Ligand-induced formation of triple helices with antiparallel third strands containing G and T

We have examined the effects of benzopyridoindole derivatives on triple helices with antiparallel third strands. Absorption spectroscopy, footprinting, and gel retardation experiments demonstrate that a benzopyridoindole derivative (BePI) is able to induce formation of a triple helix with an antiparallel (G, T)-containing third strand, which does not form in the absence of this ligand. This triple-helical complex is very stable with a half-dissociation temperature as high as 51 degrees C, and its formation is pH independent. Antiparallel oligonucleotides containing thymine and guanine bind strongly to double-helical DNA under physiological conditions in the presence of only 0.5 microM BePI. Formation of a BePI-stabilized triple helix strongly inhibits cleavage of the target duplex by DNase I.     

Synthesis, properties and biological evaluation of substituted furo[3,2-e] and pyrano[3,2-e]pyrido[4,3-b]indoles

Furo[3,2-e]- and pyrano[3,2-e]pyrido[4,3-b] indoles were synthesized from 1,4,5-trisubstituted 8-hydroxy-5H-pyrido[4,3-b]indoles. The intermediates, 10-chloro-6H-furo[3,2-e]pyrido[4,3-b]indole (11), 10-chloro-2,6-dihydro-1H-furo[3,2-e]pyrido-[4,3-b]indole (10) and 11-chloro-2,3-dihydro-3H,7H-pyrano[3,2-e]pyrido[4,3-b]indole (15), were substituted by diamines under thermal conditions (180 degrees C). In contrast, 11-chloro-3H,7H-pyrano[3,2-e]pyrido[4,3-b]indole (14), 9-allyl-1-chloro-4,5-dimethyl-5H-pyrido[4,3-b]indole (9a) and 8-propargyloxy-4,5-dimethyl-5H-pyrido[4,3-b]indole (8) led mainly to 1-aminosubstituted 8-hydroxy-5H-pyrido[4,3-b]indole derivatives resulting from an unexpected C3 unit elimination. When examined in three tumour cell lines (L1210 leukaemia, the B16 melanoma and the MCF7 breast adenocarcinoma) the new amino substituted furo[3,2-e]-, dihydrofuro[3,2-e]- and dihydropyrano[3,2-e]-pyrido[4,3-b]indole derivatives revealed cytotoxic properties, especially important for the 2,6-dihydro-1H-furo[3,2-e]pyrido[4,3-b]indole series. The most active compound (12b) significantly inhibits both DNA topoisomerases I and II, and is as potent as Adriamycin at inhibiting cell proliferation and inducing a massive accumulation of L1210 cells in the G2 + M phase of the cell cycle. However, 12b was less active than Adriamycin when tested in vivo against P388 leukaemia or the B16 melanoma tumour models.     

Interactions of DNA with a new electron-deficient tentacle porphyrin: meso-tetrakis[2,3,5,6-tetrafluoro-4-(2-t rimethylammoniumethyl-amine)phenyl]porphy rin

A new electron-deficient tentacle porphyrin meso-tetrakis[2,3,5,6-tetrafluoro-4-(2-trimethylammoniumethylamine )phenyl]porphyrin (TthetaF4TAP) has been synthesized. The binding interactions of TthetaF4TAP with DNA polymers were studied for comparison to those of an electron-deficient tentacle porphyrin and an electron-rich tentacle porphyrin; these previously studied porphyrins bind to DNA primarily by intercalative and outside-binding modes, respectively. The three tentacle porphyrins have similar size and shape. The basicity of TthetaF4TAP indicated that it has electronic characteristics similar to those of the intercalating electron-deficient tentacle porphyrin. However, TthetaF4TAP binds to calf thymus DNA, [poly(dA-dT)]2, and [poly(dG-dC)]2 in a self-stacking, outside-binding manner under all conditions. Evidence for this binding mode included a significant hypochromicity of the Soret band, a conservative induced CD spectrum, and the absence of an increase in DNA solution viscosity. As found previously for the electron-rich porphyrin, the results suggest that combinations of closely related self-stacked forms coexist. The mix of forms depended on the DNA and the solution conditions. There are probably differences in the detailed features of the self-stacking adducts for the two types of tentacle porphyrins, especially at high R (ratio of porphyrin to DNA). At low R values, the induced CD signal of TthetaF4TAP/CT DNA resembled that of TthetaF4TAP/[poly(dA-dT)]2, suggesting that TthetaF4TAP binds preferentially at AT regions. Competitive binding experiments gave evidence that TthetaF4TAP binds preferentially to [poly(dA-dT)]2 over [poly (dG-dC)]2. Thus, despite the long, positively charged, flexible substituents on the porphyrin, the binding of TthetaF4TAP is significantly affected by base-pair composition. Similar characteristics were found previously for the electron-rich tentacle porphyrin. Thus, significant changes in electron richness have relatively minor effects on this outside binding selectivity for AT regions. TthetaF4TAP is the first porphyrin with electron deficiency and shape similar to intercalating porphyrins that does not appear to intercalate. All porphyrins reported to intercalate have had pyridinium substituents. Thus, the electronic distribution in the porphyrin ring, not just the overall electron richness, may play a role in facilitating intercalation.     

