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Biochimica Et Biophysica Acta Jul 2002Cellular DNA is not a uniform target for DNA-reactive drugs. At the nucleotide level, drugs recognize and bind short motifs of a few base pairs. The location of drug... (Review)
Review
Cellular DNA is not a uniform target for DNA-reactive drugs. At the nucleotide level, drugs recognize and bind short motifs of a few base pairs. The location of drug adducts at the genomic level depends on how these short motifs are distributed in larger domains. This aspect, referred to as region specificity, may be critical for the biological outcome of drug action. Recent studies demonstrated that certain minor groove binding (MGB) drugs, such as bizelesin, produce region-specific lesions in cellular DNA. Bizelesin binds mainly T(A/T)(4)A sites, which are on average scarce, but occasionally cluster in distinct minisatellite regions (200-1000 bp of approximately 85-100% AT), herein referred to as AT islands. Bizelesin-targeted AT islands are likely to function as strong matrix attachment regions (MARs), domains that organize DNA loops on the nuclear matrix. Distortion of MAR-like AT islands may be a basis for the observed inhibition of new replicon initiation and the extreme lethality of bizelesin adducts (<10 adducts/cell for cell growth inhibition). Hence, long AT-islands represent a novel class of critical targets for anticancer drugs. The AT island paradigm illustrates the potential of the concept of regional targeting as an essential component of the rational design of new sequence-specific DNA-reactive drugs.
Topics: Antineoplastic Agents; Base Sequence; Benzofurans; Binding Sites; Cyclohexanecarboxylic Acids; Cyclohexenes; DNA Damage; DNA, Neoplasm; Distamycins; Drug Design; Duocarmycins; Genome, Human; Humans; Indoles; Nitrogen Mustard Compounds; Tandem Repeat Sequences; Tumor Cells, Cultured; Urea
PubMed: 12084472
DOI: 10.1016/s0925-4439(02)00093-5 -
European Journal of Biochemistry Jun 2002Single-crystal X-ray structure determinations of the complex between the minor-groove binder distamycin and d(GGCCAATTGG) reveal two 1 : 1 binding modes which differ in...
Single-crystal X-ray structure determinations of the complex between the minor-groove binder distamycin and d(GGCCAATTGG) reveal two 1 : 1 binding modes which differ in the orientation of the drug molecule in the minor groove. The two crystals were grown from different crystallization conditions and found to diffract to 2.38 and 1.85 A, respectively. The structures were refined to completion using SHELXL-93, resulting in a residual R factor of 20.30% for the 2.38-A resolution structure (including 46 water molecules) and 19.74% for the 1.85-A resolution structure (including 74 water molecules). In both orientations, bifurcated hydrogen bonds are formed between the amide nitrogen atoms of the drug and AT base pairs. With a binding site of at least five base pairs, close contacts between the terminal distamycin atoms and guanine amino groups are inevitable. The detailed nature of several of these interactions was further investigated by ab initio quantum chemical methods.
Topics: Adenine; Amides; Base Pairing; Binding Sites; Crystallography, X-Ray; Distamycins; Guanine; Hydrogen Bonding; Models, Molecular; Nucleic Acid Conformation; Oligodeoxyribonucleotides; Quantum Theory; Thymidine
PubMed: 12071949
DOI: 10.1046/j.1432-1033.2002.02952.x -
Biophysical Journal May 2002Fibroblast growth factor-2 (basic FGF), a potent inducer of angiogenesis, and the naphthalene sulfonic distamycin A derivative,...
Nature of Interaction between basic fibroblast growth factor and the antiangiogenic drug 7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolecarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino])-bis-(1,3-naphtalene disulfonate). II. Removal of polar interactions affects protein folding.
