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WO1992006985A1 - Inhibiteurs de methyltransferase d'adn fondes sur un mecanisme - Google Patents

Inhibiteurs de methyltransferase d'adn fondes sur un mecanisme Download PDF

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Publication number
WO1992006985A1
WO1992006985A1 PCT/US1991/007622 US9107622W WO9206985A1 WO 1992006985 A1 WO1992006985 A1 WO 1992006985A1 US 9107622 W US9107622 W US 9107622W WO 9206985 A1 WO9206985 A1 WO 9206985A1
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dna
blocks
hbo
self
enzyme
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PCT/US1991/007622
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English (en)
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Steven S. Smith
Bruce E. Kaplan
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City Of Hope
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Priority to AU89538/91A priority Critical patent/AU648851B2/en
Priority to US07/861,899 priority patent/US5503975A/en
Publication of WO1992006985A1 publication Critical patent/WO1992006985A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates

Definitions

  • This invention relates to synthetic oligomers of unique three-dimensional structure that combine the principles of polymer, peptide and synthetic DNA chemistry to provide rationally designed drugs, drug delivery systems, research tools and other products.
  • the invention also relates to unique oligonucleotide molecules in which 5-fluorodeoxycy- tidine inhibits DNA methyltrnasferase.
  • oligomer component consisting of at least three blocks.
  • Oligomer A molecule having from about 12 to about 150 monomers, e.g., carbon atoms, amino acid residues or nucleotides and comprising at least one unit.
  • Self-Association The capability shared by two or more blocks to form a mutual linkage, e.g., the capability of homologous nucleic acid sequences to hybridize and of certain peptides to interact.
  • Homologous Block One of a series of blocks where members of the series exhibit common properties, for example, one of a series of nucleic acid, peptide or organic polymer blocks.
  • Organic polymer blocks may generally comprise straight or branched chain polyolefins such as polyethylene, polypropylene or polystyrene and other kinds of polymers which are not cross-linke .
  • Heterologous Block One of a series of blocks whose members do not exhibit common properties.
  • Heterologous Block Oligomer (HBO)—An oligomer comprising at least one unit of the schematic formula A1-B-A2 in which the block B is heterologous with respect to at least one of the blocks Ai and A2 and is constrained into a generally looped configuration by the self-association energy of the blocks A ⁇ and A2 or in which the blocks Ai and A2 are heterologous and are constrained into a spatially juxtaposed position by the internal self-association of the block B.
  • HBO Heterologous Block Oligomer
  • HBO detergents can be formed with self-associated DNA blocks joined by simple linker blocks. The properties of these detergent molecules can be exploited in several ways. Micelles formed primarily of the self-associated DNA blocks may permit the passage of any short, double-stranded DNA through the bloodstream. Antisense molecules and short duplex DNA's more intrinsically resistant to DNase could be delivered in this fashion. "Suicide" substrates of duplex DNAs can be constructed to target tissues and neoplastic or virus infected cells. The inclusion of a hydrophobic linker block in these HBOs facilitates diffusion or transport across cell membranes at the requisite site.
  • FdCyd and DNA containing FdCyd are stable to chemical hydrolysis.
  • FdCyd is susceptible to enzymatic deamination to toxic 5-fluorodeoxyuridine metabolites. See, Newman et al., Proc. Natl. Acad. Sci. USA 79:6419-6423 (1982).