Interactions of the antiviral quinoxaline derivative 9-OH-B220 [2, 3-dimethyl-6-(dimethylaminoethyl)- 9-hydroxy-6H-indolo-[2, 3-b]quinoxaline] with duplex and triplex forms of synthetic DNA and RNA

The binding of an antiviral quinoxaline derivative, 2,3-dimethyl- 6 - (dimethylaminoethyl) - 9 - hydroxy - 6H - indolo - [2,3 - b]quinoxaline (9-OH-B220), to synthetic double and triple helical DNA (poly(dA).poly(dT) and poly(dA).2poly(dT)) and RNA (poly(rA). poly(rU) and poly (rA).2poly(rU)) has been characterized using flow linear dichroism (LD), circular dichroism (CD), fluorescence spectroscopy, and thermal denaturation. When either of the DNA structures or the RNA duplex serve as host polymers a strongly negative LD is displayed, consistent with intercalation of the chromophoric ring system between the base-pairs/triplets of the nucleic acid structures. Evidence for this geometry also includes weak induced CD signals and strong increments of the fluorescence emission intensities upon binding of the drug to each of these polymer structures. In agreement with intercalative binding, 9-OH-B220 is found to effectively enhance the thermal stability of both the double and triple helical states of DNA as well as the RNA duplex. In the case of poly(dA).2poly(dT), the drug provides an unusually large stabilization of its triple helical state; upon binding of 9-OH-B220 the triplex-to-duplex equilibrium is shifted towards higher temperature by 52.5 deg. C in a 10 mM sodium cacodylate buffer (pH 7.0) containing 100 mM NaCl and 1 mM EDTA. When triplex RNA serves as host structure, LD indicates that the average orientation angle between the drug chromophore plane and the helix axis of the triple helical RNA is only about 60 to 65 degrees. Moreover, the thermal stabilizing capability, as well as the fluorescence increment, CD inducing power and perturbations of the absorption envelope, of 9-OH-B220 in complex with the RNA triplex are all less pronounced than those observed for the complexes with DNA and duplex RNA. These features indicate binding of 9-OH-B220 in the wide and shallow minor groove of poly(rA).2poly(rU). Based on the present results, some implications for the applications of this low-toxic, antiviral and easily administered drug in an antigene strategy, as well as its potential use as an antiretroviral agent, are discussed.     