Fibroblast growth factor-2 (basic FGF), a potent inducer of angiogenesis, and the naphthalene sulfonic distamycin A derivative, 7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolecarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino])-bis-(1,3-naphtalene disulfonate) (PNU145156E), which exhibits in vivo antiangiogenic activity, form a tight reversible (1:1) complex. PNU145156E binds to the heparin and the selenate-binding sites on bFGF. The cis bFGF-heparin (2:1) complex, essential for the activation of the angiogenic process, is thus prevented. The nature of the forces involved in bFGF:PNU145156E complex, using the wild-type and the K128Q, K138Q, K134Q, and K128Q-K138Q point mutated bFGFs was sought. Based on thermodynamic analysis of the complexation constants, protein temperature stability profiles by ultraviolet absorption, circular dichroism measurements, fluorescence Förster energy-transfer, and anisotropy studies, in harmony with the published x-ray crystallographic structure, the following molecular interactions are proposed: reduced coulombic interactions, hence loosening of the complex by the removal of charged polar groups from the bFGF-heparin binding cleft resulted in decreased binding constants and in a change in the binding mode from polar to nonpolar. Concomitantly, upon mutation, the protein was rendered more compact, less flexible, and less aqueously exposed compared with the wild type. These were further pronounced with the double mutant: weaker dominantly nonpolar protein-drug interactions were accompanied by conspicuous folding. With heparin, however, wild-type bFGF forms a tighter complex with a more compact structure.
Topics: Amino Acid Substitution; Angiogenesis Inhibitors; Distamycins; Fibroblast Growth Factor 2; Humans; Kinetics; Models, Molecular; Mutagenesis, Site-Directed; Neovascularization, Physiologic; Protein Conformation; Protein Denaturation; Protein Folding; Thermodynamics; Tryptophan; Tyrosine
PubMed: 11964252
DOI: 10.1016/S0006-3495(02)75607-5 -
The Journal of Biological Chemistry May 2002Human fragile sites are weak staining gaps in chromosomes generated by specific culture conditions. The short CGG repeating DNA derived from folate-sensitive fragile...
Human fragile sites are weak staining gaps in chromosomes generated by specific culture conditions. The short CGG repeating DNA derived from folate-sensitive fragile sites has been shown to exclude single nucleosomes. To test whether this nucleosome exclusion model provides a general molecular mechanism for the formation of fragile sites, a different class of fragile site, the 33-base pair AT-rich repeating DNAs derived from the rare distamycin-inducible site, FRA16B, was examined for its ability to assemble single nucleosomes and nucleosome arrays using in vitro nucleosome reconstitution methods. The FRA16B DNA fragments strongly exclude nucleosome assembly only in the presence of distamycin, and increasing the number of 33-bp repeats increases the effect of distamycin in the destabilization of the nucleosome formation, suggesting a common mechanism for the formation of fragile sites.
Topics: Antiviral Agents; Base Sequence; Binding, Competitive; Chromosome Fragile Sites; Chromosome Fragility; Chromosomes, Human, Pair 16; DNA; Distamycins; Dose-Response Relationship, Drug; HeLa Cells; Humans; Molecular Sequence Data; Nucleosomes; Plasmids; Trinucleotide Repeat Expansion
PubMed: 11880377
DOI: 10.1074/jbc.M200901200 -
The Journal of Biological Chemistry May 2002Splice variants of the Nmp4 gene include nuclear matrix transcription factors that regulate the type I collagen alpha1(I) polypeptide chain (COL1A1) promoter and several...
Splice variants of the Nmp4 gene include nuclear matrix transcription factors that regulate the type I collagen alpha1(I) polypeptide chain (COL1A1) promoter and several matrix metalloproteinase (MMP) genes. To date, these are the only Cys(2)His(2) zinc finger proteins known to bind within the minor groove of homopolymeric (dA.dT) DNA. Nmp4 isoforms contain from 5 to 8 Cys(2)His(2) zinc fingers, an SH3-binding domain that overlaps with a putative AT-hook and a polyglutamine-alanine repeat (poly(QA)). To determine the mechanistic significance of Cys(2)His(2) zinc finger association with this unusual consensus DNA binding element, we identified the Nmp4 DNA-binding and transcriptional activation domains. Zinc fingers 2, 3, and 6 mediated association with the homopolymeric (dA.dT) COL1A1/MMP DNA consensus element. The N terminus of the Nmp4 protein exhibited a strong trans-activation capacity when fused to the GAL4 DNA-binding domain, but this activity was masked within the context of the full-length Nmp4-GAL4 DNA-binding domain chimera. However, upon binding to the COL1A1/MMP homopolymeric (dA.dT) element, the native Nmp4 protein up-regulated transcription, and the poly(QA) domain acquired a significant role in trans-activation. We propose that allosteric effects induced upon zinc finger association with the homopolymeric (dA.dT) minor groove confer context-specific functionality to this unusual family of Cys(2)His(2) transcription factors.
Topics: Antigens, Nuclear; Binding, Competitive; Cell Line; Collagen Type I; DNA-Binding Proteins; Distamycins; Gene Expression Regulation; Humans; Kinetics; Matrix Metalloproteinases; Methyl Green; Nuclear Matrix; Nuclear Proteins; Promoter Regions, Genetic; Protein Isoforms; Recombinant Proteins; Transcription Factors; Transcriptional Activation; Transfection; Zinc Fingers
PubMed: 11867614
DOI: 10.1074/jbc.M107496200 -
Zeitschrift Fur Naturforschung. C,... 2001The effects of DNA interacting drugs on: (1) total RNA synthesis catalyzed by E. coli and T7 RNA polymerase; (2) synthesis of the initiating dinucleotide (pppApU) by E....
The effects of DNA interacting drugs on: (1) total RNA synthesis catalyzed by E. coli and T7 RNA polymerase; (2) synthesis of the initiating dinucleotide (pppApU) by E. coli RNA polymerase ("abortive initiation"); (3) elongation of RNA chains synthesized by T7 RNA polymerase on pT7-7 plasmid DNA bearing T7 RNA polymerase promoter phi 10 with human Cu/Zn superoxide dismutase coding sequence, (4) interaction of transcription factor Sp1 and its binding site were studied. Intercalating ligands which form quickly dissociating complexes with DNA (anthracyclines, proflavine, ethidium bromide) are compared with the slowly dissociating drug of d(G x C) specificity (actinomycin D), the non-intercalating, d(A x T) specific pyrrole antibiotics (netropsin and distamycin A) and covalently binding to DNA 1-nitroacridine derivative (nitracrine). The obtained results indicate that rapidly dissociating ligands, proflavine and ethidium bromide, inhibit total RNA synthesis in vitro and the abortive initiation to a similar extent while they do not induce discrete elongation stops of RNA polymerase. Actinomycin D and nitracrine exhibit a high inhibitory effect on total RNA synthesis and induce stops of RNA polymerase while not affecting abortive initiation. Pyrrole antibiotics primarily inhibit the initiation, while no elongation stops are induced. Actinomycin D inhibits complex formation between nuclear proteins and the Sp1 binding site. Netropsin, ethidium bromide, proflavine and other intercalating acridines do not affect Sp1 binding. The results indicate that the effects primarily depend on sequence specificity and secondarily on the dissociation rate of ligands from their complexes with DNA.
Topics: Antibiotics, Antineoplastic; Antineoplastic Agents; Bacteriophage T7; Cell Line; Cell Nucleus; DNA-Directed RNA Polymerases; Endothelium, Vascular; Escherichia coli; Humans; Promoter Regions, Genetic; Protein Subunits; Receptors, Vitronectin; Sp1 Transcription Factor; Tissue Extracts; Transcription, Genetic; Viral Proteins
PubMed: 11724400
DOI: 10.1515/znc-2001-9-1034 -
Nucleic Acids Research Nov 2001The interactions of self-complementary oligonucleotides with a group of metal-mediated DNA-binding drugs, including chromomycin A(3), mithramycin and the novel compound...
The interactions of self-complementary oligonucleotides with a group of metal-mediated DNA-binding drugs, including chromomycin A(3), mithramycin and the novel compound UK-1, were examined via electrospray ionization quadrupole ion trap mass spectrometry. Both chromomycin and mithramycin were shown to bind preferentially to GC-rich oligonucleotide duplexes in a 2:1 drug:metal ratio, while UK-1 was shown to bind in a 1:1 drug:metal stoichiometric ratio without a strong sequence preference. These trends were observed in the presence of Co(2+), Ni(2+) and Zn(2+), with the exception that chromomycin-Zn(2+) complexes were not readily observed. The binding stoichiometries as well as the sequence specificities are in agreement with literature reports for solution studies. Binding selectivities and stabilities of the complexes were also probed using electrospray ionization mass spectrometry. Both of the GC-rich oligomers 5'-GCGCGC-3' and 5'-GCGCATGCGC-3' exhibited a binding preference for chromomycin over mithramycin in the presence of Co(2+) and Ni(2+). Energy-variable collisionally activated dissociation of the complexes was employed to determine the stabilities of the complexes. The relative metal-dependent binding energies were Ni(2+) > Zn(2+) > Co(2+) for UK-1-oligomer complexes and Ni(2+) > Co(2+) for both mithramycin and chromomycin complexes.
Topics: Antineoplastic Agents; Base Sequence; Benzoxazoles; Binding Sites; Chromomycin A3; DNA; Distamycins; Dose-Response Relationship, Drug; Hydrogen-Ion Concentration; Metals; Molecular Weight; Nucleic Acid Synthesis Inhibitors; Oligodeoxyribonucleotides; Plicamycin; Spectrometry, Mass, Electrospray Ionization; Streptomyces; Substrate Specificity; Temperature; Thermodynamics
PubMed: 11691940
DOI: 10.1093/nar/29.21.e103 -
Proceedings of the National Academy of... Sep 2001Developing minor groove-binding drugs to selectively inhibit transcription factor (TF)/DNA interactions and accompanying gene expression is a current goal in drug...
Developing minor groove-binding drugs to selectively inhibit transcription factor (TF)/DNA interactions and accompanying gene expression is a current goal in drug development studies. Equipping minor groove-binding agents with positively charged, major groove-contacting side chains yields microgonotropens (MGTs). Previously, we demonstrated that MGTs were superior inhibitors of TF/DNA complexes in cell-free assays compared with "classical" groove binders, but MGTs showed limited ability to inhibit gene expression. To determine what chemical characteristics contribute to or improve activity, we evaluate five MGTs for their effectiveness in inhibiting TF complex formation and resultant transcription by using the c-fos serum response element (SRE) as a target. MGT L1 binds DNA via a bisbenzimidazole equipped with a tripyrrole moiety. It is compared with analog L2, which has been functionalized with propylamines on each of the three pyrroles. L2, which binds DNA at subpicomolar concentrations, was at least three orders of magnitude more potent than L1 at inhibiting TF binding to the c-fos SRE in cell-free assays. Unlike L1 and previous MGTs, L2 also inhibited endogenous c-fos expression in NIH 3T3 cells at micromolar levels. Structure/activity relationships suggest that, although the tripyrrole/polyamine functional group of L2 may be largely responsible for its inhibition of TF complexes in cell-free assays, its bisbenzimidazole moiety appears to impart improved cellular uptake and activity. These findings make L2 a promising lead candidate for future, rational MGT design.
Topics: 3T3 Cells; Animals; Benzimidazoles; Binding Sites; Cell-Free System; DNA; DNA-Binding Proteins; Distamycins; Gene Expression Regulation; HeLa Cells; Humans; Ligands; Mice; Molecular Structure; Nuclear Proteins; Nucleic Acid Synthesis Inhibitors; Proto-Oncogene Proteins c-fos; Pyrroles; Serum Response Factor; Transcription, Genetic
PubMed: 11535831
DOI: 10.1073/pnas.191374698 -
Nucleic Acids Research Aug 2001The gene-to-drug quest will be most directly served by the discovery and development of small molecules that bind to nucleic acids and modulate gene expression at the...
The hybridization-stabilization assay: a solution-based isothermal method for rapid screening and determination of sequence preference of ligands that bind to duplexed nucleic acids.