  • This invention provides self-priming HBOs in which (1) the blocks Ai and A2 are homologous DNA sequences (2) the block designated A2 contains fewer, e.g., at least 10 fewer bases, and preferably from about 10 to about 50 fewer bases, than the block designated Ai, and in which the block A2 is a primer for the block A and may be extended, for example, by a DNA polymerase with a combination of dNTPs and FdCTP.
  • Such an HBO is depicted, for example, by the molecules designated 48L-1, 48L-2 and 48L-3 in Table 1, infra.
  • This invention further provides synthetic oligonucleotide molecules produced by the extension of such a self-priming HBO with a combination of dNTPs and FdCTP.
  • Such molecules are mechanism based inhibitors of human DNA methyltransferase.
  • FIGURES Figure 1 is a two-dimensional generalized schematic depiction of an HBO in which the Ai and A2 blocks are self-associated.
  • the A ⁇ and A2 blocks may be self-associating oligonucleotides, peptides or the like and the linker block B provides a preselected property, e.g., hydrophobicity.
  • the linker block B may be nucleic acid sequences when the Ai and A2 blocks are self-associating peptides.
  • Figure 2 depicts an HBO in which the linker block B is internally self-associating, e.g., a homologous DNA sequence, flanked by Ai and A2 blocks such as peptides or organic polymers which do not self-associate and which are constrained in juxtaposition by the self-association energy of the linker block.
  • Suitable DNA sequences for the B blocks of such HBOs include a stretch of hydrized nucleotides, generally a sequence of about 15 to 50 bases to provide the self-association energy appropriate to maintain the desired juxtaposition of the Ai and A2 blocks.
  • Suitable peptides for use as A blocks contain from about 5 to about 30 residues.
  • Suitable A block polymers include RNA, DNA, peptides or mixed RNA-DNA polymers having 12 to 150 nucleotides.
  • Figure 3 is a copy of an autoradiograph illustrating the utility of an HBO as a human methyl transferase substrate.
  • Figure 4 illustrates a three nucleotide rule DNA methylation with DNA methyl transferase.
  • Figure 5 depicts the Methyl-Directed DNA Methyltransferase Reaction.
  • Figure 5A Space-filling models of the asymmetrically methylated B-DNA molecule and the symmetrically methylated B-DNA molecule produced by the enzyme, depict the reaction. Methyl groups on 5-methyleytosine residues at the CG dinucleotide pair are shown.
  • Figure 5B Restriction analysis of the enzymatically labelled product. Labelling pattern predicted for the product of the methyl-directed reaction is shown on the left. The position of the 5-methylcytosine residue introduced synthetically is indicated with an (m) ; the predicted position of the 5-methylcytosine generated enzymatically is indicated with an (*) . Cleavage sites for the indicated restriction enzymes are depicted as gaps, the autoradiograph on the right shows the sizes of the labelled restriction fragments observed after enzymatic labelling with f 3 H-methyl1AdoMet. Figure 6 depicts consequences of the Enzyme Mechanism.
  • Figure 6A Mechanism of Human DNA(cytosine-5) methyl-transferases: Nucleophilic attack at C-6 of cytosine by a group at the active site of the DNA. Methyltransferase activates C-5 of cytosine as a methyl acceptor by saturating the 5-6 double bond. AdoMet donates a methyl group to C-5 of cytosine to form AdoHcy and the bracketed dihyrocytosine intermediate shown. Dissociation of the enzyme from the complex results in the regeneration of the 5-6 double bond to produce 5-methylcytosine.
  • Figure 7 depicts mechanism-based labelling of the human DNA(cytosine-5)methyltransferase.
  • Figure 7A As starting material, a 47mer was synthesized from ⁇ -cyanoethylphophoramidite precursors using an ABI DNA synthesizer. The 47mer was constructed so as to link a long block of DNA to a shorter complementary block of DNA through a tether consisting of five thy ine residues. A 5-methylcyto- sine residue was introduced during synthesis at position 17 (indicated with m) . Molecules of this type form unimolecular foldbacks and are self-priming substrates for DNA polymerase I. In order to generate an oligodeoxynucleotide in which position 48 was occupied by 5FC, 5-FdCTP synthesized by the method of Ruth, et al. Mol. Pharm.
