ligand-free structures. This behavior strongly indicates that the release of water molecules upon ligand binding leads to a gain of entropy and explains why this effect is especially pronounced for A-tract B-DNA sequences.

597 POSTER Novel Topoisomerase 1 mutations in colorectal carcinoma cell lines are involved in SN38 resistance

Polymorphic RNA conformations may serve as potential targets for structure specific antiviral agents. As an initial step in the development of such drugs, the interaction of a wide variety of compounds which are characterized to bind to DNA through classical or partial intercalation or by mechanism of groove binding, with the A-form and the protonated form of poly(rC)·poly(rG), been evaluated by multifaceted spectroscopic and viscometric techniques. Results of this study suggest that (i) ethidium intercalates to the A-form of RNA, but does not intercalate to the protonated form, (ii) methylene blue intercalates to the protonated form of the RNA but does not intercalate to the A-form, (iii) actinomycin D does not bind to either conformations of the RNA, and (iv) berberine binds to the protonated form by partial intercalation process, while its binding to the A-form is very weak. The DNA groove binder distamycin A has much higher affinity to the protonated form of the RNA compared to the A-form and binds to both structures by non-intercalative mechanism. We conclude that the binding affinity characteristics of these DNA binding molecules to the RNA conformations are vastly different and may serve as data for the development of RNA based antiviral drugs.

Synthesis, structure, DNA binding and DNA cleavage activity of oxovanadium(IV) N-salicylidene-S-methyldithiocarbazate complexes of phenanthroline bases

Ternary oxovanadium(IV) complexes [VO(salmdtc)(B)] (1–3), where salmdtc is dianionic N-salicylidene-S-methyldithiocarbazate and B is N,N-donor phenanthroline bases like 1,10-phenanthroline (phen, 1), dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq, 2) and dipyrido[3,2-a:2′,3′-c]phenazine (dppz, 3), are prepared, characterized and their DNA binding and DNA cleavage activity studied. Complex 3 is structurally characterized by single-crystal X-ray crystallography. The molecular structure shows the presence of a vanadyl group in six-coordinate VN3O2S coordination geometry. The S-methyldithiocarbazate Schiff base acts as a tridentate NSO-donor ligand in a meridional binding mode. The N,N-donor heterocyclic base displays a chelating mode of binding with an N-donor site trans to the vanadyl oxo-group. The complexes show a d–d band in the range of 675–707nm in DMF. They exhibit an irreversible oxidative cyclic voltammetric response near 0.9V due to the V(V)/V(IV) couple and a quasi-reversible reductive V(IV)/V(III) redox couple near −1.0V vs. SCE in DMF-0.1M TBAP. The complexes show good binding propensity to calf thymus DNA giving binding constant values in the range of 7.4×104–2.3×105M−1. The thermal denaturation and viscosity binding data suggest DNA surface and/or groove binding nature of the complexes. The complexes show poor chemical nuclease activity in dark in the presence of 3-mercaptopropionic acid (MPA) or hydrogen peroxide. The dpq and dppz complexes show efficient DNA cleavage activity in UV-A light of 365nm via a type-II mechanistic pathway involving formation of singlet oxygen (1O2) as the reactive species.

Pyrazolyl–Diamine Ligands That Bear Anthracenyl Moieties and Their Rhenium(I) Tricarbonyl Complexes: Synthesis, Characterisation and DNA‐Binding Properties

Two novel families of pyrazolyl-diamine ligands that bear an anthracen-9-yl group as a DNA-binding fragment, pz*(CH2)2NH(CH2)2NHCH2-9-anthryl (pz*=pz (L(1)), 3,5-Me2pz (L2)) and pz*(CH2)2NH(CH2)2NH(2 (pz*=4-(9-anthrylmethyl)pz (L3), 3,5-Me2-4-(9-anthrylmethyl)pz (L4)), have been prepared and fully characterised. In the case of L2-L4, the evaluation of their coordination capability towards the fac-[Re(CO)3]+ core led to the synthesis of the organometallic complexes fac-[Re(CO)(3){3,5-Me(2)pz(CH2)2NH(CH2)2NHCH2-9-anthryl}]Br (7) and fac-[Re(CO)3{4-(9-anthrylmethyl)pz*(CH2)2NH(CH2)2NH2}]Br (pz*=pz (8), 3,5-Me2pz 9). The interaction of the novel pyrazole-diamine ligands and the rhenium(I) complexes with calf thymus (CT) DNA has been investigated with a variety of spectroscopic techniques (UV-visible, fluorescence, circular dichroism (CD) and linear dichroism (LD)). All of the evaluated compounds have a moderate affinity to CT DNA (3.46x10(3)<K(b)<1.95x10(4)), but the binding mode depends on the position of the chromophore in the framework of the pyrazolyl-diamine ligands. LD measurements have shown that L1 and L2 act as DNA intercalators, but complex 7 intercalates only partially. By contrast, the compounds with the anthracenyl group at the 4-position of the azolyl ring (L(3), L4 and 9) do not intercalate, and behave more like DNA groove binders. Fluorescence microscopy studies have demonstrated that complexes 7 and 9 can target the nucleus of murine B16-F1 melanoma cells, and appear to be promising platforms for the further design of radiopharmaceuticals for targeted radiotherapy.