Activation of chemically diverse procarcinogens by human cytochrome P-450 1B1

A human cytochrome P-450 (P450) 1B1 cDNA was expressed in Saccharomyces cerevisiae and the microsomes containing P450 1B1 were used to examine the selectivity of this enzyme in the activation of a variety of environmental carcinogens and mutagens in Salmonella typhimurium TA1535/pSK1002 or NM2009 tester strains, using the SOS response as an end point of DNA damage. We also determined and compared these activities of P450 1B1 with those catalyzed by recombinant human P450s 1A1 and 1A2, which were purified from membranes of Escherichia coli. The carcinogenic chemicals tested included 27 polycyclic aromatic hydrocarbons and their dihydrodiol derivatives, 17 heterocyclic and aryl amines and aminoazo dyes, three mycotoxins, two nitroaromatic hydrocarbons, N-nitrosodimethylamine, vinyl carbamate, and acrylonitrile. Among the three P450 enzymes examined here, P450 lB1 was found to have the highest catalytic activities for the activation of 11,12-dihydroxy-11,12-dihydrodibenzo[a,l]pyrene, 1,2-dihydroxy-1,2-dihydro-5-methylchrysene, (+)-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, 11,12-dihydroxy-11,12-dihydrobenzo[g]chrysene, 3,4-dihydroxy-3,4-dihydrobenzo[c]phenanthrene, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole, 2-aminoanthracene, 3-methoxy-4-aminoazobenzene, and 2-nitropyrene. P450 1B1 also catalyzed the activation of 2-amino-3,5-dimethylimidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, 2-amino-3-methylimidazo[4,5-f]quinoline, 2-aminofluorene, 6-aminochrysene and its 1,2-dihydrodiol, (-)-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, 1,2-dihydroxy-1,2-dihydrochrysene, 1,2-dihydroxy-1,2-dihydro-5,6-dimethylchrysene, 2,3-dihydroxy-2,3-dihydrofluoranthene, 3,4-dihydroxy-3,4-dihydro-7,12-dimethylbenz[a]anthracene, and 6-nitrochrysene to appreciable extents. However, P450 1B1 did not produce genotoxic products from benzo[a]pyrene, trans- 3,4-dihydroxy-3,4-dihydrobenzo[a]anthracene, trans-8,9-dihydroxy-8,9-dihydrobenzo[a]anthracene, 7,12-dimethylbenz[a]anthracene and its cis-5,6-dihydrodiol, 5-methylchrysene, 11,12-dihydroxy-11,12-dihydro-3-methylcholanthrene, 1,2-dihydroxy-1,2-dihydro-6-methylchrysene, benzo[c]phenanthrene, 2-amino-6-methyldipyridol[1,2-a:3',2'-d]imidazole, 2-acetylaminofluorene, benzidine, 2-naphthylamine, aflatoxin B1, aflatoxin G1, sterigmatocystin, N-nitrosodimethylamine, vinyl carbamate, or acrylonitrile in this assay system. P450 1B1 is expressed constitutively in extrahepatic organs, including fetal tissue samples, and is highly inducible in various organs by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds in experimental animal models. Thus, activation of procarcinogens by P450 lB1 may contribute to human tumors of extrahepatic origin.     

Induction of DNA adducts and mutations in spleen, liver and lung of XPA-deficient/lacZ transgenic mice after oral treatment with benzo[a]pyrene: correlation with tumour development

We were interested to study the relationship between DNA lesions, DNA repair, mutation fixation, and tumour development. Therefore, mice harbouring lacZ reporter genes and being either wild-type or defective in the DNA excision repair gene XPA, were treated with the genotoxic carcinogen benzo[a]pyrene at an oral dose of 13 mg/kg b.w. (3 times/week). At different time points, i.e. 1, 5, 9 or 13 weeks after start of the oral administration, levels of BPDE-N2-dG adducts (the major formed DNA adduct by benzo[a]pyrene in mice), and lacZ mutation frequencies were measured both in target (spleen) and non-target (lung and liver) tissues. Both in wild-type and XPA-deficient mice, benzo[a]pyrene treatment resulted in increased BPDE-N2-dG adduct levels in all three tissues analysed. In XPA-deficient mice, BPDE-N2-dG adduct levels still increased up to 13 weeks of oral benzo[a]pyrene treatment, whereas in DNA repair proficient mice steady-state levels were reached after 5 weeks of treatment. After 13 weeks, the BPDE-N2-dG adduct levels observed in XPA-/- mice, were 2- to 3-fold higher than the steady state levels observed in XPA+/+ mice in the same tissues. Mutation frequencies in the lacZ reporter gene were the same in wild-type and XPA-deficient mice that were treated with the solvent only. Oral benzo[a]pyrene treatment resulted in an increase in mutation frequency in the lacZ marker gene in all three tissues, but this increase was most profound in the spleen. After 13 weeks of treatment, a 7-fold increase in lacZ mutation frequency was detected in the spleen of wild-type mice as compared to mutation frequencies in control mice. At the same time point, a 15-fold increase in lacZ mutation frequency was observed in the spleen of XPA-deficient mice. The data presented here show, that a defect in NER mainly results in enhanced mutation frequencies in lymphocytic cells after oral treatment with the genotoxic compound benzo[a]pyrene. Interestingly, as we established in a previously performed carcinogenicity assay, the same oral treatment with benzo[a]pyrene induced lymphomas residing in the spleen of XPA-deficient mice.     