  • Figure 7B Primer extension products were end-labelled using T4-polynucleotide kinase and incubated with a crude DNA methyltransferase preparation under the conditions given below. The product was concentrated by precipitation with trichloroacetic acid and resuspended in buffer containing 0-mercaptoethanol and sodium dodecylsulphate. After heating to 95°C for 5 minutes the sample was separated by electrophoresis through sodium dodecylsulphate containing polyacrylamide gels. 32 P was visualized by autoradiography.
  • Lane 1 5FdC-65mer + complete reaction mixture: 50 mM4-[2-hydroxyethyl]-1- piperazine-ethanesulfonic acid (pH 7.0) (HEPES) , 50 mM NaCl, 2 m_M dithiothreitol, 75 ⁇ " spermine, 10% (v/v) glycerol, and 6.0 / iM r 3 H-methyl1AdoMet. Lane 2: 5FdC-containing 65mer + reaction mixture lacking AdoMet. Lane 3: dC-containing 65mer + complete reaction mixture.
  • Figure 7C Unlabelled primer extension products were incubated with the methyltransferase preparation and separated by electrophoresis through sodium dodecyl sulphate-polyacrylamide gels as in A except that the tritiated methyl-groups applied to the foldback were visualized by fluorography after impregnating the gels with En 3 Hance. See, Smith, et al., J. Mol. Biol. 217:39 (1991). Lane 1: complete reaction mixture + 5FdC-containing 65mer. Lane 2: Complete reaction mixture + dC-containing 65mer.
  • Figure 8 depicts chromatographic Analysis of Enzymatically Labelled DNA.
  • a duplex oligodeoxynucleotide 30mer (inset) was enzymatically methylated in the complete reaction mixture containing [ 3 H-methy_l]AdoMet labelled in the methyl-moiety. After digestion of the labelled oligodeoxynucleotide with nuclease PI and bacterial alkaline phosphatase, the liberated nucleosides were separated by HPLC on a ⁇ bondapack C18 column. (Palmgren et al, Biochem. Biophys. Acta 1049:293 (1990)) Fractions we collected and tritium was quantified by scintillation counting. 8(A) Tritium profile. 8(B) Absorbance profile.
  • Formula I schematically represents one form of an HBO of the kind depicted by Figure 1: k) ic n sequence)
  • R is an alkyl or aryl group of from about 1 to about 10 carbon atoms and x may be from about 3 to about 12. When x is greater than about 10, these HBO's are surfactive.
  • n The number, n, of B block moieties depends upon the properties desired in the HBO. For many purposes n is from about 5 to about 20.
  • Ai, B and A 2 blocks yield bioengineered catalysts in which catalysis is carried out by an appropriately constrained peptide, protein or RNA block.
  • hydrophobic B blocks e.g., amino-alkyl phosphonates, amino-aryl phosphonates, yield HBOs which are surfactants, particularly when the self-associating blocks are DNA.
  • This example demonstrates the scope and significance of the invention. To do so, it identifies a specific question which has arisen in scientific research, describes the design of an HBO for use in answering the question, exemplifies the synthesis of the postulated HBO and shows that the synthesized HBO functions as intended.
  • the question addressed concerns the substrate specificity of the human DNA methyltransferase, i.e., whether the enzyme is capable of methylating across a gap in duplex DNA.
  • a 30 mer and a 13 mer were selected to provide a gapped duplex DNA as depicted by Formula II:
  • linker block raised two questions: (1) what moiety should be used to construct it, and (2) how long should it be? Hydrophobic moieties were rejected to preclude any test of the capability of the enzyme to interact with a detergent.
  • L is HN—(CH 2 ) 3 —O- -p.
