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Journal of Chemical Information and... Aug 2008The molecular docking tools Autodock and Surflex accurately reproduce the crystallographic structures of a collection of small molecule ligands that have been shown to...
The molecular docking tools Autodock and Surflex accurately reproduce the crystallographic structures of a collection of small molecule ligands that have been shown to bind nucleic acids. Docking studies were performed with the intercalators daunorubicin and ellipticine and the minor groove binders distamycin and pentamidine. Autodock and Surflex dock daunorubicin and distamycin to their nucleic acid targets within a resolution of approximately 2 A, which is similar to the limit of the crystal structure resolution. However, for the top ranked poses, Autodock and Surflex both dock ellipticine into the correct site but in a different orientation compared to the crystal structure. This appears not only to be partly related to the symmetry of the target nucleic acid, as ellipticine is able to dock from either side of the intercalation site, but also due to the shape of the ligand and docking accuracy. Surflex docks pentamidine in a symmetrically equivalent orientation relative to the crystal structure, while Autodock was able to dock this molecule in the original orientation. In the case of the Surflex docking of pentamidine, the initial rmsd is misleading, given the symmetrical structure of pentamidine. Importantly, the ranking functions of both of these programs are able to return a top pose within approximately 2 A rmsd for daunorubicin, distamycin, and pentamidine and approximately 3 A rmsd for ellipticine compared to their respective crystal structures. Some docking challenges and potential pitfalls are explored, such as the importance of hydrogen treatment on ligands as well as the scoring functions of Autodock and Surflex. Overall for this set of complexes, Surflex is preferred over Autodock for virtual screening, as although the results are comparable, Surflex has significantly faster performance and ease of use under the optimal software conditions tested. These experiments show that molecular docking techniques can be successfully extended to include nucleic acid targets, a finding which has important implications for virtual screening applications and in the design of new small molecules to target therapeutically relevant morphologies of nucleic acids.
Topics: Crystallography, X-Ray; Ligands; Models, Molecular; Molecular Structure; Nucleic Acids
PubMed: 18642866
DOI: 10.1021/ci800063v -
PloS One Jul 2008Tau, an important microtubule associated protein, has been found to bind to DNA, and to be localized in the nuclei of both neurons and some non-neuronal cells. Here,...
Tau, an important microtubule associated protein, has been found to bind to DNA, and to be localized in the nuclei of both neurons and some non-neuronal cells. Here, using electrophoretic mobility shifting assay (EMSA) in the presence of DNA with different chain-lengths, we observed that tau protein favored binding to a 13 bp or a longer polynucleotide. The results from atomic force microscopy also showed that tau protein preferred a 13 bp polynucleotide to a 12 bp or shorter polynucleotide. In a competitive assay, a minor groove binder distamycin A was able to replace the bound tau from the DNA double helix, indicating that tau protein binds to the minor groove. Tau protein was able to protect the double-strand from digestion in the presence of DNase I that was bound to the minor groove. On the other hand, a major groove binder methyl green as a negative competitor exhibited little effect on the retardation of tau-DNA complex in EMSA. This further indicates the DNA minor groove as the binding site for tau protein. EMSA with truncated tau proteins showed that both the proline-rich domain (PRD) and the microtubule-binding domain (MTBD) contributed to the interaction with DNA; that is to say, both PRD and MTBD bound to the minor groove of DNA and bent the double-strand, as observed by electron microscopy. To investigate whether tau protein is able to prevent DNA from the impairment by hydroxyl free radical, the chemiluminescence emitted by the phen-Cu/H(2)O(2)/ascorbate was measured. The emission intensity of the luminescence was markedly decreased when tau protein was present, suggesting a significant protection of DNA from the damage in the presence of hydroxyl free radical.
Topics: Binding Sites; DNA; DNA Damage; Electrophoretic Mobility Shift Assay; Humans; Hydroxyl Radical; Microscopy, Atomic Force; Microscopy, Electron; Models, Molecular; Nucleic Acid Conformation; Proline; tau Proteins
PubMed: 18596978
DOI: 10.1371/journal.pone.0002600 -
Molecular Biology of the Cell Sep 2008Assembly of the nuclear pore, gateway to the genome, from its component subunits is a complex process. In higher eukaryotes, nuclear pore assembly begins with the...