Genotoxicity of coke-oven and urban air particulate matter in in vitro acellular assays coupled with 32P-postlabeling and HPLC analysis of DNA adducts

This study is an in vitro part of the ongoing biomarker studies with population from a polluted region of Northern Bohemia and coke-oven workers from Czech and Slovak Republics. The aim of this study is to compare DNA adduct forming ability of chemical compound classes from both the urban and coke-oven extractable organic mass (EOM) of airborne particles. The crude extracts were fractionated into seven fractions by acid-base partitioning and silica gel column chromatography. In in vitro acellular assays we used calf thymus DNA (CT DNA) with oxidative (+S9) and reductive activation mediated by xanthine oxidase (+XO) under anaerobic conditions. Both the butanol and nuclease P1 versions of 32P-postlabeling for detection of bulky aromatic and/or hydrophobic adducts were used. The results showed that the spectra of major DNA adducts resulting from both the in vitro assays are within the fractions similar for both the urban and coke-oven samples. The highest DNA adduct levels with S9-activation were detected for the neutral aromatic fraction, followed by slightly polar and acidic fractions for both samples. With XO-mediated metabolism, the highest DNA adduct levels were detected for both the acidic fractions. Assuming additivity of compound activities, then the acidic fraction, which in the urban sample comprises a major portion of EOM mass (28%), may contain the greatest activity in both in vitro assays (39 and 69%, +S9 and +XO, respectively). In contrast, the aromatic fraction constituting only 8% of total urban EOM mass may account for comparable activity (34%) with organic acids. The highest DNA adduct forming activity of the coke-oven sample accounts for the aromatic fraction (82 and 63%, +S9 and +XO, respectively) that also contains the greatest portion of the total EOM (48%). To characterize some of the specific DNA adducts formed, we coupled TLC on 20x20 cm plates with HPLC analysis of 32P-postlabeled adducts. In both S9-treated samples of the aromatic fraction, we tentatively identified DNA adducts presumably diolepoxide-derived from: 9-hydroxy-benzo[a]pyrene (9-OH-B[a]P), benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide[+/-] (anti-BPDE), benzo[b,j,k]fluoranthenes (B[b]F, B[j]F, B[k]F), chrysene (CHRY), benz[a]-anthracene (B[a]A) and indeno[cd]pyrene (I[cd]P). These DNA adducts accounted for about 57% of total DNA adducts detected in both S9-treated samples of the aromatic fraction. DNA adducts of XO-treated samples were sensitive to nuclease P1 and HPLC profiles of the major adducts were markedly different from the major adducts of S9-treated samples. However, the combination of TLC and HPLC did not confirm the presence of DNA adducts derived from 1-nitropyrene (1 NP), 9-nitroanthracene (9 NA) and 3-nitrofluoranthene (3 NF) that were detected by GC-MS in the slightly polar fraction. We concluded that the chemical fractionation procedure facilitates the assessing of DNA adduct forming ability of different chemical compound classes. However, based on the results obtained with the whole extracts, it does not fulfil a task of the actual contribution of individual fractions within the activity of the whole extracts. Our results are the first in detecting of DNA adducts derived from urban air and coke-oven particulate matter.     