  • Oligodeoxynucleotide Preparation and Characterization Single strand oligodeoxynucleotides were synthesized using the phosphoramidite method (Sinha, N.D., et al., Nucleic Acids Res. 12:4539-4557 (1984)). The single stranded products were purified by polyacrylamide gel electrophoresis and high performance liquid chromatography as described by Tan, et al., Cold Spring Harbor Symp. on Quantitative Biology XLVII 383-391 (1982) . The sequence of each of the oligodeoxynucleotides was verified using the method of Maxam and Gilbert.
  • the HBO was synthesized in a manner similar to the standard production of oligodeoxynucleotides.
  • the amino group is protected by a monomethyoxyl trityl MMT or dimethoryl trityl DMT group and the phosphate group is simultaneously activated. After the MMT or DMT group was removed, the amino group was neutralized after each addition so that the next monomer could be added.
  • the HBO product of this example is an excellent substrate for human DNA methyltransferase as evidenced by the following test which is dependent on the spatial conformation of the molecule.
  • DNA(cytosine-5)methyltransferase was purified from human placentas as described in Smith, supra. When stored under the conditions described there, the enzyme loses less than 50% of its activity per year at -70 ⁇ C. Two sets of assay conditions were employed. The unit of enzyme activity is defined in terms of the assay conditions used during enzyme purification with heat-denatured Micrococcus lysodeikticus DNA substrate (Smith supra) . A unit of enzyme activity is the amount required to catalyze the incorporation of 1 pmole of methyl groups into TCA insoluble DNA in one hour at 37 " C under those conditions.
  • the enzyme was dialyzed for 3 hours in a Hoefer microdialyzer (Health Products Inc., Rockford, 111.) against 38 mM glycine, 17% v/v glycerol, 5 mM Tris, 10 mM yS-mercaptoethanol, pH 7.8 at 4°C.
  • the final reaction volume of 100 ⁇ l contained: 0.4 ⁇ g total DNA, 50 mM HEPES pH 7.0, 50 mM NaCl, 2 mM DTT, 75 ⁇ M Spermine, 10% v/v glycerol and 6.0 mM [ 3 H]AdoMet (Amersham, 15 Ci/mmole) .
  • Reaction mixtures were pre-incubated at 37°C for 15 minutes before the addition of 44 U of DNA methyltransferase to initiate the reaction. The reaction rate was linear under these conditions for 30 minutes. After 20 minutes of incubation, the reactions were stopped by the addition of 5 ml of cold TCA (5% w/v TCA containing 5 mM potassium pyrophosphate) . Tritium incorporated into TCA insoluble DNA was determined as previously described (Smith, S.S., supra) .
  • the labelled duplexes were cleaved with restriction endonucleases Mbol and Mspl.
  • the products were electrophoretically separated and 3 H labelled DNA fragments were detected by fluorescence enhanced autoradiography as previously described in Smith, et al.
  • the HBO molecule is refractory to digestion by Mspl, consistent with the fact that the looped molecule cannot generate a complete duplex Mspl site.
  • the same molecule is cleaved by Mbol to about 70% completion.
  • the cleavage product is only slightly shorter than the uncut molecule suggesting that Mbol cleavage occurs on the cut site on the unmethylated portion of the molecule to produce a molecule that is six nucleotides shorter (on the 3' end) than the parent molecule.
  • This partial cleavage pattern could be produced by the presence of the linkers in the loop, or it could mean that the fold-back structure does not form in a way that provides the enzyme with a completely recognizable cleavage site.
  • the DNA methyltransferase recognizes the structure and actively methylates it.
  • Table I shows that the presence of a methyl group at the end of the short arm of the loop stimulates the reaction more than 100 fold (48L-1) , while the presence of the methyl group at position 17 (bases numbered from the 5' end of the molecule) (48L-2) does not stimulate the reaction rate.
  • Enzymatic methylation of the HBO 48L-2 is demonstrated by the strong tritium signal in lanes 2 and 3 of the Figure 3 autoradiograph.