Assembly of the nuclear pore, gateway to the genome, from its component subunits is a complex process. In higher eukaryotes, nuclear pore assembly begins with the binding of ELYS/MEL-28 to chromatin and recruitment of the large critical Nup107-160 pore subunit. The choreography of steps that follow is largely speculative. Here, we set out to molecularly define early steps in nuclear pore assembly, beginning with chromatin binding. Point mutation analysis indicates that pore assembly is exquisitely sensitive to the change of only two amino acids in the AT-hook motif of ELYS. The dependence on AT-rich chromatin for ELYS binding is borne out by the use of two DNA-binding antibiotics. AT-binding Distamycin A largely blocks nuclear pore assembly, whereas GC-binding Chromomycin A(3) does not. Next, we find that recruitment of vesicles containing the key integral membrane pore proteins POM121 and NDC1 to the forming nucleus is dependent on chromatin-bound ELYS/Nup107-160 complex, whereas recruitment of gp210 vesicles is not. Indeed, we reveal an interaction between the cytoplasmic domain of POM121 and the Nup107-160 complex. Our data thus suggest an order for nuclear pore assembly of 1) AT-rich chromatin sites, 2) ELYS, 3) the Nup107-160 complex, and 4) POM121- and NDC1-containing membrane vesicles and/or sheets, followed by (5) assembly of the bulk of the remaining soluble pore subunits.
Topics: Cell Nucleus; Chromatin; Chromomycin A3; DNA; DNA Mutational Analysis; DNA-Binding Proteins; Humans; Membrane Glycoproteins; Nuclear Pore; Nuclear Pore Complex Proteins; Point Mutation; S Phase; Saccharomyces cerevisiae; Schizosaccharomyces; Transcription Factors; Xenopus Proteins
PubMed: 18596237
DOI: 10.1091/mbc.e08-01-0012 -
Molecular Microbiology Jul 2008EhMLBP has been identified as a protein that specifically binds to methylated long interspersed element (LINE) retrotransposons and rDNA in Entamoeba histolytica. EhMLBP... (Comparative Study)
Comparative Study
EhMLBP has been identified as a protein that specifically binds to methylated long interspersed element (LINE) retrotransposons and rDNA in Entamoeba histolytica. EhMLBP is unique to Entamoeba parasites, which makes this protein a possible drug target for treating amebiasis. In the work described here, we evaluated this potential. Downregulation of EhMLBP using antisense technology resulted in trophozoites with impaired growth and cytopathic activity. This indicated that EhMLBP is an essential protein. With a view to identifying new antiamebic agents, we tested the effect of distamycin A, a drug with known antimalarial activity, on the growth of the parasite and on the ability of EhMLBP to bind to DNA. Distamycin A (IC(50) = 13 microM) efficiently inhibited the growth of E. histolytica. Indeed, distamycin A at a concentration of 5-20 microM inhibited the binding of EhMLBP to methylated LINE DNA in vitro. As an additional approach to identify molecules that inhibit EhMLBP activity, a selective biopanning assay was performed using the DNA-binding domain of EhMLBP and the Ph.D.-12 phage display peptide library. Remarkably, four out of the 11 phages selected after three rounds of biopanning expressed the peptide 'SYFDQNERWGAP' (Pept3) at their surface. The binding of EhMLBP to Pept3 was confirmed by ELISA. Phage expressing Pept3 inhibited the binding of EhMLBP to RT LINE DNA. The growth of E. histolytica transfectants expressing Pept3 was significantly impaired compared with that of trophozoites expressing a scrambled version of Pept3. These results highlight EhMLBP as an essential constituent of the parasite E. histolytica and a novel target for antiamebic chemotherapy.
Topics: Animals; Antimalarials; DNA Methylation; DNA, Protozoan; DNA-Binding Proteins; Distamycins; Down-Regulation; Entamoeba histolytica; Entamoebiasis; Epigenesis, Genetic; Gene Expression; Humans; Long Interspersed Nucleotide Elements; Peptide Library; Protein Binding; Protozoan Proteins; RNA, Antisense; Trophozoites
PubMed: 18484949
DOI: 10.1111/j.1365-2958.2008.06258.x -
Molecular Cancer Therapeutics Apr 2008We recently identified a polyamide-chlorambucil conjugate, 1R-Chl, which alkylates and down-regulates transcription of the human histone H4c gene and inhibits the growth...
