DNA intercalation and inhibition of topoisomerase II. Structure-activity relationships for a series of amiloride analogs

Among its many properties, amiloride is a DNA intercalator and topoisomerase II inhibitor. Previous work has indicated that the most stable conformation for amiloride is a planar, hydrogen-bonded, tricyclic structure. To determine whether the ability of amiloride to intercalate into DNA and to inhibit DNA topoisomerase II was dependent on the ability to assume a cyclized conformation, we studied the structure-activity relationship for 12 amiloride analogs. These analogs contained structural modifications which could be expected to allow or impede formation of a cyclized conformation. Empirical assays consisting of biophysical, biochemical, and cell biological approaches, as well as computational molecular modeling approaches, were used to determine conformational properties for these molecules, and to determine whether they intercalated into DNA and inhibited topoisomerase II. Specifically, we measured the ability of these compounds to 1) alter the thermal denaturation profile of DNA, 2) modify the hydrodynamic behavior of DNA, 3) inhibit the catalytic activity of purified DNA topoisomerase II in vitro, 4) promote the topoisomerase II-dependent cleavage of DNA, and 5) inhibit functions associated with DNA topoisomerase II in intact cells. Results indicated that only those analogs capable of cyclization could intercalate into DNA and inhibit topoisomerase II. Thus, the ability of amiloride and the 12 analogs studied to intercalate into DNA and to inhibit topoisomerase II appears dependent on the ability to exist in a planar, hydrogen-bonded, tricyclic conformation.     

Cross-resistance of an amsacrine-resistant human leukemia line to topoisomerase II reactive DNA intercalating agents. Evidence for two topoisomerase II directed drug actions.

HL-60/AMSA is a human leukemia cell line that is 50-100-fold more resistant than its drug-sensitive HL-60 parent line to the cytotoxic actions of the DNA intercalator amsacrine (m-AMSA). HL-60/AMSA topoisomerase II is also resistant to the inhibitory actions of m-AMSA. HL-60/AMSA cells and topoisomerase II are cross-resistant to anthracycline and ellipticine intercalators but relatively sensitive to the nonintercalating topoisomerase II reactive epipodophyllotoxin etoposide. We now demonstrate that HL-60/AMSA and its topoisomerase II are cross-resistant to the DNA intercalators mitoxantrone and amonafide, thus strongly indicating that HL-60/AMSA and its topoisomerase II are resistant to topoisomerase II reactive intercalators but not to nonintercalators. At high concentrations, mitoxantrone and amonafide were also found to inhibit their own, m-AMSA's, and etoposide's abilities to stabilize topoisomerase II-DNA complexes. This appears to be due to the ability of these concentrations of mitoxantrone and amonafide to inhibit topoisomerase II mediated DNA strand passage at a point in the topoisomerization cycle prior to the acquisition of the enzyme-DNA configuration that yields DNA cleavage and topoisomerase II-DNA cross-links. In addition, amonafide can inhibit the cytotoxic actions of m-AMSA and etoposide. Taken together, these results suggest that the cytotoxicity of m-AMSA and etoposide is initiated primarily by the stabilization of the topoisomerase II-DNA complex. Other topoisomerase II reactive drugs may inhibit the enzyme at other steps in the topoisomerization cycle, particularly at elevated concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epolactaene, a novel neuritogenic compound in human neuroblastoma cells, selectively inhibits the activities of mammalian DNA polymerases and human DNA topoisomerase II

We chemically synthesized epolactaene, a neuritogenic compound in human neuroblastoma cells, and investigated its biochemical action in vitro. Epolactaene and its derivatives selectively inhibited the activities of mammalian DNA polymerase alpha and beta and human DNA topoisomerase II, with IC(50) values of 25, 94, and 10 microM, respectively. By comparison with its structural derivatives, the long alkyl side chain in epolactaene seemed to have an important role in this inhibitory effect. The compound did not influence the activities of plant or prokaryotic DNA polymerases or of other DNA metabolic enzymes such as telomerase, RNA polymerase, and deoxyribonuclease I. Epolactaene did not intercalate into DNA. These results suggested that the neuritogenic compound epolactaene influences both DNA polymerases and topoisomerase II despite the dissimilarity in both structure and properties of these two enzymes and that inhibition of these enzymes could be related to the neuritogenic effect in human neuroblastoma cells. The relationship between the neuritogenic mechanism and cell cycle regulation by epolactaene was also discussed.     

