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Antiviral Research Aug 2011Human papillomavirus (HPV) causes cervical cancer and other hyperproliferative diseases. There currently are no approved antiviral drugs for HPV that directly decrease...
Human papillomavirus (HPV) causes cervical cancer and other hyperproliferative diseases. There currently are no approved antiviral drugs for HPV that directly decrease viral DNA load and that have low toxicity. We report the potent anti-HPV activity of two N-methylpyrrole-imidazole polyamides of the hairpin type, polyamide 1 (PA1) and polyamide 25 (PA25). Both polyamides have potent anti-HPV activity against three different genotypes when tested on cells maintaining HPV episomes. The compounds were tested against HPV16 (in W12 cells), HPV18 (in Ker4-18 cells), and HPV31 (in HPV31 maintaining cells). From a library of polyamides designed to recognize AT-rich DNA sequences such as those in or near E1 or E2 binding sites of the HPV16 origin of replication (ori), four polyamides were identified that possessed apparent IC(50)s≤150nM with no evidence of cytotoxicity. We report two highly-active compounds here. Treatment of epithelia engineered in organotypic cultures with these compounds also causes a dose-dependent loss of HPV episomal DNA that correlates with accumulation of compounds in the nucleus. Bromodeoxyuridine (BrdU) incorporation demonstrates that DNA synthesis in organotypic cultures is suppressed upon compound treatment, correlating with a loss of HPV16 and HPV18 episomes. PA1 and PA25 are currently in preclinical development as antiviral compounds for treatment of HPV-related disease, including cervical dysplasia. PA1, PA25, and related polyamides offer promise as antiviral agents and as tools to regulate HPV episomal levels in cells for the study of HPV biology. We also report that anti-HPV16 activity for Distamycin A, a natural product related to our polyamides, is accompanied by significant cellular toxicity.
Topics: Antiviral Agents; Binding Sites; Bromodeoxyuridine; Cell Line, Tumor; DNA, Viral; Distamycins; Female; Human papillomavirus 16; Human papillomavirus 18; Human papillomavirus 31; Humans; Immunohistochemistry; Inhibitory Concentration 50; Microbial Sensitivity Tests; Nylons; Papillomavirus Infections; Plasmids; Pyrroles; Replication Origin; Uterine Cervical Neoplasms; Viral Load
PubMed: 21669229
DOI: 10.1016/j.antiviral.2011.05.014 -
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 -
Nucleic Acids Research Oct 2008We have used metadynamics to investigate the mechanism of noncovalent dissociation from DNA by two representatives of alkylating and noncovalent minor groove (MG)...
We have used metadynamics to investigate the mechanism of noncovalent dissociation from DNA by two representatives of alkylating and noncovalent minor groove (MG) binders. The compounds are anthramycin in its anhydrous form (IMI) and distamycin A (DST), which differ in mode of binding, size, flexibility and net charge. This choice enables to evaluate the influence of such factors on the mechanism of dissociation. Dissociation of IMI requires an activation free energy of approximately 12 kcal/mol and occurs via local widening of the MG and loss of contacts between the drug and one DNA strand, along with the insertion of waters in between. The detachment of DST occurs at a larger free energy cost, approximately 16.5 or approximately 18 kcal/mol depending on the binding mode. These values compare well with that of 16.6 kcal/mol extracted from stopped-flow experiments. In contrast to IMI, an intermediate is found in which the ligand is anchored to the DNA through its amidinium tail. From this conformation, binding and unbinding occur almost at the same rate. Comparison between DST and with kinetic models for the dissociation of Hoechst 33258 from DNA uncovers common characteristics across different classes of noncovalent MG ligands.
Topics: Alkylating Agents; Anthramycin; Computational Biology; Computer Simulation; DNA; Distamycins; Models, Molecular; Nucleic Acid Conformation
PubMed: 18801848
DOI: 10.1093/nar/gkn561 -
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 -
PloS One 2011Distamycin A is a prototype minor groove binder, which binds to B-form DNA, preferentially at A/T rich sites. Extensive work in the past few decades has characterized...
BACKGROUND
Distamycin A is a prototype minor groove binder, which binds to B-form DNA, preferentially at A/T rich sites. Extensive work in the past few decades has characterized the binding at the level of double stranded DNA. However, effect of the same on physiological DNA, i.e. DNA complexed in chromatin, has not been well studied. Here we elucidate from a structural perspective, the interaction of distamycin with soluble chromatin, isolated from Sprague-Dawley rat.