We recently identified a polyamide-chlorambucil conjugate, 1R-Chl, which alkylates and down-regulates transcription of the human histone H4c gene and inhibits the growth of several cancer cell lines in vitro and in a murine SW620 xenograft model, without apparent animal toxicity. In this study, we analyzed the effects of 1R-Chl in the chronic myelogenous leukemia cell line K562 and identified another polyamide conjugate, 6R-Chl, which targets H4 genes and elicits a similar cellular response. Other polyamide conjugates that do not target the H4 gene do not elicit this response. In a murine model, both 1R-Chl and 6R-Chl were found to be highly effective in blocking K562 xenograft growth with high-dose tolerance. Unlike conventional and distamycin-based alkylators, little or no cytotoxicities and animal toxicities were observed in mg/kg dosage ranges. These results suggest that these polyamide alkylators may be a viable treatment alternative for chronic myelogenous leukemia.
Topics: Alkylation; Animals; Base Sequence; Blotting, Western; Cell Cycle; Cell Proliferation; Cell Survival; Chlorambucil; Female; Histones; Humans; Imidazoles; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Mice, Nude; Molecular Sequence Data; Nylons; Pyrroles; RNA, Messenger; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured; Xenograft Model Antitumor Assays
PubMed: 18413791
DOI: 10.1158/1535-7163.MCT-08-0130 -
FEBS Letters Mar 2008Nitric oxide synthase (NOS)2, an inducible enzyme that produces NO during inflammation, is transcriptionally regulated. Our goal was to determine whether high mobility...
Nitric oxide synthase (NOS)2, an inducible enzyme that produces NO during inflammation, is transcriptionally regulated. Our goal was to determine whether high mobility group (HMG)A1 contributes to human (h)NOS2 gene regulation. Using a small molecule inhibitor of HMGA1 binding to DNA, or a dominant-negative form of HMGA1, we blunted the induction of hNOS2 by pro-inflammatory stimuli. Binding of HMGA1 in the region -3506 to -3375 of the hNOS2 promoter, a region not previously known to be involved in hNOS2 regulation, contributed to the induction of hNOS2 promoter in conjunction with upstream enhancer regions. We demonstrate a previously unknown role for HMGA1 in the regulation of hNOS2.
Topics: Base Sequence; Cell Line; Cytokines; Distamycins; Gene Expression Regulation, Enzymologic; Genes, Dominant; HMGA1a Protein; Humans; Molecular Sequence Data; Nitric Oxide Synthase Type II; Promoter Regions, Genetic; RNA, Messenger; Sequence Deletion
PubMed: 18279675
DOI: 10.1016/j.febslet.2008.02.008 -
Brazilian Journal of Biology = Revista... Dec 2007The taxonomy/systematics of the Erythrinidae fish is still imprecise, with several doubts on their relationships. Karyotypes and chromosomal characteristics of some...
The taxonomy/systematics of the Erythrinidae fish is still imprecise, with several doubts on their relationships. Karyotypes and chromosomal characteristics of some species of the Hoplias lacerdae group (Erythrinidae), from different Brazilian hydrographic basins and pisciculture stations, were analyzed in the present study, using conventional Giemsa staining, C-banding, silver staining, Mithramycin and Distamycin/DAPI fluorochromes, and fluorescent in situ hybridization (FISH). A diploid chromosome number of 2n = 50 and karyotypes composed of meta- and submetacentric chromosomes without sex-related differences were found. Only one active NOR (Nucleolar Organizer Region) site was found, which was identified by silver staining (Ag-NOR) and FISH, located on the chromosome pair 11, although additional 45S rDNA sites were also mapped on other chromosome pairs only by FISH. The Ag-NOR of the chromosome pair 11 was found to be GC-rich, appearing positive after Mithramycin staining. Mithramycin-positive/DAPI-negative sites were also observed in the centromeric/pericentomeric regions of the chromosome pairs 4, 6, 15, and 19, which have also affinity to silver nitrate. However, these four sites were not detected by FISH with the rDNA probe, indicating to be only argentophilic GC-rich heterochromatic regions. Chromosome data show that the karyotype evolution in Hoplias lacerdae group is relatively conserved and follows a particular pathway concerning the other Erythrinidae fishes, such as Hoplias malabaricus, Hoplerythrinus unitaeniatus, and Erythrinus erythrinus, in which polytypic karyotypes are found. Thus, the H. lacerdae group shows chromosome features that are not closely related to those of the congeneric H. malabaricus group. These finds, together with genetic and morphologic data, are important tools to be considered in a major revision of the Erythrinidae family, as well as for conservation programs.