Synthesis of new amonafide analogues via coupling reaction and their cytotoxic evaluation and DNA-binding studies

A series of 5-alkylamino substituted amonafide analogues were synthesized from naphthalic anhydride by three steps including bromization, amination and CuI/proline catalyzed coupling reaction. The CuI/L-proline catalyzed coupling reaction was first applied to the naphthalimide system. These new amonafide analogues showed potential anticancer activities against HeLa and P388D1 cell lines in vitro, and 4a, 4b, and 4h exhibited better activity than amonafide against HeLa cell under the same experimental conditions. More importantly, the new analogues could avoid the side effect of amonafide due to their structure, in which lacks a primary amine at the 5 position. Moreover, the DNA-binding of the analogues was also investigated.     

5-Non-amino aromatic substituted naphthalimides as potential antitumor agents: Synthesis via Suzuki reaction, antiproliferative activity, and DNA-binding behavior

Amonafide is a naphthalimide derivative with antitumor activity and has failed to enter clinical phase III, because of its high-variable and unpredictable toxicity. In order to develop selective, efficient, and safe drugs, applying the ‘nonfused’ aromatic system strategy, a series of 5-non-amino aromatic substituted naphthalimides as replacement for amonafide were designed and were synthesized from naphthalic anhydride by three steps including bromination, amination, and Pd(PPh₃)₄ catalyzed Suzuki reaction. These new naphthalimide derivatives, except 4b, not only exhibited better activity than amonafide against HeLa and P388D1 cell lines in vitro under the same experimental conditions, but also could avoid the side effect of amonafide due to their structure, which lacks an easy acetylated arylamine at the 5 position. The DNA-binding behavior of the naphthalimide derivatives was also investigated, and the results suggested that they bind to DNA via intercalation and 4a and 4g intercalated into DNA in different fashion.     

Antibacterial activity of novel naphthoquiones derivatives

l,4-naphthoquinone moiety is Known to confer numerous molecules with distinct-hiological activities including anti -mycobacterial, anticancer and anti-inflammatory activities. Vitamin K2, doxorubicin and mitomycin are among the few examples of this class of chemicals used in the treatment of bleeding, lymphoma and carcinoma respectively. Although the exact action mechanism of these molecules is still under investigation, proposed mechanisms include their interact ion with DNA inhibition of topoisomerase II, and production of ROS, contributing individually or in combination with DNA damage and cell death . In the present study, 6 naphthoquinones were synthesized and assayed for their antimicrobial activity againstStaphylococcus aureus by disc diffusion assay and spectrophotometric analys is. DNA topoisomerase II activity was also measured to investigate the degreee of conformation change of a test plasmid DNA affected by the naphthoquinone deriva tives.     

Correlation of the cytotoxic activity of four different alkaloids, from Chelidonium majus (greater celandine), with their DNA intercalating properties and ability to induce breaks in the DNA of NK/Ly murine lymphoma cells

The purpose of this study was to examine the relationship between the DNA intercalating characteristics and the DNA damaging capacity of four alkaloids extracted from Chelidonium majus L, as well as their toxicity towards murine NK/Ly lymphoma cells. Chelerythrine, sanguinarine and coptisine were found to be intercalated into the DNA isolated from NK/Ly cells, meanwhile, chelidonine exhibited no affinity to DNA. Sanguinarine exhibited the greatest toxicity toward NK/Ly cells, and the toxicity of the other three decreased in descending order: chelerythrine, coptisine and chelidonine. Chelerythrine and sanguinarine caused DNA damage, illustrated by the formation of comets of the third class. Coptisine was less toxic than chelerythrine and sanguinarine, and affected the formation the same class of comets in higher concentration. The quantity of comets induced by chelidonine were negligible, a finding consistent with its inability to intercalate into DNA structure. The ability of four main alkaloids of Chelidonium majus L., to intercalate into DNA isolated from murine NK/Ly lymphoma cells, correlated with their ability to induce breaks in cellular DNA and with their toxic effect towards those cells.     

DNA binding,cytotoxicity, apoptotic inducing activity,and molecular modeling study of quercetin zinc(II) complex

DNA cleavage activity of quercetin zinc(II) complex has been studied, but little attention has been devoted to the relationship between antitumor activity of this complex and DNA-binding properties. DNA-binding properties of quercetin zinc(II) complex were studied using UV–vis spectra, fluorescence measurements, and viscosity measurements. The results obtained indicate that quercetin zinc(II) complex can intercalate into the stacked base pairs of DNA, and compete with the strong intercalator ethidium bromide for the intercalative binding sites with Stern–Volmer quenching constant, K_sq = 1.24. The complex was subjected to biological tests in vitro using three tumor cell lines (HepG2, SMMC7721, and A549), which showed significant cytotoxicity against three tumor cell lines. Moreover, Hoechst33258 staining showed HepG2 cells underwent the typical morphologic changes of apoptosis characterized by nuclear shrinkage, chromatin condensation, or fragmentation after exposure to the complex. In addition, Molecular modeling was performed to learn the complex could be preferentially bound to DNA in GC region. Our results suggest that antitumor activity of quercetin zinc(II) complex might be related to its intercalation into DNA.     

