Synthesis, biological evaluation, and molecular modeling studies of rebeccamycin analogues modified in the carbohydrate moiety

A new series of indolocarbazole glycosides containing disaccharides were synthesized and their in vitro antiproliferative activity was evaluated against three human cancer cell lines (A2780, H460, and GLC4). Cytotoxicity appeared to be remarkably affected by the regio- and stereochemical features of the disaccharide moiety. In vivo antitumor activity of the compounds studied, two of which having IC(50)<100 nm, was determined using ovarian cancer cell line A2780 xenografted on nude mice. One compound showed an efficacy similar to that of the reference compound edotecarin, though with a lower long-lasting activity. The topoisomerase I inhibitory properties of some compounds were also examined. Molecular dynamics simulations of the ternary topoisomerase I-DNA-ligand complexes were performed to analyze the structural features of topoisomerase I poisoning with this class of indolocarbazoles. A plausible explanation of their biological behavior was provided. These theoretical results were compared with the recently published crystal structure of an indolocarbazole monosaccharide bound to the covalent human topoisomerase I-DNA complex.     

Novel dications with unfused aromatic systems: trithiophene and trifuran derivatives of furimidazoline

We report the synthesis, interaction with DNA, topoisomerase II inhibition, and cytotoxicity of two novel unfused aromatic dications derived from the antimicrobial agent furimidazoline. The central diphenylfuran core of furimidazoline has been replaced with a trithiophene (DB358) or a trifuran (DB669) unit and the terminal imidazoline groups were preserved. The strength and mode of binding of the drugs to nucleic acids were investigated by complementary spectroscopic techniques including spectrophotometric, surface plasmon resonance, circular and linear dichroism measurements. The trifuran derivative forms intercalation complexes with double-stranded DNA, whereas the mode of binding of the trithiophene derivative varies depending on the drug/DNA ratio, as independently confirmed by NMR spectroscopic studies performed with (A-T)7 and (G-C)7 oligomers. Two-dimensional NMR data provided a molecular model for the binding of DB358 within the minor groove of the AATT sequence of the decanucleotide d(GCGAATTCGC)(2). DNase I footprinting experiments confirmed the sequence-dependent binding of DB358 to DNA. The trithiophene derivative interacts preferentially with AT-rich sequences at low concentrations, but can accomodate GC sites at higher concentrations. DNA relaxation assays revealed that DB358 stimulated DNA cleavage by topoisomerase II, in contrast to DB669. The substitution of N-alkylamidines for the imidazoline terminal groups abolished the capacity of the drug to poison topoisomerase II. At the cellular level, flow cytometry analysis indicated that DB358, which is about six times more cytotoxic than the trifuran analogue, induced a significant accumulation of HL-60 human leukemia cells in the G2/M phase. The incorporation of thiophene heterocycles appears as a convenient procedure to limit the strict AT selectivity of dications containing an extended unfused aromatic system and to design cytotoxic DNA intercalating agents acting as poisons for human topoisomerase II.     

Structure-activity relationship study of 16 a-thiocamptothecins: an integrated in vitro and in silico approach

The instability of the hydroxylactone E ring represents a critical drawback of camptothecins, because the lactone ring is recognized to be essential for stabilization of topoisomerase I‐mediated DNA cleavage. In an attempt to investigate the effect of the thiopyridone pharmacophofore on the molecular and pharmacological features of the drug, we prepared a series of novel 16 a‐thiocamptothecin analogues. Due to the sulfur atom, a destabilization of the hydrogen bond between the hydroxy group in position 17 of the opened E ring and the carbonyl of the pyridone moiety is predicted, thus shifting the equilibrium toward the closed lactone form and increasing the lipophilic properties of the compounds. This feature was associated with superior antiproliferative potency, with reduced interaction with the human serum albumin and with substantial increase of the persistence of the topoisomerase I–DNA cleavable complex. These effects were prominent for thio‐SN38, the most active compound of the series. The favorable interactions at the molecular and cellular level of the reported thiocamptothecins confer promising features, and these compounds warrant preclinical development.     