5-substituted 2-bromoindolo[3,2-b]quinoxalines. A class of potential antitumor agents with cdc25 phosphatase inhibitory properties

Several derivatives of 5-substituted 2-bromoindolo[3,2-b]quinoxaline were synthesized and characterized. The synthesized compounds were evaluated for their antitumor activity using the National Cancer Institute-in vitro-disease oriented antitumor screen and two biochemical mechanism-based screens (cdc2 kinase and cdc25 phosphatase). Compound 19 showed broad spectrum antitumor activity with full panel (MG-MID) GI50. TGI, and LC50 of 14.2, 31.6- and 66.2 microM, respectively. In addition it inhibited cdc2 kinase and cdc25 phosphatase with IC50's of 70 and 25 microM, respectively. Thus, compound 19 represents a model for compounds with potential antitumor activity and cdc25 phosphatase inhibitory properties.     

Homologous pairing in stretched supercoiled DNA

By using elastic measurements on single DNA molecules, we show that stretching a negatively supercoiled DNA activates homologous pairing in physiological conditions. These experiments indicate that a stretched unwound DNA locally denatures to alleviate the force-driven increase in torsional stress. This is detected by hybridization with 1 kb of homologous single-stranded DNA probes. The stretching force involved (approximately 2 pN) is small compared with those typically developed by molecular motors, suggesting that this process may be relevant to DNA processing in vivo. We used this technique to monitor the progressive denaturation of DNA as it is unwound and found that distinct, stable denaturation bubbles formed, beginning in A+T-rich regions.     

Bioactivation of the cooked food mutagen N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine by estrogen sulfotransferase in cultured human mammary epithelial cells

Cooked food mutagens from fried meat and fish have recently been suggested to contribute to the etiology of breast cancer. Thus, the most prevalent of these compounds, i.e. 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, or rather its more mutagenic N-hydroxylated metabolite (N-OH-PhIP), forms DNA adducts in mammary cells, including human mammary epithelial (HME) cells. The objective of this study was to determine the involvement of estrogen sulfotransferase (EST), the only sulfotransferase identified in HME cells, in the further bioactivation of N-OH-PhIP. These studies were done in vitro using human recombinant EST and in intact HME cells. Human recombinant EST increased the covalent binding of [3H]N-OH-PhIP to calf thymus DNA approximately 3.5-fold in the presence of the sulfotransferase co-substrate 3'-phosphoadenosine-5'-phosphosulfate at each N-OH-PhIP concentration (1, 10 and 100 microM) (n = 6, P < 0.001). In contrast, EST did not catalyze the DNA binding of two other cooked food mutagens, N-hydroxy-2-amino-3-methylimidazo[4,5-f]quinoline and N-hydroxy-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, which are mainly hepatocarcinogens. Cultured HME cells displayed high EST activity, which could be completely inhibited by 1 microM estrone. When the cells were incubated with [3H]N-OH-PhIP, binding to native DNA occurred at 60-240 pmol/mg DNA. This binding was inhibited to 55% of control by 1 microM estrone (P < 0.01, n = 8), suggesting that EST plays a significant role in carcinogen bioactivation in human breast tissue.     

Spectroscopic investigation of an intramolecular DNA triplex containing both G.G:C and T.A:T triads and its complex with netropsin

The triple helix formation by the oligonucleotide 5'd(G4T4G4-[T4]-G4A4G4-[T4]-C4T4C4) ([T4] represents a stretch of 4 thymine residues) has been investigated by UV absorption spectroscopy and circular dichroism. In a 10 mM sodium cacodylate, 0.2 mM disodium EDTA (pH 7) buffer, we show the following significant results: i) In the absence of MgCl2, the oligonucleotide adopts a hairpin duplex structure with the dangling tail 5'd(G4T4G4-[T4]). This 5' extremity, which contains separated runs of four guanine residues, does not assume the expected tetraplex conformation observed when this sequence is free. ii) In the presence of MgCl2, the oligonucleotide folds back on itself twice to give a triple helix via a double hairpin formation, with [T4] single-strand loops. iii) The addition of high concentration of KCl to the preformed triplex does not disrupt the structure. Nevertheless, if the oligonucleotide is allowed to fold back in the presence of K+, triplex formation is inhibited. Circular dichroism studies demonstrate that the oligonucleotide adopts a dimeric conformation, resulting from the association of two hairpin duplexes, via the formation of an antiparallel G-quadruplex by the telomeric 5'd(G4T4G4-[T4]) extremities. iv) Under the experimental conditions used in this report, the triplex melts in a monophasic manner. v) Netropsin, a DNA minor groove ligand, binds to the central site A4/T4 of the duplex and to that of the triplex in an equimolar stoichiometry. In contrast with previous studies concerning pyr.pur:pyr triplexes, thermal denaturation experiments demonstrate that the netropsin binding stabilizes the intramolecular triplex.     