Synthesis,DNA and protein interactions and human topoisomerase inhibition of novel Spiroacridine derivatives

Nine new spiroacridine derivatives were synthetized by introducing cyano-N-acylhydrazone group between the acridine and phenyl-substituted rings followed by spontaneous cyclization. The new compounds were assayed for their DNA binding properties, human topoisomerase IIα inhibition and bovine serum albumin (BSA) interaction. Besides, docking analysis were performed in order to better understanding the biomolecule-compounds interactions. All compounds interacted with BSA which was demonstrated by the fluorescence suppression constant of 10⁴?M^?1. Compounds with chloro and NO₂ substituents at that para-position on phenyl ring demonstrated the best results for BSA interaction. DNA binding constant determined by UV–vis data demonstrated high values for AMTAC-11 and AMTAC-14, 1.1?×?10⁸?M^?1 and 4.8?×?10⁶?M^?1, respectively, and all others presented constant values of 10⁵?M^?1. AMTAC-06 with chloro at para-position on phenyl ring presented a topoisomerase II inhibition of 84.34% in comparison to the positive controls used. Docking studies indicated that AMTAC-06 is able to intercalate the DNA base pairs at topoisomerase IIα active site, preventing DNA connection after break, in a process known as poisoning. Topoisomerase enzyme inhibition result was correlated to BSA interaction profile, since AMTAC-06 showed the best results in both analysis. The findings obtained here proved that methoxy or chloro substitution on phenyl ring at para-position is fundamental for in vitro activity of new spiroacridine derivatives, and indicates that AMTAC-06 is a promising entity and should serve as a lead compound in the development of new DNA and protein binders, as well as human topoisomerase II inhibitors.     

Diimine Platinum(II) and Palladium(II) Complexes of Dithiocarbamate Derivative as Potential Antitumor Agents: Synthesis,Characterization, Cytotoxicity,and Detail DNA-Binding Studies

Abstract

The synthesis and chemical characterization of two structurally related platinum(II) and palladium(II) complexes, [M(2,2′-bipyridine)(morpholinedithiocarbamate)]NO₃ or [M(bpy) (mor-dtc)]NO₃, where M = Pt(II) or Pd(II), are described. Studies of anti-tumor activities of these complexes against human cell tumor lines (K₅₆₂) have been carried out. They show 50% cytotoxic concentration (Cc₅₀) values much lower than that of cisplatin. Both of these water soluble complexes have been shown to interact with calf thymus DNA (ct-DNA) using difference absorption-, fluorescence-, and circular dichroism-titration techniques. These studies showed that both complexes exhibit cooperative binding and presumably intercalate in DNA. These complexes unexpectedly denature DNA at very low concentrations (50–100 μM). Several binding and thermodynamic parameters are also described.     

1,2,3-Triazole-derived naphthalimides as a novel type of potential antimicrobial agents: Synthesis,antimicrobial activity,interaction with calf thymus DNA and human serum albumin

A series of 1,2,3-triazole-derived naphthalimides as a novel type of potential antimicrobial agents were synthesized and characterized by IR, NMR and HRMS spectra. All the new compounds were screened for their antimicrobial activity against four Gram-positive bacteria, four Gram-negative bacteria and three fungi. Bioactive assay manifested that 3,4-dichlorobenzyl compound 9e and its corresponding hydrochloride 11e showed better anti-Escherichia coli activity than Norfloxacin and Chloromycin. Preliminary research revealed that compound 9e could effectively intercalate into calf thymus DNA to form compound 9e–DNA complex which might block DNA replication and thus exert antimicrobial activities. Human serum albumin could effectively store and carry compound 9e by electrostatic interaction.     

Interplay of DNA repair,homologous recombination,and DNA polymerases in resistance to the DNA damaging agent 4-nitroquinoline-1-oxide in Escherichia coli

Escherichia coli has three DNA damage-inducible DNA polymerases: DNA polymerase II (Pol II), DNA polymerase IV (Pol IV), and DNA polymerase V (Pol V). While the in vivo function of Pol V is well understood, the precise roles of Pol IV and Pol II in DNA replication and repair are not as clear. Study of these polymerases has largely focused on their participation in the recovery of failed replication forks, translesion DNA synthesis, and origin-independent DNA replication. However, their roles in other repair and recombination pathways in E. coli have not been extensively examined. This study investigated how E. coli's inducible DNA polymerases and various DNA repair and recombination pathways function together to convey resistance to 4-nitroquinoline-1-oxide (NQO), a DNA damaging agent that produces replication blocking DNA base adducts. The data suggest that full resistance to this compound depends upon an intricate interplay among the activities of the inducible DNA polymerases and recombination. The data also suggest new relationships between the different pathways that process recombination intermediates.     