Intramolecular Interaction with the E6 Region Stabilizes the Closed Conformation of the N-SH2 Domain and Concurs with the Self-Inhibitory Docking in Downregulating the Activity of the SHP2 Tyrosine Phosphatase: A Molecular Dynamics Study

The localization and activity of the SHP2 tyrosine phosphatase across different cellular compartments to the target substrates are steered by the binding of phosphotyrosine (pY) peptides to the tandem SH2 domains. The most N-terminal domain (N-SH2) can also keep the enzyme inactive by intramolecular occlusion of the catalytic site. Enzyme activity can be recovered by an allosteric disruption of this self-inhibitory docking upon the binding of pY peptides to the N-SH2 domain. Prior to this, the N-SH2 domain must abandon the closed conformation because it impedes the access of pY peptides to the binding cleft. Although it cooperates with the self-inhibitory docking in the negative regulation of the phosphatase activity, the structural determinants of the stability of the closed conformation in the self-inhibited phosphatase are still elusive. To address this issue, a molecular dynamics simulation study is carried out. It is shown that the closed conformation is stabilized by the interaction of the N-SH2 domain with a conserved peptide portion in the region encoded by PTPN11 exon 6 (E6).     

DNA Interaction Studies of Selected Polyamine Conjugates

The interaction of polyamine conjugates with DNA double helix has been studied. Binding properties were examined by ethidium bromide (EtBr) displacement and DNA unwinding/topoisomerase I/II (Topo I/II) activity assays, as well as dsDNA thermal stability studies and circular dichroism spectroscopy. Genotoxicity of the compounds was estimated by a comet assay. It has been shown that only compound 2a can interact with dsDNA via an intercalative binding mode as it displaced EtBr from the dsDNA-dye complex, with K_app = 4.26 × 10⁶ M⁻¹; caused an increase in melting temperature; changed the circular dichroism spectrum of dsDNA; converted relaxed plasmid DNA into a supercoiled molecule in the presence of Topo I and reduced the amount of short oligonucleotide fragments in the comet tail. Furthermore, preliminary theoretical study has shown that interaction of the discussed compounds with dsDNA depends on molecule linker length and charge distribution over terminal aromatic chromophores.     

Unusually strong positive cooperativity in binding of peptides to latent membrane protein-1 DNA fragments of the Epstein-Barr viral gene

The DNA-binding preferences of two oligopeptide amides, (His-Pro-Arg-Lys)(3)NH(2) (HR-12) and (Ser-Pro-Arg-Lys)(3)NH(2) (SP-12), have been examined by quantitative DNase I footprinting studies. Two different DNA fragments were investigated: a pair of 5'-(32)P-labeled duplexes from pBR322 with one or other of the complementary strands labeled and a corresponding pair of 5'-(32)P-labeled duplexes representing fragments of the latent membrane protein (LMP-1) gene from a pathogenic Epstein-Barr virus variant derived from nasopharyngeal carcinoma. The major objective was to examine molecular recognition and cooperative features associated with sequence-selective binding of synthetic peptides to the LMP-1 fragments. At various binding sites on the pBR322 fragments, Hill coefficients (n(H)) ranging from 1.9-2.2 were observed; these results indicate modest positive cooperativity between binding sites for both peptides. By contrast, unusually high values of n(H), ranging from 4.0-9.3, were observed at various binding sites on the LMP-1 fragments. Allosteric models can be constructed to interpret the observed cooperative interactions between different DNA recognition sites in the LMP-1 gene upon binding of the peptide ligands. It is noteworthy that these models feature a novel network of cooperativity interconnecting multiple DNA allosteric sites. The evidence of sequence selectivity and strong cooperativity discovered in this work may prove to be a general feature of peptide interactions with some nucleic acids.     

Structure‐Based Mutational Study of an Archaeal DNA Ligase towards Improvement of Ligation Activity

DNA ligases catalyze the joining of strand breaks in duplex DNA. The DNA ligase of Pyrococcus furiosus (PfuLig), which architecturally resembles the human DNA ligase I (hLigI), comprises an N‐terminal DNA‐binding domain, a middle adenylylation domain, and a C‐terminal oligonucleotide‐binding (OB)‐fold domain. Here we addressed the C‐terminal helix in the OB‐fold domain of PfuLig by mutational analysis. The crystal structure of PfuLig revealed that this helix stabilizes a closed conformation of the enzyme by forming several ionic interactions with the adenylylation domain. The C‐terminal helix is oriented differently in hLigI when DNA is bound; this suggested that disruption of its interaction with the adenylylation domain might facilitate the binding of DNA substrates. We indeed identified one of its residues, Asp540, as being critical for ligation efficiency. The D540R mutation improved the overall ligation activity relative to the wild‐type enzyme, and at lower temperatures; this is relevant to applications such as ligation amplification reactions. Physical and biochemical analyses indicated that the improved ligation activity of the D540R variant arises from effects on the ligase adenylylation step and on substrate DNA binding in particular.     