METHODOLOGY/PRINCIPAL FINDINGS
Chromatin is a hierarchical assemblage of DNA and protein. Therefore, in order to characterize the interaction of the same with distamycin, we have classified the system into various levels, according to the requirements of the method adopted, and the information to be obtained. Isothermal titration calorimetry has been employed to characterize the binding at the levels of chromatin, chromatosome and chromosomal DNA. Thermodynamic parameters obtained thereof, identify enthalpy as the driving force for the association, with comparable binding affinity and free energy for chromatin and chromosomal DNA. Reaction enthalpies at different temperatures were utilized to evaluate the change in specific heat capacity (ΔCp), which, in turn, indicated a possible binding associated structural change. Ligand induced structural alterations have been monitored by two complementary methods--dynamic light scattering, and transmission electron microscopy. They indicate compaction of chromatin. Using transmission electron microscopy, we have visualized the effect of distamycin upon chromatin architecture at di- and trinucleosome levels. Our results elucidate the simultaneous involvement of linker bending and internucleosomal angle contraction in compaction process induced by distamycin.
CONCLUSIONS/SIGNIFICANCE
We summarize here, for the first time, the thermodynamic parameters for the interaction of distamycin with soluble chromatin, and elucidate its effect on chromatin architecture. The study provides insight into a ligand induced compaction phenomenon, and suggests new mechanisms of chromatin architectural alteration.
Topics: Animals; Chromatin; DNA; Distamycins; Nucleic Acid Conformation; Nucleic Acid Synthesis Inhibitors; Rats; Rats, Sprague-Dawley; Thermodynamics
PubMed: 22046291
DOI: 10.1371/journal.pone.0026486 -
Antimicrobial Agents and Chemotherapy Feb 1972Equine abortion virus, a member of the herpesvirus group, produces a lethal infection in hamsters. With this system, the protective effect of certain inhibitors of...
Equine abortion virus, a member of the herpesvirus group, produces a lethal infection in hamsters. With this system, the protective effect of certain inhibitors of deoxyribonucleic acid viruses, inducers of interferon and exogenous interferon, was evaluated. Of the various agents studied, 9-beta-d-arabinofuranosyladenine markedly suppressed mortality, and 5-iodo-2'-deoxyuridine, distamycin A, and N-ethylisatin beta-thiosemicarbazone were inactive. Of the inducers tested, statolon, ultraviolet-irradiated Newcastle disease virus, and polyriboinosinic:polyribocytidylic acid (poly I:C) were protective, and endotoxin, polyacrylic acid, and polymethacrylic acid did not protect. Administration of exogenous interferon did not afford protection. Statolon and ultraviolet-irradiated Newcastle disease virus induced circulating interferon in hamsters, whereas poly I:C, endotoxin, and polyacrylic acid did not produce interferon. Because of the severity of the disease produced in hamsters by equine abortion virus, lack of protective activity by an agent in this system should not preclude possible efficacy against other members of the herpesvirus group.
Topics: Animals; Antiviral Agents; Cricetinae; Herpesviridae; Herpesviridae Infections; Herpesvirus 1, Equid; Interferon Inducers; Interferons; Male
PubMed: 4376907
DOI: 10.1128/AAC.1.2.143 -
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 -
Molecular and Cellular Biology Oct 2003Fragile sites are specific loci that form gaps, constrictions, and breaks on chromosomes exposed to partial replication stress and are rearranged in tumors. Fragile...
Fragile sites are specific loci that form gaps, constrictions, and breaks on chromosomes exposed to partial replication stress and are rearranged in tumors. Fragile sites are classified as rare or common, depending on their induction and frequency within the population. The molecular basis of rare fragile sites is associated with expanded repeats capable of adopting unusual non-B DNA structures that can perturb DNA replication. The molecular basis of common fragile sites was unknown. Fragile sites from R-bands are enriched in flexible sequences relative to nonfragile regions from the same chromosomal bands. Here we cloned FRA7E, a common fragile site mapped to a G-band, and revealed a significant difference between its flexibility and that of nonfragile regions mapped to G-bands, similar to the pattern found in R-bands. Thus, in the entire genome, flexible sequences might play a role in the mechanism of fragility. The flexible sequences are composed of interrupted runs of AT-dinucleotides, which have the potential to form secondary structures and hence can affect replication. These sequences show similarity to the AT-rich minisatellite repeats that underlie the fragility of the rare fragile sites FRA16B and FRA10B. We further demonstrate that the normal alleles of FRA16B and FRA10B span the same genomic regions as the common fragile sites FRA16C and FRA10E. Our results suggest that a shared molecular basis, conferred by sequences with a potential to form secondary structures that can perturb replication, may underlie the fragility of rare fragile sites harboring AT-rich minisatellite repeats and aphidicolin-induced common fragile sites.