The gene-to-drug quest will be most directly served by the discovery and development of small molecules that bind to nucleic acids and modulate gene expression at the level of transcription and/or inhibit replication of infectious agents. Full realization of this potential will require implementation of a complete suite of modern drug discovery technologies. Towards this end, here we describe our initial results with a new assay for identification and characterization of novel nucleic acid binding ligands. It is based on the well recognized property of stabilization of hybridization of complementary oligonucleotides by groove and/or intercalation binding ligands. Unlike traditional thermal melt methodologies, this assay is isothermal and, unlike gel-based footprinting techniques, the assay also is performed in solution and detection can be by any number of highly sensitive, non-radioisotopic modalities, such as fluorescence resonance energy transfer, described herein. Thus, the assay is simple to perform, versatile in design and amenable to miniaturization and high throughput automation. Assay validation was performed using various permutations of direct and competitive binding formats and previously well studied ligands, including pyrrole polyamide and intercalator natural products, designed hairpin pyrrole-imidazole polyamides and furan-based non-polyamide dications. DNA specific ligands were identified and their DNA binding site size and sequence preference profiles were determined. A systematic approach to studying the relationship of binding sequence specificity with variation in ligand structure was demonstrated, and preferred binding sites in longer DNA sequences were found by pseudo-footprinting, with results that are in accord with established findings. This assay methodology should promote a more rapid discovery of novel nucleic acid ligands and potential drug candidates.
Topics: Base Pairing; Base Sequence; Binding, Competitive; DNA Footprinting; Dactinomycin; Distamycins; Drug Evaluation, Preclinical; Energy Transfer; Fluorescence; Intercalating Agents; Kinetics; Ligands; Netropsin; Nucleic Acid Hybridization; Nucleic Acids; Nylons; Oligonucleotides; Promoter Regions, Genetic; Pyrroles; Reproducibility of Results; Solutions; Substrate Specificity; Temperature; Thermodynamics
PubMed: 11504893
DOI: 10.1093/nar/29.16.e85 -
British Journal of Haematology Jun 2001Human leukaemic K562 cells can be induced in vitro to erythroid differentiation by a variety of chemical compounds, including haemin, butyric acid, 5-azacytidine,...
Human leukaemic K562 cells can be induced in vitro to erythroid differentiation by a variety of chemical compounds, including haemin, butyric acid, 5-azacytidine, cytosine arabinoside, mithramycin and chromomycin, cisplatin and cisplatin analogues. Differentiation of K562 cells is associated with an increase of expression of embryo-fetal globin genes, such as the zeta-, epsilon- and gamma-globin genes. The K562 cell line has been proposed as a very useful in vitro model system to determine the therapeutic potential of new differentiating compounds as well as to study the molecular mechanism(s) regulating changes in the expression of embryonic and fetal human globin genes. Inducers of erythroid differentiation stimulating gamma-globin synthesis could be considered for possible use in the therapy of haematological diseases associated with a failure in the expression of normal beta-globin genes. We have analysed the effects of tallimustine and distamycin on cell growth and differentiation of K562 cells. The results demonstrated that tallimustine is a potent inducer, while distamycin is a weak inducer, of K562 cell erythroid differentiation. Erythroid differentiation was associated with an increase of accumulation of gamma-globin mRNA and of production of both haemoglobin (Hb) Gower 1 and Hb Portland. In addition, tallimustine-mediated erythroid induction occurred in the presence of activation of the apoptotic pathway. The reasons for proposing tallimustine as an inducer of gamma-globin gene expression are strongly sustained by the finding that this compound stimulates fetal haemoglobin production in human erythroid precursor cells from normal subjects.
Topics: Apoptosis; Blotting, Northern; Cell Differentiation; Cell Division; Cytarabine; DNA-Binding Proteins; Distamycins; Erythrocytes; Fetal Hemoglobin; Globins; Hematopoietic Stem Cells; Hemoglobins; Humans; Hydroxyurea; K562 Cells; Nitrogen Mustard Compounds; RNA, Messenger; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 11442489
DOI: 10.1046/j.1365-2141.2001.02843.x