  • HBO 48L-2 was exposed to human DNA methyltransferase in the presence of S adenosyl methionine as the methyl group donor.
  • the reaction product was separated by polyacrylamide gel electrophoresis and the presence of enzymatically tritiated DNA was demonstrated by fluorescence enhanced autoradiography.
  • lanes 1 and 7 include markers corresponding to 18, 24 and 30 bases.
  • Mbol and Mspl identify restriction enzymes.
  • HBO's of all types may be synthesized by techniques known to the skilled person.
  • Linker blocks B may be added to pre-self associated j and A2 blocks as typified by the example.
  • all of the blocks may be separately synthesized and the desired HBO constructed from these prefabricated blocks. Such a procedure is preferred for the production of HBO's which involve the linkage of peptide and DNA blocks.
  • Example II illustrates one method for producing an HBO of schematic formula (DNA-2)-peptide-(DNA-1) .
  • MMT-C1 monomethoxytrityl chloride
  • MMT-C1 monomethoxytrityl chloride
  • the MMT-amino alkanol product is purified by chromatography and then reacted with an appropriate phosphitylating agent forming a cyanoethyl- diisopropylamino phosphite or a hydrogen phosphonate reagent or any other phosphite reagent known to the skilled worker.
  • the product of this reaction is an activated phosphite reagent useful in any standard DNA synthesis machine.
  • This activated phosphate reagent is coupled to the 5'OH of a growing DNA molecule synthesized in known manner on a solid support such as controlled pore glass (CPG) .
  • CPG controlled pore glass
  • the MMT group is then removed with dichloroacetic acid or trichloroacetic acid and the amino group is neutralized to permit coupling to the next incoming phosphite reagent.
  • Neutralization is accomplished by treating the growing DNA molecule with a dilute basic solution such as 1% triethyl amine in acetonitrile for a few seconds to convert the protonated amino group into a free amino group.
  • a DNA fragment with a free carboxylic acid on the 3' end is synthesized on a solid support, for example, by connecting a DMT protected hydroxy carboxylic acid such as the DMT protected 6-hydroxyhexanoic acid to an amino-propyl CPG using a carbodiimide. After the coupling, the DMT group is removed in known manner using dichloroacetic acid in dichloromethane. A second reaction with a DMT protected hydroxy carboxylic acid is completed. The DMT group is again removed and again coupled with the DMT-protected 6-hydroxyhexanoic acid using a carbodiimide and dimethylaminopyridine to provide controlled pore glass as a support for the synthesis of DNA-2 in known manner. These reactions are illustrated by the following equations: DMT-O-(CH 2 ) x - [CPG
  • Example III illustrates the preparation of an oligonucleotide containing 5FC residues. This oligonucleotide is shown to be a mechanism based inhibitor of DNA methyl transferase.
  • methylated pyrimidines are generated from the dihydropyrimidine intermediates by elimination of the hydrogen atom at C5 and the enzyme moiety at C6 to regenerate the 5-6 double bond.
  • DNA polymerase I was used to extend a self-priming 47mer in the presence of 5FdCTP as shown in Figure 7A.
  • Tritium fluorography could also be used to visualize the complexes when methyl groups were transferred to DNA by the enzyme from r 3 H-methyl1AdoMet ( Figure 7C) .
  • Figure 7C Tritium fluorography
  • the 5FC containing oligodeoxynucleotide was used, more than 80% of the label was observed in complexes.
  • the small amount of label at the position of the oligodeoxynucleotide 65mer could be attributed to nonspecific breakdown of the complex.
  • the oligodeoxynucleotide 65mer lacking 5FC was incubated with the enzyme and r 3 H-methyl1AdoMet, no label was observed in complexes.
  • the bulk of the tritium label was observed at the position of the 65mer.