Mixed ligand copper(II) complexes of phenanthroline/bipyridyl and curcumin diketimines as DNA intercalators and their electrochemical behavior under Nafion and clay modified electrodes

The mode of binding of copper(II) mixed ligand complexes of phen/bpy and Knoevenagel condensate of curcumin (4-salicylidene-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) and 4-X-anilines with herring sperm DNA has been investigated using spectral and electrochemical techniques in Tris-HCl buffer pH 7.1. On titration with DNA, usual hypochromism and unusual (large) red shift (30-35 nm) for some of these complexes were observed in their absorption spectra of intense intraligand (IL) pi-pi* transition around 420 nm. Variations in the absorbance due to their interaction with DNA on time scale were also investigated, under fixed concentrations of complex and DNA. On interaction with DNA, the quasi-reversible CuII/I redox couple slightly improves its reversibility with considerable decrease in current intensity. The intercalation of these copper complexes into the DNA base pairs was also investigated by gel retardation assay method. All the experimental results indicate that the phen mixed copper(II) complexes intercalate more effectively into the DNA base pairs than their bpy counterparts. Significant differences in the redox behavior of these copper(II) complexes under electrochemically modified GC electrodes with Nafion and K10 Montmorillonite clay have also been investigated and discussed.     

Modulation of oxidative DNA damage and DNA-crosslink formation induced by cis-diammine-tetrachloro-platinum(IV) in the presence of endogenous reductants

Platinum(IV) [Pt(IV)] complex, satraplatin, is currently in clinical trials for the treatment of various cancers. As a key step of the anti-cancer effect exertion, satraplatin is supposed to be reduced by endogenous reductants to platinum(II) [Pt(II)] complex. In this study, we investigated the interaction of DNA, Pt(IV), and the endogenous reductants such as ascorbic acid (AsA) and glutathione (GSH). As a model Pt(IV) compound, cis-diammine-tetrachloro-Pt(IV) [cis-Pt(IV)], which is a prodrug of cisplatin [cis-diammine-dichloro-Pt(II), cis-Pt(II)], was incubated with calf thymus DNA in the presence of AsA or GSH. In the presence of AsA, cis-Pt(IV) induced oxidative DNA damage. Hydroxyl radical scavengers suppressed the AsA-associated oxidative damage, thereby suggesting that hydroxyl radicals are involved in the DNA oxidation. cis-Pt(II)-like CD spectral change and crosslink formation in calf thymus DNA were also observed during this DNA oxidation, suggesting cis-Pt(IV) reduction by AsA and DNA conformational change induced by the newly formed cis-Pt(II) binding to DNA. GSH did not induce oxidative DNA damage likely due to its own hydroxyl radical scavenging ability. Further, GSH suppressed the Pt(II)-mediated DNA conformational change and crosslink formation, suggesting that GSH sequesters the cis-Pt(II) away from DNA by GSH-cis-Pt(II) complex formation.     

Oxocrebanine: A Novel Dual Topoisomerase inhibitor,Suppressed the Proliferation of Breast Cancer Cells MCF-7 by Inducing DNA Damage and Mitotic Arrest