Evaluation of Interactions of Selected Olivacine Derivatives with DNA and Topoisomerase II

Olivacine and ellipticine are model anticancer drugs acting as topoisomerase II inhibitors. Here, we present investigations performed on four olivacine derivatives in light of their antitumor activity. The aim of this study was to identify the best antitumor compound among the four tested olivacine derivatives. The study was performed using CCRF/CEM and MCF-7 cell lines. Comet assay, polarography, inhibition of topoisomerase II activity, histone acetylation, and molecular docking studies were performed. Each tested compound displayed interaction with DNA and topoisomerase II, but did not cause histone acetylation. Compound 2 (9-methoxy-5,6-dimethyl-1-({[1-hydroxy-2-(hydroxymethyl)butan-2-yl]amino}methyl)-6H-pyrido[4,3-b]carbazole) was found to be the best candidate as an anticancer drug because it had the highest affinity for topoisomerase II and caused the least genotoxic damage in cells.     

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.     

Novel Antitumor L‐Arabinose Derivative of Indolocarbazole with High Affinity to DNA

Novel indolocarbazole derivative 12‐(α‐L ‐arabinopyranosyl)indolo[2,3‐α]pyrrolo[3,4‐c]carbazole‐5,7‐dione (AIC) demonstrated high potency (at submicromolar concentrations) against the NCI panel of human tumor cell lines and transplanted tumors in vivo. In search of tentative targets for AIC, we found that the drug formed high affinity intercalative complexes with d(AT)₂₀, d(GC)₂₀ and calf thymus DNA (binding constants (1.6×10⁶) M ^?1≤K_a≤(3.3×10⁶) M ^?1). The drug intercalated preferentially into GC pairs of the duplex. Importantly, the concentrations at which AIC formed the intercalative complexes with DNA (C≤1 μM ) were identical to the concentrations that triggered p53‐dependent gene reporter transactivation, the replication block, the inhibition of topoisomerase I‐mediated DNA relaxation and death of HCT116 human colon carcinoma cells. We conclude that the formation of high affinity intercalative complexes with DNA is an important factor for anticancer efficacy of AIC.     

Microbial Type IA Topoisomerase C-Terminal Domain Sequence Motifs,Distribution and Combination

Type IA topoisomerases have highly conserved catalytic N-terminal domains for the cleaving and rejoining of a single DNA/RNA strand that have been extensively characterized. In contrast, the C-terminal region has been less covered. Two major types of small tandem C-terminal domains, Topo_C_ZnRpt (containing C4 zinc finger) and Topo_C_Rpt (without cysteines) were initially identified in Escherichia coli and Mycobacterium tuberculosis topoisomerase I, respectively. Their structures and interaction with DNA oligonucleotides have been revealed in structural studies. Here, we first present the diverse distribution and combinations of these two structural elements in various bacterial topoisomerase I (TopA). Previously, zinc fingers have not been seen in type IA topoisomerases from well-studied fungal species within the phylum Ascomycota. In our extended studies of C-terminal DNA-binding domains, the presence of zf-GRF and zf-CCHC types of zinc fingers in topoisomerase III (Top3) from fungi species in many phyla other than Ascomycota has drawn our attention. We secondly analyze the distribution and combination of these fungal zf-GRF- and zf-CCHC-containing domains. Their potential structures and DNA-binding mechanism are evaluated. The highly diverse arrangements and combinations of these DNA/RNA-binding domains in microbial type IA topoisomerase C-terminal regions have important implications for their interactions with nucleic acids and protein partners as part of their physiological functions.     

Ortho Effects. I. The Mechanisms of the H3O and CH3O2 Elimination from the Molecular Ion of N-Methyl Ortho-Nitro-Para-Toluidine

The molecular ion of the N-methyl ortho-nitro-para-toluidine (I) readily eliminates H₃O. The sources of the H atoms lost were proved by deuterium labeling to be an HNCH₃ group as well as, probably, the C-methyl group of the aromatic ring. The interaction between the N-methyl and nitro groups in I also leads to the expulsion of CH₃O₂ from the molecular ion.     