Characterization of DNA adducts formed by anti-benzo[g]chrysene 11,12-dihydrodiol 13,14-epoxide

The anti-11,12-dihydrodiol 13,14-epoxide of benzo[g]chrysene, a fjord-region-containing hydrocarbon, was found to react with DNA in vitro to yield, as the major product, an adduct in which the epoxide of the 11R, 12S, 13S, 14R enantiomer was opened trans by the amino group of deoxyadenosine. The structures of this adduct and other deoxyadenosine and deoxyguanosine adducts were established by spectroscopic methods. In reactions with deoxyguanylic acid, a product tentatively identified as a 7-substituted guanine was also detected. The mutagenic properties of this dihydrodiol epoxide in shuttle vector pSP189 showed that mutation at AT pairs accounted for 39% of base change mutations whereas chemical findings indicated that about 60% of adducts formed in calf thymus DNA involved adenines. Since calf thymus DNA is 56% AT and the target supF gene is 41% AT, the findings represent a fairly close relationship between adduct formation and mutagenic response. Overall, the chemical and mutagenic selectivities for the two purine bases in DNA were similar, though not identical, to those for the only other fjord-region-containing hydrocarbon studied in depth, i.e., benzo[c]phenanthrene. A major difference for these two hydrocarbon derivatives, however, is that benzo[c]phenanthrene dihydrodiol epoxides react to much higher extents (approximately 4-fold) with DNA than did the benzo[g]chrysene derivative.     

Tumour-initiating activities on mouse skin of dihydrodiols derived from benzo[a]pyrene

Three dihydrodiols that are metabolites of benzo[a]pyrene and benzo[a]-pyrene itself have been tested in a comparative experiment for their activities as initiators of tumours in mouse skin. A single application (25 mug) of 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene, of 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, of 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene, or of benzo[a]pyrene was made to the shaved dorsal skin of adult female CDI mice; this was followed 2 weeks later by multiple thrice-or twice-weekly applications (1 mug) of 12-O-tetradecanoyl-phorbol-13-acetate as promoting agent. A control group of 30 mice received the promoting agent alone. The experiments were terminated 52 weeks after initiation. At this stage, all the groups contained mice bearing skin papillomas, some of which had progressed to malignancy. Quantitatively the results show that the 7,8-dihydrodiol is almost as active an initiator of mouse skin tumours as benzo[a]pyrene itself; the 4,5- and 9,10-dihydrodiols were significantly less active. The significance of these results is discussed in relation to the hypothesis that diol-epoxides are important in the metabolic activation of polycyclic hydrocarbons like benzo[a]pyrene.     

Structural determination of a mutagenic aminophenylnorharman produced by the co-mutagen norharman with aniline

Norharman (9H-pyrido[3,4-b]indole), widely distributed in our environment, including cigarette smoke and cooked foodstuffs, is not mutagenic to Salmonella strains, but becomes mutagenic to S.typhimurium TA98 and YG1024 with S9 mix in the presence of non-mutagenic aromatic amines such as aniline and o-toluidine. To elucidate the mechanisms of co-mutagenicity, we tried to isolate the mutagen(s) produced by a reaction between norharman and aniline with S9 mix. By HPLC purification, two mutagenic compounds (I and II), one (I) showing mutagenicity with and the other (II) without S9 mix, were isolated. The structure of compound I was deduced to be a coupled compound of norharman and aniline, 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman), by a variety of spectrometry techniques and this was confirmed by its chemical synthesis. The mutagenic activity of this novel heterocyclic amine was tested using the pre-incubation method and was found to induce 187,000 revertants in TA98 and 1,783,000 revertants in YG1024 per microg with S9 mix. Compound II was shown to be hydroxyaminophenylnorharman. Formation of the same DNA adducts was observed in YG1024 when aminophenylnorharman or a mixture of norharman plus aniline was incubated with S9 mix. The hydroxyamino derivative also yielded the same DNA adducts in YG1024. Thus, the appearance of mutagenicity by norharman with aniline in the presence of S9 mix suggests that the coupled mutagenic compound, aminophenylnorharman, is formed from norharman and aniline, then converted to the hydroxyamino derivative and forms DNA adducts to induce mutations in TA98 and YG1024.     