BackgroundDNA topoisomerase (Topo) inhibition plays key role in breast cancer treatment. Stephania hainanensis H. S. Lo et Y. Tsoong (S. hainanensis), a Li nationality plant that has abundant aporphine alkaloids, can inhibit Topo.PurposeTo identify a dual Topo inhibitor, a deep and systematic study of active aporphine alkaloids in S. hainanensis and their mechanisms of inhibiting breast cancer proliferation and Topo activity are essential.Study designThis study aimed to assess the anti-breast cancer and Topo inhibitory activities of oxocrebanine and explore the underlying mechanisms.MethodsThe growth inhibitory activities of 12 compounds in S. hainanensis were screened by MTT assay in MCF-7, SGC-7901, HepG-2 cells, and compared with the effects on human normal mammary epithelial MCF-10A cells as non cancer control cells. The Topo inhibitory activity was assessed by DNA relaxation and unwinding assays, kDNA decatenation assay and western blot. Cell cycle and autophagy analyses were carried out with flow cytometry and staining. Acridine orange staining and α-tubulin morphology were observed by fluorescence microscopy. Western blot was used to examine microtubule assembly dynamics and the expression levels of key proteins associated with DNA damage, autophagy and mitotic arrest.ResultsOxocrebanine was the anti-breast cancer active alkaloid in S. hainanensis. It exhibited the best inhibitory effect on MCF-7 cells with an IC₅₀ of 16.66 μmol/l, and had only weak effect on the proliferation of MCF-10A cells. Oxocrebanine inhibited Topo I and II α in a cell-free system and in MCF-7 cells. The DNA unwinding assay suggested that oxocrebanine intercalated with DNA as a catalytic inhibitor. Oxocrebanine regulated the levels of Topo I and IIα and DNA damage-related proteins. Oxocrebanine led to the mitotic arrest, and these effects occurred through both p53-dependent and p53-independent pathways. Oxocrebanine induced autophagy, abnormal α-tubulin morphology and stimulated enhanced microtubule dynamics.ConclusionOxocrebanine was the anti-breast cancer active aporphine alkaloid in S. hainanensis. Oxocrebanine was a Topo I/IIα dual inhibitor, catalytic inhibitor and DNA intercalator. Oxocrebanine caused DNA damage, autophagy, and mitotic arrest in MCF-7 cells. Oxocrebanine also disrupted tubulin polymerization. Accordingly, oxocrebanine held a great potential for development as a novel dual Topo inhibitor for effective breast cancer treatment.     

Bacteriophage Orphan DNA Methyltransferases: Insights from Their Bacterial Origin,Function, and Occurrence

Type II DNA methyltransferases (MTases) are enzymes found ubiquitously in the prokaryotic world, where they play important roles in several cellular processes, such as host protection and epigenetic regulation. Three classes of type II MTases have been identified thus far in bacteria which function in transferring a methyl group from S-adenosyl-l-methionine (SAM) to a target nucleotide base, forming N-6-methyladenine (class I), N-4-methylcytosine (class II), or C-5-methylcytosine (class III). Often, these MTases are associated with a cognate restriction endonuclease (REase) to form a restriction-modification (R-M) system protecting bacterial cells from invasion by foreign DNA. When MTases exist alone, which are then termed orphan MTases, they are believed to be mainly involved in regulatory activities in the bacterial cell. Genomes of various lytic and lysogenic phages have been shown to encode multi- and mono-specific orphan MTases that have the ability to confer protection from restriction endonucleases of their bacterial host(s). The ability of a phage to overcome R-M and other phage-targeting resistance systems can be detrimental to particular biotechnological processes such as dairy fermentations. Conversely, as phages may also be beneficial in certain areas such as phage therapy, phages with additional resistance to host defenses may prolong the effectiveness of the therapy. This minireview will focus on bacteriophage-encoded MTases, their prevalence and diversity, as well as their potential origin and function.     

Molecular design, chemical synthesis, and biological evaluation of '4-1' pentacyclic aryl/heteroaryl-imidazonaphthalimides

A novel series of '4-1' pentacyclic naphthalimides, where the chromophore consists of a naphthalimide moiety, fused to an imidazole ring containing an unfused aryl or heteroaryl ring, were synthesized and evaluated for in vitro antitumor activity. In general, the new derivatives showed an improved cytotoxic activity over amonafide. DNA binding experiments supported that this class of compounds behaves as effective DNA-intercalating agents.     

Accelerated decline in lung function in cigarette smokers is associated with TP53/MDM2 polymorphisms

In vitro studies have shown that p53 mediates a protective response against DNA damage by causing either cell-cycle arrest and DNA repair, or apoptosis. These responses have not yet been demonstrated in humans. A common source of DNA damage in humans is cigarette smoke, which should activate p53 repair mechanisms. As the level of p53 is regulated by MDM2, which targets p53 for degradation, the G-allele of a polymorphism in intron 1 of MDM2 (rs2279744:G/T), that results in higher MDM2 levels, should be associated with a reduced p53 response and hence more DNA damage and corresponding tissue destruction. Similarly, the alleles of rs1042522 in TP53 that encode arginine (G-allele) or proline (C-allele) at codon 72, which cause increased pro-apoptotic (G-allele) or cell-cycle arrest activities (C-allele), respectively, may moderate p53’s ability to prevent DNA damage. To test these hypotheses, we examined lung function in relation to cumulative history of smoking in a population-based cohort. The G-alleles in MDM2 and TP53 were found to be associated with accelerated smoking-related decline in lung function. These data support the hypothesis that p53 protects from DNA damage in humans and provides a potential explanation for the variation in lung function impairment amongst smokers.