Dynophore-Based Approach in Virtual Screening: A Case of Human DNA Topoisomerase IIα

In this study, we utilized human DNA topoisomerase IIα as a model target to outline a dynophore-based approach to catalytic inhibitor design. Based on MD simulations of a known catalytic inhibitor and the native ATP ligand analog, AMP-PNP, we derived a joint dynophore model that supplements the static structure-based-pharmacophore information with a dynamic component. Subsequently, derived pharmacophore models were employed in a virtual screening campaign of a library of natural compounds. Experimental evaluation identified flavonoid compounds with promising topoisomerase IIα catalytic inhibition and binding studies confirmed interaction with the ATPase domain. We constructed a binding model through docking and extensively investigated it with molecular dynamics MD simulations, essential dynamics, and MM-GBSA free energy calculations, thus reconnecting the new results to the initial dynophore-based screening model. We not only demonstrate a new design strategy that incorporates a dynamic component of molecular recognition, but also highlight new derivates in the established flavonoid class of topoisomerase II inhibitors.     

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)相似文献   

Steric Determinants of Pt/DNA Interactions and Anticancer Activity

Studies directed at establishing the structural features that control Pt/DNA interactions and the anticancer activity of Pt drugs are described. [(1)H, (15)N]-HSQC 2D NMR spectroscopic studies of the reactions of cisplatin with oligonucleotides containing ApG and GpA binding sites reveal dramatic differences in the rates of formation of monofunctional adducts at the two sites. When the reactant is cis-[Pt(NH(3))(2)(OH(2))(2)](2+) no such differences are observed suggesting that outer-sphere interactions between the reactant and the oligonucleotide may play a substantial role in determining the rates. Rates of closure to the bifunctional adducts are similar to those observed for cisplatin. Studies of the adduct profiles formed by sterically bulky and/or optically active complexes reveal that steric interactions play a major role in mediating the binding of Pt(ll) to DNA but that hydrogen bonds play less of a role. In vitro cytotoxic activities for these complexes do not always follow the trends that would be expected on the basis of the adduct profiles.     

一种吡喃并噻吩并嘧啶酮衍生物与牛血清白蛋白的相互作用研究

采用荧光光谱法,研究了不同温度下、pH值为7.4时,3-对氯苯基-2-(四氢吡咯-1-基)-5,6,8-三氢吡喃并[3′,4′:4,5]噻吩并[2,3-d]嘧啶-4(3H)-酮(PPTP)与牛血清白蛋白的相互作用;计算了不同温度下二者的结合常数和结合位点数;并用同步荧光技术讨论了PPTP衍生物对牛血清白蛋白构象的影响....     

Probing the Ion Binding Site in a DNA Holliday Junction Using F?rster Resonance Energy Transfer (FRET)

Holliday Junctions are critical DNA intermediates central to double strand break repair and homologous recombination. The junctions can adopt two general forms: open and stacked-X, which are induced by protein or ion binding. In this work, fluorescence spectroscopy, metal ion luminescence and thermodynamic measurements are used to elucidate the ion binding site and the mechanism of junction conformational change. Förster resonance energy transfer measurements of end-labeled junctions monitored junction conformation and ion binding affinity, and reported higher affinities for multi-valent ions. Thermodynamic measurements provided evidence for two classes of binding sites. The higher affinity ion-binding interaction is an enthalpy driven process with an apparent stoichiometry of 2.1 ± 0.2. As revealed by Eu³⁺ luminescence, this binding class is homogeneous, and results in slight dehydration of the ion with one direct coordination site to the junction. Luminescence resonance energy transfer experiments confirmed the presence of two ions and indicated they are 6–7 Å apart. These findings are in good agreement with previous molecular dynamics simulations, which identified two symmetrical regions of high ion density in the center of stacked junctions. These results support a model in which site-specific binding of two ions in close proximity is required for folding of DNA Holliday junctions into the stacked-X conformation.     