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Abstract

Procédé permettant de produire des composés d'oligonucléotides bicaténaires comprenant l'utilisation d'un fragment d'ADN à auto-amorçage et l'allongement dudit ADN bicaténaire avec une polymérase d'ADN et une combinaison de dNTP et de 5-FdCTP, et composés destinés à ce procédé.
PCT/US1991/007622 1989-03-01 1991-10-21 Inhibiteurs de methyltransferase d'adn fondes sur un mecanisme WO1992006985A1 (fr)

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AU89538/91A AU648851B2 (en) 1990-10-23 1991-10-21 Mechanism based inhibitors of DNA methyltransferase
US07/861,899 US5503975A (en) 1989-03-01 1991-10-21 Mechanism based inhibitors of DNA methyltransferase

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US59866590A 1990-10-23 1990-10-23
US598,665 1990-10-23

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995015373A2 (fr) * 1993-11-30 1995-06-08 Mcgill University Inhibition de la methyltransferase de l'adn
WO1995015378A1 (fr) * 1993-12-01 1995-06-08 Hybridon, Inc. Oligonucleotides antisens inhibant l'oncogenicite
EP0756008A2 (fr) * 1995-07-28 1997-01-29 Health Research, Inc. Substrat pour la détection de 5-C-ADN méthyltransférase de mammifères
EP0771359A1 (fr) * 1992-06-22 1997-05-07 City Of Hope Structure d'ensembles a base de nucleoproteines comprenant des composants adressables pour ensemble a nano-echelle et nanoprocesseurs
WO1997044346A2 (fr) * 1996-05-22 1997-11-27 Mcgill University Inhibiteurs specifiques d'adn metyltransferase
US6268137B1 (en) 1996-05-22 2001-07-31 Methylgene, Inc. Specific inhibitors of DNA methyl transferase
WO2002103052A1 (fr) * 2001-06-15 2002-12-27 Quiatech Ab Sonde amplifiable
WO2003031648A2 (fr) * 2001-10-05 2003-04-17 Epigenomics Ag Procede pour detecter la methylation de l'adn au moyen d'analogues marques de la s-adenosylmethionine
US7125857B2 (en) 1997-08-29 2006-10-24 The Regents Of The University Of California Modulators of DNA cytosine-5 methyltransferase and methods for use thereof
US7342108B2 (en) 1998-06-25 2008-03-11 The General Hospital Corporation De novo DNA cytosine methyltransferase genes, polypeptides and uses thereof
US7368551B2 (en) 1998-06-25 2008-05-06 The General Hospital Corporation De novo DNA cytosine methyltransferase genes, polypeptides and uses thereof

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US4963662A (en) * 1986-07-24 1990-10-16 Akademie Der Wissenschaften Der Ddr Fluorinated nucleosides and method for treating retrovirus infections therewith
US4990499A (en) * 1989-09-29 1991-02-05 University Of Saskatchewan Anti-herpes simplex virus activity of 5-alkoxymethyl-2'-deoxycytidimes and their 5-monophosphates
US5002868A (en) * 1988-07-20 1991-03-26 Atom Sciences, Inc. DNA sequencing process using stable isotopes

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AU5192190A (en) * 1989-02-24 1990-09-26 City Of Hope Heterologous block oligomers

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US4963662A (en) * 1986-07-24 1990-10-16 Akademie Der Wissenschaften Der Ddr Fluorinated nucleosides and method for treating retrovirus infections therewith
US4921794A (en) * 1987-01-14 1990-05-01 President And Fellows Of Harvard College T7 DNA polymerase
US5002868A (en) * 1988-07-20 1991-03-26 Atom Sciences, Inc. DNA sequencing process using stable isotopes
US4990499A (en) * 1989-09-29 1991-02-05 University Of Saskatchewan Anti-herpes simplex virus activity of 5-alkoxymethyl-2'-deoxycytidimes and their 5-monophosphates

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See also references of EP0506944A4 *

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0771359A1 (fr) * 1992-06-22 1997-05-07 City Of Hope Structure d'ensembles a base de nucleoproteines comprenant des composants adressables pour ensemble a nano-echelle et nanoprocesseurs
EP0771359A4 (fr) * 1992-06-22 2000-11-15 Hope City Structure d'ensembles a base de nucleoproteines comprenant des composants adressables pour ensemble a nano-echelle et nanoprocesseurs
US6184211B1 (en) 1993-11-30 2001-02-06 Methylgene Inc. Inhibition of DNA methyltransferase
WO1995015373A3 (fr) * 1993-11-30 1995-08-10 Univ Mcgill Inhibition de la methyltransferase de l'adn
WO1995015373A2 (fr) * 1993-11-30 1995-06-08 Mcgill University Inhibition de la methyltransferase de l'adn
EP1352956A1 (fr) * 1993-11-30 2003-10-15 McGILL UNIVERSITY Inhbition d' une méthyltransferase d' ADN
WO1995015378A1 (fr) * 1993-12-01 1995-06-08 Hybridon, Inc. Oligonucleotides antisens inhibant l'oncogenicite
US5919772A (en) * 1993-12-01 1999-07-06 Mcgill University Antisense oligonucleotides having tumorigenicity-inhibiting activity
EP0756008A2 (fr) * 1995-07-28 1997-01-29 Health Research, Inc. Substrat pour la détection de 5-C-ADN méthyltransférase de mammifères
EP0756008A3 (fr) * 1995-07-28 1998-10-07 Health Research, Inc. