Indolocarbazole glycosides in inactive conformations

Indolocarbazole glycosides related to rebeccamycin represent a promising category of antitumor agents targeting DNA and topoisomerase I. These drugs prefer to adopt a closed conformation with an intramolecular hydrogen bond between the indole NH group and the pyranose oxygen atom. Three pairs of indolocarbazole monoglycosides bearing an NH or an N-methyl indole moiety were synthesized and their biological properties investigated at the molecular and cellular level. Replacing the indole NH proton with a methyl group reduces DNA interaction and abolishes activity against DNA topoisomerase I. Surface plasmon resonance studies performed with a pair of water-soluble indolocarbazole glycosides and two hairpin oligonucleotides containing an [AT]4 or a [CG]4 sequence indicate that both the NH and the N-methyl derivative maintain a relatively high affinity for DNA (Keq = 2 - 6 x 10(5) M(-1)) but the incorporation of the methyl group restricts access to the DNA. The number of ligand binding sites (n) on the oligonucleotides is about twice as high for the NH compound compared to its N-methyl analogue. Modeling and 1H NMR studies demonstrate that addition of the N-methyl group drives a radical change in conformation in which the orientation of the aglycone relative to the beta-glucoside is reversed. The loss of the closed conformation by the N-methyl derivatives perturbs thir ability to access DNA binding sites and prevents the drug from inhibiting topoisomerase I. As a consequence, the NH compounds exhibit potent cytotoxicity against CEM leukemia cells with an IC50 value in the 1 microM range, whereas the N-methyl analogues are 10 to 100 times less cytotoxic. These studies offer circumstantial evidence supporting the importance of the closed conformation in the interaction of indolocarbazole glycosides with their molecular targets, DNA and topoisomerase I.     

Highly selective glycosylated prodrugs of cytostatic CC-1065 analogues for antibody-directed enzyme tumor therapy

Novel prodrugs of the cytotoxic antibiotic CC-1065 for an antibody-directed enzyme prodrug therapy (ADEPT) were prepared that show an excellent selectivity with a high toxicity of the corresponding drug. In particular, the seco-CBI analogue of CC-1065, 1-chloromethyl-5-hydroxy-1,2-dihydro-3H-benz[e]indole, as well as the novel methyl-seco-CBI analogue 1-(1'-chloroethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole, were synthesized and transformed into their galactosides 10 a and 10 b, respectively. These galactosides can be cleaved with beta-D-galactosidase to give the free cytotoxic compounds. They were tested in in vitro cytotoxicity assays by using human bronchial carcinoma cells of line A549 in the presence and in the absence of beta-D-galactosidase. While the seco-CBI prodrugs revealed only modest selectivity, prodrugs of the methyl-seco-CBI analogue bearing an anti orientation of the substituents at the two stereogenic centers of the N-heterocycle displayed an excellent selectivity with an ED(50) quotient of about 750. The cytotoxicity of the corresponding phenol was rather high, with an ED(50) of 1.3 nM. The diastereomer with a syn orientation at the stereogenic centers was much less toxic.     

A novel bis-aziridinylnaphthoquinone with anti-solid tumor activity in which induced apoptosis is associated with altered expression of Bcl-2 protein

Aziridine-containing compounds have been of interest as anticancer agents since the late 1970s. The design, synthesis, and study of aziridinylnaphthoquinone analogues to obtain compounds with enhanced activity/toxicity profiles are an ongoing research effort in our group. A series of bis-aziridinylnaphthoquinone derivatives has been prepared, and the cytotoxic activities of these synthetic bis-aziridinylnaphthoquinone derivatives has been investigated. The synthetic derivatives displayed significant cytotoxicity against human carcinoma cell lines and weak cytotoxic activities against skin fibroblasts (SF). The bis-aziridinylnaphthoquinone 1 c was the most effective of the tested analogues at reducing the viability of Hep2 cells, with an LD(50) value of 5.23 microM, and also exhibited weak cytotoxic activity against SF cells, with an LD(50) value of 54.12 microM. The DNA alkylation and DNA interstrand cross-linking abilities of 1 c were also investigated. Bis-aziridinylnaphthoquinone 1 c was an effective agent for alkylation of DNA after chemical reduction in vitro, and its bifunctional alkylating moieties were able to cross-link DNA. We also report here our efforts to determine direct antitumor effects of 1 c on Hep2 cells. Growth arrest in Hep2 cells was preceded by early induction of G(2)-M cell cycle arrest at 0.75 microM of 1 c after culture for 24 h, and was then followed by apoptosis after 60 h. This was associated with decreased expression of antiapoptotic bcl2 protein (by 78 %) upon culture with 3.0 microM of 1 c after 60 h. Our results suggest that 1 c is a novel antitumor aziridinylnaphthoquinone with therapeutic potential against solid tumors.     