Synthesis and structure study of copolymers from thiadiazole fused indolocarbazole and dithienosilole

We report synthesis and characterization of two D-A polymers (PSDT-C12 and PSDT-EH) with different side chains. Both polymers are based on alternate 12,13-dioctyl-indolo[2,3-a][1,2,5]thiadiazolo[3,4-c]carbazole (TDZIC) and dithienosilole derivative units in polymer main chain. We used TDZIC to enlarge the 2D conjugated plane of acceptor monomers by fusing benzothiadiazole (BT) unit with an indole unit having alkyl groups. PSDT-C12 exhibited 10 nm redshift compared to PSDT-EH in solid films, while their absorption spectra were almost identical in solutions. Since the backbone and side chains on the indolocarbazole group are the same, the redshift on PSDT-C12 could be resulted from the dodecyl (C12) side chain on the dithienosilole unit and different molecular weight between these two polymers. PSDT-C12 has a larger molecular weight than PSDT-EH. Therefore possibly both side chains and molecular weight contributed to the difference in the absorption spectra in solid films. The straight C-12 side chain has less steric hindrance than the branched EH side chain in solid films. PSDT-C12 has a longer main chain (larger molecule weight) than PSDT-EH, which can favour a more extended main chain interaction. The vibronic peak at 519 nm and shoulder at 563 nm in the PSDT-C12 film further confirmed stronger main chain interaction. Geometry optimization showed that head–tail (HT)-PSDT had a more twisting conjugated backbone with larger dihedral angle between dithienosilole unit and thiadiazole-fused ring compared to head–head/tail–tail (HH/TT)-PSDT.     

Crystal Structures of a Bioactive 6′‐Hydroxy Variant of Sisomicin Bound to the Bacterial and Protozoal Ribosomal Decoding Sites

Parasitic infections recognized as neglected tropical diseases are a source of concern for several regions of the world. Aminoglycosides are potent antimicrobial agents that have been extensively studied by biochemical and structural studies in prokaryotes. However, the molecular mechanism of their potential antiprotozoal activity is less well understood. In the present study, we have examined the in vitro inhibitory activities of some aminoglycosides with a 6′‐hydroxy group on ring I and highlight that one of them, 6′‐hydroxysisomicin, exhibits promising activity against a broad range of protozoan parasites. Furthermore, we have conducted X‐ray analyses of 6′‐hydroxysisomicin bound to the target ribosomal RNA A‐sites in order to understand the mechanisms of both its antibacterial and antiprotozoal activities at the molecular level. The unsaturated ring I of 6′‐hydroxysisomicin can directly stack on G1491, which is highly conserved in bacterial and protozoal species, through π–π interaction and fits closer to the guanidine base than the typically saturated and hydroxylated ring I of other structurally related aminoglycosides. Consequently, the compound adopts a lower energy conformation within the bacterial and protozoal A‐sites and makes pseudo pairs to either A or G at position 1408. The A‐site‐selective binding mode strongly suggests that 6′‐hydroxysisomicin is a potential lead for the design of next‐generation aminoglycosides targeting a wide variety of infectious diseases.     

Homology modeling of NR2B modulatory domain of NMDA receptor and analysis of ifenprodil binding

NMDA receptors are glutamate-gated ion channels (iGluRs) that are involved in several important physiological functions such as neuronal development, synaptic plasticity, learning, and memory. Among iGluRs, NMDA receptors have been perhaps the most actively investigated for their role in chronic neurodegeneration such as Alzheimer's, Parkinson's, and Huntington's diseases. Recent studies have shown that the NTD of subunit NR2B modulates ion channel gating through the binding of allosteric modulators such as the prototypical compound ifenprodil. In the present paper, the construction of a three-dimensional model for the NR2B modulatory domain is described and docking calculations allow, for the first time, definition of the ifenprodil binding pose at an atomic level and fully explain all the available structure-activity relationships. Moreover, in an attempt to add further insight into the ifenprodil mechanism of action, as it is not completely clear if it binds and stabilizes an open or a closed conformation of the NR2B modulatory domain, a matter, which is fundamental for the rational design of NMDA antagonists, MD simulations followed by an MM-PBSA analysis were performed. These calculations reveal that the closed conformation of the R1-R2 domain, rather than the open, constitutes the high affinity binding site for ifenprodil and that a profound stabilization of the closed conformation upon ifenprodil binding occurs. Thus, for a rational design and/or for virtual screening experiments, the closed conformation of the R1-R2 domain should be taken into account and our 3D model can provide valuable hints for the design of NR2B-selective antagonists.