Different effects of nonintercalative antitumor drugs on DNA triple helix stability: SN-18071 promotes triple helix formation

The interaction of the nonintercalating bisquaternary ammonium heterocyclic drugs SN-18071 and SN-6999 with a DNA triple helix has been studied using thermal denaturation and CD spectroscopy. Our data show, that both minor groove binders can bind to the triple helix of poly(dA).2poly(dT) under comparable ionic conditions, but they influence the stability of the triplex relative to the duplex structure of poly(dA).poly(dT) in a different manner. SN-18071, a ligand devoid of forming hydrogen bonds, can promote triplex formation and thermally stabilizes it up to 500 mM Na+ concentration. SN-6999 destabilizes the triplex to duplex equibilirium whereas it stabilizes the duplex. The binding constant of SN-18071 is found to be greater than that to the duplex. The stabilizing effect of SN-18071 is explained by electrostatic interactions of three ligand molecules with the three grooves of the triple stranded structure. From the experiments it is concluded that SN-6999 binds to the triplex minor groove thereby destabilizing the triplex similar as previously reported for netropsin.     

New indole and pyridazinoindole analogs--synthesis and study as inhibitors of phosphodiesterases and as inhibitors of blood platelet aggregation

This paper presents the synthesis of new indole, pyridazino[4,5-b]-indole, and pyridazino[4,5-a]indole analogs as well as a study of their "in vitro" activity as inhibitors of different phosphodiesterases isolated from dog cardiac tissue, dog aorta, and bovine platelets; the study of their activity as inhibitors of platelet aggregation in guinea pig whole blood, with ADP and arachidonic acid (AA) as pro-aggregants, is also included. The selected compounds 8-benzyloxy-3,4-dihydro-1-(3,4,5-trimethoxy)benzylideneaminopyridazin o[4,5- b]indole 14g, and 8-benzyloxy-4-[(3,4-dimethyl)pyrazolyl]pyridazino[4,5-b]indo le 20 present an interesting profile as potential inodilators, with a complementary, beneficial activity as inhibitors of the aggregation, activities which could possibly be related to the inhibition of the PDE's. Among the other compounds studied, 8-benzyloxy-3,4-dihydro-1-[4-(methyl)piperazino]acetamidopyrida zino[4,5- b]indol-4-one 16c and 8-benzyloxy-3,4-dihydro-1-[4-(2- methoxyphenyl)piperazino]acetamidopyridazino[4,5-b]indol-4-o ne 16f stood out as inhibitors of platelet aggregation, with a mechanism that could possibly be related to the AA cascade.     

TROSY in triple-resonance experiments: new perspectives for sequential NMR assignment of large proteins

The NMR assignment of 13C, 15N-labeled proteins with the use of triple resonance experiments is limited to molecular weights below approximately 25,000 Daltons, mainly because of low sensitivity due to rapid transverse nuclear spin relaxation during the evolution and recording periods. For experiments that exclusively correlate the amide proton (1HN), the amide nitrogen (15N), and 13C atoms, this size limit has been previously extended by additional labeling with deuterium (2H). The present paper shows that the implementation of transverse relaxation-optimized spectroscopy ([15N,1H]-TROSY) into triple resonance experiments results in several-fold improved sensitivity for 2H/13C/15N-labeled proteins and approximately twofold sensitivity gain for 13C/15N-labeled proteins. Pulse schemes and spectra recorded with deuterated and protonated proteins are presented for the [15N, 1H]-TROSY-HNCA and [15N, 1H]-TROSY-HNCO experiments. A theoretical analysis of the HNCA experiment shows that the primary TROSY effect is on the transverse relaxation of 15N, which is only little affected by deuteration, and predicts sensitivity enhancements that are in close agreement with the experimental data.