DNA Cleavage by New Oxovanadium(IV) Complexes of N-Salicylidene α-Amino Acids and Phenanthroline Bases in the Photodynamic Therapy Window

Oxovanadium(IV) complexes [VO(salmet)(B)] (1-3) and [VO(saltrp)(B)] (4-6), where salmet and saltrp are N-salicylidene-l-methionate and N-salicylidene-l-tryptophanate, respectively, and B is a N,N-donor heterocyclic base (viz. 1,10-phenanthroline (phen, 1, 4), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq, 2, 5), and dipyrido[3,2-a:2',3'-c]phenazine (dppz, 3, 6)) are prepared and characterized and their DNA binding and photoinduced DNA cleavage activity studied. Complexes 1, 2, and 4 are structurally characterized by single-crystal X-ray crystallography. The molecular structure shows the presence of a vanadyl group in the VO3N3 coordination geometry. The dianionic alpha-amino acid Schiff base acts as a tridentate O,N,O-donor ligand in a meridional binding mode. The N,N-donor heterocyclic base displays a chelating mode of bonding with a N-donor site trans to the oxo group. The complexes show a d-d band in the range of 680-710 nm in DMF with a shoulder near 840 nm. They exhibit an irreversible oxidative cyclic voltammetric response near 0.8 V assignable to the V(V)/V(IV) couple and a quasi-reversible V(IV)/V(III) redox couple near -1.1 V vs SCE in DMF-0.1 M TBAP. The complexes show good binding propensity to calf thymus DNA giving binding constant values in the range from 5.2 x 10(4) to 7.2 x 10(5) M(-1). The binding site size, thermal melting, and viscosity data suggest DNA surface and/or groove binding nature of the complexes. The complexes show poor "chemical nuclease" activity in the dark in the presence of 3-mercaptopropionic acid or hydrogen peroxide. The dpq and dppz complexes show efficient DNA cleavage activity on irradiation with UV-A light of 365 nm via a mechanistic pathway involving formation of singlet oxygen as the reactive species. They also show significant DNA cleavage activity on photoexcitation in red light (>750 nm) by (1)O2 species. Observation of red-light-induced cleavage of DNA is unprecedented in the vanadium chemistry. The DNA cleavage activity is metal promoted as the ligands or vanadyl sulfate alone are cleavage inactive on photoirradiation at these wavelengths.

DNA as a target for anticancer compounds: methods to determine the mode of binding and the mechanism of action

Small molecules that bind to DNA are extremely useful as biochemical tools for the visualization of DNA both in vitro and inside the cell. Additionally, the clinical significance of DNA-binding compounds can hardly be overstated, as many anticancer regimens include a compound that binds to and/or modifies DNA. Although many of the important DNA-binding anticancer drugs were discovered in phenotypic, cell-based screens, in vitro experiments have been developed that enable a precise determination of how a compound interacts with DNA. This review provides a summary of the assays that should be performed when it is suspected that DNA may be a target for a given small molecule. A battery of in vitro assays readily distinguishes between DNA intercalation, DNA groove binding, and the inhibition of topoisomerases. Further cell-based investigations can implicate a direct effect of a compound on DNA within the cell. Together, these assays are powerful tools to determine the mechanism of previously discovered molecules, and will be crucial to the discovery of the next generation of DNA-binding anticancer drugs.

Structure of 4'-thioDNA which exhibits endonuclease resistance

4'-thio DNA consisting of 2'-deoxy-4'-thionucleosides exhibits resistance to both endonuclease and 3'-exonuclease cleavages. Interestingly, we found that 4'-thioDNA duplex behaved like RNA molecules in hybridization properties and structural aspects. Here, we have determined the structure of 4'-thioDNA duplex in solution by NMR. Most residues take on C3'-endosugar puckering, which is characteristic to A-form. The major groove of 4'-thioDNA duplex is narrow and deep, while the minor groove is wide and shallow. These features are also characteristic to A-form. Thus, although DNA duplex usually takes on B-form in solution, 4'-thioDNA takes on A-form, like RNA molecules. A-form-like groove features of 4'-thioDNA can account for the fact that 4'-thioDNA duplex interacts with an RNA major groove binder, but not with DNA groove binder. Interaction of 4'-thioDNA with RNase V1 and resistance to endonuclease may also be accounted for in the same context.

Relationship between the Structure and the DNA Binding Properties of Diazoniapolycyclic Duplex- and Triplex-DNA Binders: Efficiency, Selectivity, and Binding Mode

The association of dicationic polycyclic ligands, namely, four diazoniapentaphene derivatives, three diazoniaanthra[1,2-a]anthracenes, diazoniahexaphene, and a partly saturated hydroxy-substituted diazoniapentaphene with double-stranded and triple-helical DNA, was investigated by spectrophotometric and viscosimetric titrations, CD and LD spectroscopy, DNA melting experiments, and molecular modeling studies. All experimental and theoretical data reveal an intercalative DNA-binding mode of the diazoniapentaphenes and diazoniaanthra[1,2-a]anthracenes; the latter have approximately 10-fold higher affinity for the DNA duplex. CD spectroscopic investigations and molecular modeling studies show that only one azonianaphthalene part of the ligand is intercalated between the DNA base pairs, whereas the remaining part of the ligand points outside the intercalation pocket. In contrast, the diazoniahexaphene is a DNA groove binder, which binds selectively to [poly(dAdT)]2. At low ligand-to-DNA ratios (r < 0.15), the diazoniahexaphene also behaves as an intercalator; however, all spectroscopic and viscosimetric data are consistent with significant groove binding of this ligand at r > 0.2. Studies of the interaction of diazoniapolycyclic ions with triplex DNA reveal a preferential binding of both diazoniapentaphenes and diazoniaanthra[1,2-a]anthracenes to the triplex and stabilization thereof. These properties are more pronounced in the case of the hexacyclic diazoniaanthra[1,2-a]anthracenes; however, the diazoniahexaphene shows no preferential binding to the triplex. The DNA binding properties of the diazoniapentaphene derivatives remain essentially the same upon variation of the positions of nitrogen atoms or substitution with methyl groups. In contrast, the interactions of the diazoniaanthra[1,2-a]anthracence isomers with triplex DNA are slightly different. Notably, the 14a,16a-diazoniaanthra[1,2-a]anthracene is among the most efficient triplex stabilizers, with a 9-fold larger binding affinity for the triplex than for the DNA duplex. Moreover, the diazoniapentaphene and diazoniaanthra[1,2-a]anthracene derivatives represent the first examples of triplex-DNA binders that do not require additional aminoalkyl side chains for efficient triplex stabilization.

Toxicogenomics: Overview and potential applications for the study of non-covalent DNA interacting chemicals

Non-covalent DNA interacting agents, DNA-groove binding chemicals and DNA intercalators, are generally considered less cytotoxic than agents producing covalent DNA adducts and other DNA damage. Although the impact of non-covalent compound–DNA interactions on convoluted molecular and biochemical pathways is not well characterized, the most prominent effects include DNA conformational and related structural perturbations, interference with normal DNA protein interactions, such as topoisomerases, as well as effects on mitochondrial DNA and function. The cellular responses to such perturbations would be expected to include changes in transcription of a variety of genes. The emerging field of toxicogenomics seeks to exploit gene responses to define expression profiling signatures for various types of drugs and toxicants, and to provide mechanistic insight into their cellular effects. There are a variety of examples whereby different classes of genotoxicants and non-genotoxic agents can be distinguished by gene expression profiling using functional genomics approaches, which survey global transcriptional responses. In this review, we will discuss the promises and precautions in the use of functional genomics approaches to characterize stress agents including non-covalent DNA interacting agents.