Topics: Animals; Biological Evolution; Brazil; Chromosomes; Female; Fishes; In Situ Hybridization, Fluorescence; Karyotyping; Male; Staining and Labeling
PubMed: 18278357
DOI: 10.1590/s1519-69842007000500013 -
Acta Poloniae Pharmaceutica 2007A DNA-binding affinity and the effect on restriction enzymes activity of seven carbocyclic mono- and bis-lexitropsins and two analogues of pentamidine with unsubstituted...
A DNA-binding affinity and the effect on restriction enzymes activity of seven carbocyclic mono- and bis-lexitropsins and two analogues of pentamidine with unsubstituted N-terminal amine group were investigated. DNA association constants (Kapp) show that DNA affinity of mono-compounds is much weaker than netropsin and distamycin. Bis-analogues of netropsin bind DNA more strongly than mono-ligands, but without sequence-selectivity. Only pentamidine derivatives reveal preference to AT-rich sequence. The studied compounds can inhibit catalytical action of endonucleases recognizing sequence of four AT base pairs following one another.
Topics: Acids, Carbocyclic; Binding Sites; Binding, Competitive; Bisbenzimidazole; DNA; Distamycins; Endonucleases; Molecular Structure; Netropsin; Pentamidine; Structure-Activity Relationship
PubMed: 17665860
DOI: No ID Found -
Journal of the American Chemical Society Jul 2006The four Watson-Crick base pairs of DNA can be distinguished in the minor groove by pairing side-by-side three five-membered aromatic carboxamides, imidazole (Im),...
The four Watson-Crick base pairs of DNA can be distinguished in the minor groove by pairing side-by-side three five-membered aromatic carboxamides, imidazole (Im), pyrrole (Py), and hydroxypyrrole (Hp), four different ways. On the basis of the paradigm of unsymmetrical paired edges of aromatic rings for minor groove recognition, a second generation set of heterocycle pairs, imidazopyridine/pyrrole (Ip/Py) and hydroxybenzimidazole/pyrrole (Hz/Py), revealed that recognition elements not based on analogues of distamycin could be realized. A new set of end-cap heterocycle dimers, oxazole-hydroxybenzimidazole (No-Hz) and chlorothiophene-hydroxybenzimidazole (Ct-Hz), paired with Py-Py are shown to bind contiguous base pairs of DNA in the minor groove, specifically 5'-GT-3' and 5'-TT-3', with high affinity and selectivity. Utilizing this technology, we have developed a new class of oligomers for sequence-specific DNA minor groove recognition no longer based on the N-methyl pyrrole carboxamides of distamycin.
Topics: Base Pairing; DNA; Nucleic Acid Conformation; Nylons; Oligonucleotides; Protein Binding; Pyrroles
PubMed: 16834381
DOI: 10.1021/ja0621795 -
Biophysical Journal Aug 2006Binding of a small molecule to a macromolecular target reduces its conformational freedom, resulting in a negative entropy change that opposes the binding. The goal of...
Binding of a small molecule to a macromolecular target reduces its conformational freedom, resulting in a negative entropy change that opposes the binding. The goal of this study is to estimate the configurational entropy change of two minor-groove-binding ligands, netropsin and distamycin, upon binding to the DNA duplex d(CGCGAAAAACGCG).d(CGCGTTTTTCGCG). Configurational entropy upper bounds based on 10-ns molecular dynamics simulations of netropsin and distamycin in solution and in complex with DNA in solution were estimated using the covariance matrix of atom-positional fluctuations. The results suggest that netropsin and distamycin lose a significant amount of configurational entropy upon binding to the DNA minor groove. The estimated changes in configurational entropy for netropsin and distamycin are -127 J K(-1) mol(-1) and -104 J K(-1) mol(-1), respectively. Estimates of the configurational entropy contributions of parts of the ligands are presented, showing that the loss of configurational entropy is comparatively more pronounced for the flexible tails than for the relatively rigid central body.
Topics: Binding Sites; Computer Simulation; DNA; Distamycins; Entropy; Models, Chemical; Models, Molecular; Netropsin; Nucleic Acid Conformation; Protein Binding; Protein Conformation; Thermodynamics
PubMed: 16731550
DOI: 10.1529/biophysj.105.074617