Gold(I) N‐Heterocyclic Carbene Complexes with Naphthalimide Ligands as Combined Thioredoxin Reductase Inhibitors and DNA Intercalators

Organometallic conjugates consisting of a gold(I) N‐heterocyclic carbene (NHC) moiety and a naphthalimide were prepared and investigated as cytotoxic agents that interact with both DNA and the disulfide reductase enzyme thioredoxin reductase (TrxR). The complexes were potent DNA intercalators related to their naphthalimide partial structure, inhibited TrxR as a consequence of the incorporation of the gold(I) moiety, and triggered efficient cytotoxic effects in MCF‐7 breast and HT‐29 colon adenocarcinoma cells. Strong effects on tumor cell metabolism were noted for the most cytotoxic complex, chlorido[1‐(3′‐(4′′‐ethylthio‐1′′,8′′‐naphthalimid‐N′′‐yl))‐propyl‐3‐methyl‐imidazol‐2‐ylidene]gold(I) ( 4 d ). In conclusion, the conjugation of naphthalimides with gold(I) NHC moieties provided a useful strategy for the design of bioorganometallic anticancer agents with multiple modes of action.     

Receptor‐Based Virtual Screening and Biological Characterization of Human Apurinic/Apyrimidinic Endonuclease (Ape1) Inhibitors

The endonucleolytic activity of human apurinic/apyrimidinic endonuclease (AP endo, Ape1) is a major factor in maintaining the integrity of the genome. Conversely, as an undesired effect, Ape1 overexpression has been linked to resistance to radio‐ and chemotherapeutic treatments in several human tumors. Inhibition of Ape1 using siRNA or the expression of a dominant negative form of the protein has been shown to sensitize cells to DNA‐damaging agents, including various chemotherapeutic agents. Therefore, inhibition of the enzymatic activity of Ape1 might result in a potent antitumor therapy. A number of small molecules have been described as Ape1 inhibitors; however, those compounds are in the early stages of development. Herein we report the identification of new compounds as potential Ape1 inhibitors through a docking‐based virtual screening technique. Some of the compounds identified have in vitro activities in the low‐to‐medium micromolar range. Interaction of these compounds with the Ape1 protein was observed by mass spectrometry. These molecules also potentiate the cytotoxicity of the chemotherapeutic agent methyl methanesulfonate in fibrosarcoma cells. This study demonstrates the power of docking and virtual screening techniques as initial steps in the design of new drugs, and opens the door to the development of a new generation of Ape1 inhibitors.     

Molecular recognition of T:G mismatched base pairs in DNA as studied by electrospray ionization mass spectrometry

Postreplicative mismatch repair (MMR) is a cellular system involved in the recognition and correction of DNA polymerase errors that escape detection in proofreading. Of the various mismatched bases, T:G pairing in DNA is one of the more common mutations leading to the formation of tumors in humans. In addition, the absence of the MMR system can generate resistance to several chemotherapeutic agents, particularly DNA-damaging substances. The main purpose of this study was the setup and validation of an electrospray ionization (ESI) mass spectrometry method for the identification of small molecules that are able to recognize T:G mismatches in DNA targets. These findings could be useful for the discovery of new antitumor drugs. The analytical method is based on the ability of electrospray to preserve the noncovalent adducts present in solution and transfer them to the gas phase. Lexitropsin derivatives (polyimidazole compounds) have been previously described as selective for T:G mismatch binding by NMR and ITC studies. We synthesized and tested various polyimidazole derivatives, one of which in particular (NMS-057) showed a higher affinity for an oligonucleotide DNA sequence containing a T:G mismatched base pair. To rationalize these findings, molecular docking studies were performed using available NMR structures. Moreover, ESI-MS experiments, performed on an orbitrap mass spectrometer, highlighted the formation of heterodimeric complexes between DNA sequences, distamycin A, and polyimidazole compounds. Our results confirm that this ESI method could be a valuable tool for the identification of new molecules able to specifically recognize T:G mismatched base pairs.     

[Ce(LA)_2(NO_3)_3]的晶体结构、抗肿瘤活性以及与DNA作用研究

以鹅掌楸碱为配体与稀土金属铈反应合成配合物[Ce(LA)_2(NO_3)_3],通过X-射线单晶衍射对该配合物进行结构表征,并对其进行肿瘤细胞的体外抑制活性筛选。采用紫外-可见光谱、粘度实验研究该金属配合物与DNA的作用机制。     

DNA Interstrand Crosslinks by H‐pin Polyamide (S)‐seco‐CBI Conjugates

Although DNA interstrand crosslinking (ICL) agents are widely used as antitumor drugs, DNA sequence‐specific ICL agents are quite rare. In this study, H‐pin imidazole‐pyrrole polyamide 1‐(chloromethyl)‐2,3‐dihydro‐1H‐benzo[e]indol‐5‐ol (seco‐CBI) conjugates that produce sequence‐specific DNA ICLs were designed and synthesized. Conjugates with H‐pin polyamide and seco‐CBI moieties were constructed to recognize a 7 bp DNA sequence, and their reactivity and selectivity in DNA alkylation were evaluated by using high‐resolution denaturing gel electrophoresis and sequence‐specific plasmid cleavage. One conjugate ( 6 ), which contained a chiral (S)‐seco‐CBI, exhibited greater sequence‐specific ICL activity toward the target DNA sequence and was cytotoxic to a cancer cell line. Molecular modeling studies indicated that the greater activity of 6 resulted from the relative orientation of the cyclopropane group in the (S)‐CBI unit.     

DNA Nanobarrel-Based Drug Delivery for Paclitaxel and Doxorubicin

Co-delivery of anticancer drugs and target agents by endogenous materials is an inevitable approach towards targeted and synergistic therapy. Employing DNA base pair complementarities, DNA nanotechnology exploits a unique nanostructuring method and has demonstrated its capacity for nanoscale positioning and templated assembly. Moreover, the water solubility, biocompatibility, and modifiability render DNA structure suitable candidate for drug delivery applications. We here report single-stranded DNA tail conjugated antitumor drug paclitaxel (PTX), and the co-delivery of PTX, doxorubicin and targeting agent mucin 1 (MUC-1) aptamer on a DNA nanobarrel carrier. We investigated the effect of tail lengths on drug release efficiencies and dual drug codelivery-enabled cytotoxicity. Owing to the rapidly developing field of structural DNA nanotechnology, functional DNA-based drug delivery is promising to achieve clinical therapeutic applications.     

Synthesis of Fluorinated Imidazole[4,5f][1,10]phenanthroline Derivatives as Potential Inhibitors of Liver Cancer Cell Proliferation by Inducing Apoptosis via DNA Damage

Phenanthroline derivatives containing fluorinated imidazole ring are effective anti-neoplastic agents. Herein, a series of four fluorinated imidazole[4,5f][1,10]phenanthroline derivatives were synthesized and investigated as potential inhibitors to fight against the growth of liver cancer cells. The in vitro antitumor activity of targeted compounds have been evaluated by using MTT assay, and results showed that compound 4 (2-(2,3-difluorophenyl)-1H-imidazo[4,5-f][1,10]phenanthroline) exhibited excellent inhibitory effect against the growth of various tumor cells, particularly for HepG2 cells, with IC₅₀ value of approximately 0.29 μM. This result has been further confirmed by colony formation assay, showing that compound 4 suppressed the proliferation of HepG2 cells. Moreover, cell apoptosis (AO/PI dual staining and flow cytometry) analyses as well as comet assay showed that compound 4 may induce apoptosis of HepG2 cells through triggering DNA damage. Furthermore, the in vivo anti-tumor activity were evaluated on zebrafish bearing HepG2 cells showed that compound 4 can observably block the growth of liver cancer cells. All in together, these compounds, particularly compound 4 , may be developed as a potential agent to treat liver cancer in the future.     

Incorporation of Nucleoside Probes Opposite O6‐Methylguanine by Sulfolobus solfataricus DNA Polymerase Dpo4: Importance of Hydrogen Bonding

O⁶‐Methylguanine (O⁶‐MeG) is a mutagenic DNA lesion, arising from the action of methylating agents on guanine (G) in DNA. Dpo4, an archaeal low‐fidelity Y‐family DNA polymerase involved in translesion DNA synthesis (TLS), is a model for studying how human Y‐family polymerases bypass DNA adducts. Previous work showed that Dpo4‐mediated dTTP incorporation is favored opposite O⁶‐MeG rather than opposite G. However, factors influencing the preference of Dpo4 to incorporate dTTP opposite O⁶‐MeG are not fully defined. In this study, we investigated the influence of structural features of incoming dNTPs on their enzymatic incorporation opposite O⁶‐MeG in a DNA template. To this end, we utilized a new fluorescence‐based primer extension assay to evaluate the incorporation efficiency of a panel of synthetic dNTPs opposite G or O⁶‐MeG by Dpo4. In single‐dNTP primer extension studies, the synthetic dNTPs were preferentially incorporated opposite G, relative to O⁶‐MeG. Moreover, pyrimidine‐based dNTPs were generally better incorporated than purine‐based syn‐conformation dNTPs. The results suggest that hydrophobicity of the incoming dNTP appears to have little influence on the process of nucleotide selection by Dpo4, with hydrogen bonding capacity being a major influence. Additionally, modifications at the C2‐position of dCTP increase the selectivity for incorporation opposite O⁶‐MeG without a significant loss of efficiency.     

Highly fluorescent 5-(5,6-dimethoxybenzothiazol-2-yl)-2'-deoxyuridine 5'-triphosphate as an efficient substrate for DNA polymerases

We herein describe the synthesis of fluorescent 5-(5,6-dimethoxybenzothiazol-2-yl)-2'-deoxyuridine 5'-triphosphate (d(bt)UTP) and primer extension reactions using d(bt)UTP. We also carried out primer extension reactions using the (bt)U template. B family DNA polymerases, such as KOD, Deep Vent (exo-), and 9°N(m) DNA polymerases, were effective for elongation with d(bt)UTP. Deep Vent (exo-) and KOD DNA polymerases have excellent fidelity for incorporating d(bt)UTP only at the site opposite the adenine template and only dATP when using the (bt)U template. Therefore, d(bt)UTP is an excellent fluorescent nucleotide that can be incorporated into DNA by DNA polymerases.     

PAMAM-calix-dendrimers: Synthesis and Thiacalixarene Conformation Effect on DNA Binding

A convenient method for the synthesis of the first generation PAMAM dendrimers based on the thiacalix[4]arene has been developed for the first time. Three new PAMAM-calix-dendrimers with the macrocyclic core in cone, partial cone, and 1,3-alternate conformations were obtained with high yields. The interaction of the obtained compounds with salmon sperm DNA resulted in the formation of the associates of the size up to 200 nm, as shown by the UV-Vis spectroscopy, DLS, and TEM. It was demonstrated by the CD method that the structure of the DNA did not undergo significant changes upon binding. The PAMAM-calix-dendrimer based on the macrocycle in cone conformation stabilized DNA and prevented its degradation.     

Synthesis, Crystal Structure and Interaction With DNA of N,N'-(Butane-1,4-Diyl)Bis(Guanidinium) Tetrachloroplatinate (II)

The design, synthesis, crystal structure and interaction with DNA of the N,N'-(butane-1,4-diyl)bis(guanidinium) tetrachloroplatinate(ll) are described. Crystal data: a = 8.152(1), b = 8.889(4), c = 10.700(3) A , alpha = 81.59(3), beta = 87.99(5), gamma = 78.48(6) degrees , V = 752(1) A(3), Z = 2 , space group P-1. The structure was refined to R = 0.039 and Rw = 0.046 from 1853 reflections (I > 3sigma(I)). This compound, named PtC(4)Gua, does not exhibit a center of symmetry and the center linker chain C(2) - C(3) - C(4) - C(5) is in gauche conformation. The cation is bisprotonated with the H(+) attached to the imine group of each terminal guanidinium function. The presence of the platinum moiety reinforces the binding of the butane(bis)guanidinium structure with double stranded DNA as judged from thermal denaturation studies and DNA unwinding experiments.     

1,7(1,5)-二芳基庚(戊)二烯类姜黄素衍生物、类似物结构与活性的关系研究

大量研究结果证明,姜黄素具有抗肿瘤,抗氧化,消炎的作用与它们的结构有密切关系,且大部分姜黄素衍生物与类似物的药理活性较姜黄素本身有所提高.这为进一步开发新的姜黄素衍生物与类似物药理功效奠定了理论基础.本文就近年来对1,7(1,5)-二芳基庚(戊)二烯类姜黄素衍生物与类似物结构的改造、修饰与其活性之间关系的研究进行探讨.     

Altered DNA Binding and Amplification of Human Breast Cancer Suppressor Gene BRCA1 Induced by a Novel Antitumor Compound, [Ru(��6-p-phenylethacrynate)Cl2(pta)]

The ruthenium-based complex [Ru(η⁶-p-phenylethacrynate)Cl₂(pta)] (pta = 1,3,5-triaza-7-phosphatricyclo-[3.3.1.1]decane), termed ethaRAPTA, is an interesting antitumor compound. The elucidation of the molecular mechanism of drug activity is central to the drug development program. To this end, we have characterized the ethaRAPTA interaction with DNA, including probing the sequence specific modified DNA structural stability and DNA amplification using the breast cancer suppressor gene 1 (BRCA1) of human breast and colon adenocarcinoma cell lines as models. The preference of ethaRAPTA base binding is in the order A > G > T > C. Once modified, the ethaRAPTA-induced BRCA1 structure has higher thermal stability than the modified equivalents of its related compound, RAPTA-C. EthaRAPTA exhibits a higher efficiency than RAPTA-C in inhibiting BRCA1 amplification. With respect to both compounds, the inhibition of BRCA1 amplification is more effective in an isolated system than in cell lines. These data provide evidence that will help to understand the process of elucidating the pathways involved in the response induced by ethaRAPTA.     

Mechanism of Action of AminoCBIs: Highly Reactive but Highly Cytotoxic Analogues of the Duocarmycins

Duocarmycins are highly cytotoxic natural products that have potential for development into anticancer agents. Herein we describe proposed but previously unidentified NH analogues of the DNA‐alkylating subunit and characterise these by solvolysis studies, NMR and computational modelling. These compounds are shown to be the exclusive intermediates in the solvolysis of their seco precursors and to possess very similar structural features to the widely studied O‐based analogues, apart from an unusually high basicity. The measured pK_a of 10.5 implies that the NH compounds are fully protonated under physiological conditions. Remarkably, their extremely high reactivity (calculated hydrolysis rate 10⁸ times higher for protonated NH compared to the neutral O analogue) is still compatible with potent cytotoxicity, provided the active species is formed in the presence of cells. These surprising findings are of relevance to the design of duocarmycin‐based tumour‐selective therapies.     

The DNA helical biopolymer: A template for the binding, assembly, and reactivity of metal ions and complexes

The helical DNA polymer provides many structural features that facilitate the binding of metal ions or complexes. DNA binding inorganic agents have proven invaluable, with demonstrated applications ranging from chemotherapeutic agents to probes of DNA structure. This broad range of applications attests to the utility of inorganic agents in the design of compounds which interact with the DNA helix and is due, in part, to the ability of inorganic species to define a particular ligand geometry complementary to the DNA structure, bind, or chemically react along the polymer strand. While a diverse array of novel inorganic compounds that interact with DNA have already been studied, many opportunities still exist to exploit inorganic agents in the design of new molecules that will interact uniquely with the DNA polymer. This review examines the structure of the DNA polymer, emphasizing aspects which promote the binding of inorganic agents. Along with a structural overview, the binding modes available to an inorganic element or complex are reviewed, in combination with a discussion of the ability of the DNA to act as a template for the organized binding of inorganic agents. In addition, chemical alteration of the DNA polymer structure by inorganic agents is discussed, along with the potential utility of such modifications.     

A comprehensive study of the interactions between DNA and poly(3,4-ethylenedioxythiophene)

The interaction between poly(3,4-ethylenedioxythiophene), a conducting polymer with excellent electrical and electrochemical properties, and plasmid DNA has been investigated using electrophoresis, UV-visible and CD spectroscopy, and quantum mechanical calculations. Analyses of mixtures with different DNA:polymer mass ratios indicate that, in all cases, interactions form immediately and induce structural alterations in DNA. Furthermore, the existence of interactions between poly(3,4-ethylenedioxythiophene) and specific nucleotides sequences has been evidenced by adding restriction enzymes to the mixtures. In contrast, interactions between DNA and poly(3-methylthiophene), a similar polyheterocyclic conducting polymer but without hydrogen bonding acceptors, are weak or do not exist. These results suggest that, in addition to non-specific electrostatic interactions between the charged phosphate groups of DNA and the positively charged fragments of the conducing polymers, specific hydrogen bonding interactions play a crucial role. The ability of 3,4-ethylenedioxythiophene units to form hydrogen bonds with the methylated analogues of DNA bases has been examined in different environments using MP2/6-31G(d) and MP2/6-311++G(d,p) calculations. Results indicate that, in environments with low polarity, the formed interactions are significantly stronger than those reached by unsubstituted thiophene and similar to those established by pyrrole. However, in polar environments (aqueous solution) 3,4-ethylenedioxythiophene provides stronger interactions with nucleic acids than both thiophene and pyrrole. These theoretical results are fully consistent with experimental observations.     

Mass Spectral Analysis of Unstable N7-Aralkyl DNA Adducts Resulting from Reaction of 7-Sulfooxymethyl-12-methylbenz[ a ]anthracene (SMBA) with DNA and Deoxynucleotides

The unified hypothesis for PAH activation predicts that SMBA plays a role in the metabolic activation and carcinogenicity of 7,12-dimethylbenz[ a ]anthracene (DMBA). SMBA and closely related aralkylating agents are derived from 7-hydroxymethyl-12-methylbenz[ a ]anthracene (HMBA), a direct metabolite of DMBA, and react with bases in nucleic acids. This occurs by generation of a benzylic carbonium ion owing to the fact that sulfate is a good leaving group. Previous characterization of reaction products with deoxynucleotide-3'-monophosphates detected stable adducts as primarily resulting from reaction with adenine at the N6- and guanine at the N2-amino groups, respectively. Pyrimidine adducts were also found; however, examination of SMBA-reacted DNA confirmed that the purine bases were the major targets for reaction. We now report evidence for the formation of unstable DNA adducts, most likely by reaction with purine N-7 positions. Treatment of SMBA-reacted DNA with formic acid at 70°C for 60 min and examination of acidified reaction products with electrospray-positive mode mass spectrometry (ESI + MS) disclosed m/z 390 and 406 products corresponding to 12-methylbenz[ a ]anthracene-7-methylene-N7-adenine or -guanine, respectively. Release of these adducts is expected to be accompanied by generation of apurinic sites in DNA structure. Examination of alternative leaving groups on the aralkylating agent suggest the following relative reactivities with deoxyguanosine or deoxyadenosine: chloro > sulfooxy > acetoxy > iodo. It is our expectation that development of these analytical methodologies will enable us to assign a role for the participation of aralkylating agents in PAH carcinogenicity.