Topics: Alleles; Antiviral Agents; Base Sequence; Bromodeoxyuridine; Cell Line, Transformed; Chromosome Banding; Chromosome Fragility; Chromosome Mapping; Cytogenetics; DNA; Databases, Genetic; Distamycins; Fibroblasts; Genome; Humans; In Situ Hybridization, Fluorescence; Minisatellite Repeats; Molecular Sequence Data; Nucleic Acid Conformation; Phylogeny; Physical Chromosome Mapping; Polymerase Chain Reaction; Software
PubMed: 14517285
DOI: 10.1128/MCB.23.20.7143-7151.2003 -
Nucleic Acids Research Jun 2003Distamycin binds the minor groove of duplex DNA at AT-rich regions and has been a valuable probe of protein interactions with double-stranded DNA. We find that...
Distamycin binds the minor groove of duplex DNA at AT-rich regions and has been a valuable probe of protein interactions with double-stranded DNA. We find that distamycin can also inhibit protein interactions with G-quadruplex (G4) DNA, a stable four-stranded structure in which the repeating unit is a G-quartet. Using NMR, we show that distamycin binds specifically to G4 DNA, stacking on the terminal G-quartets and contacting the flanking bases. These results demonstrate the utility of distamycin as a probe of G4 DNA-protein interactions and show that there are (at least) two distinct modes of protein-G4 DNA recognition which can be distinguished by sensitivity to distamycin.
Topics: Base Sequence; Binding Sites; Binding, Competitive; DNA; Distamycins; G-Quadruplexes; Guanine; Ligands; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Phosphoproteins; Protein Binding; Protein Structure, Tertiary; RNA-Binding Proteins; Repetitive Sequences, Nucleic Acid; Nucleolin
PubMed: 12771220
DOI: 10.1093/nar/gkg392 -
The Journal of Biological Chemistry Nov 2000The terminase of bacteriophage SPP1, constituted by a large (G2P) and a small (G1P) subunit, is essential for the initiation of DNA packaging. A hexa-histidine G2P...
The terminase of bacteriophage SPP1, constituted by a large (G2P) and a small (G1P) subunit, is essential for the initiation of DNA packaging. A hexa-histidine G2P (H6-G2P), which is functional in vivo, possesses endonuclease, ATPase, and double-stranded DNA binding activities. H6-G2P introduces a cut with preference at the 5'-RCGG downward arrowCW-3' sequence. Distamycin A, which is a minor groove binder that mimics the architectural structure generated by G1P at pac, enhances the specific cut at both bona fide 5'-CTATTGCGG downward arrowC-3' sequences within pacC of SPP1 and SF6 phages. H6-G2P hydrolyzes rATP or dATP to the corresponding rADP or dADP and P(i). H6-G2P interacts with two discrete G1P domains (I and II). Full-length G1P and G1PDeltaN62 (lacking domain I) stimulate 3.5- and 1.9-fold, respectively, the ATPase activity of H6-G2P. The results presented suggest that a DNA structure, artificially promoted by distamycin A or facilitated by the assembly of G1P at pacL and/or pacR, stimulates H6-G2P cleavage at both target sites within pacC. In the presence of two G1P decamers per H6-G2P monomer, the H6-G2P endonuclease is repressed, and the ATPase activity stimulated. Based on these results, we propose a model that can account for the role of terminase in headful packaging.
Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Bacillus subtilis; Bacteriophages; Base Sequence; DNA; DNA, Superhelical; DNA-Binding Proteins; Distamycins; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Endodeoxyribonucleases; Endonucleases; Escherichia coli; Hydrolysis; Kinetics; Models, Biological; Models, Genetic; Molecular Sequence Data; Plasmids; Promoter Regions, Genetic; Protein Binding; Protein Structure, Tertiary; Time Factors
PubMed: 10930407
DOI: 10.1074/jbc.M004309200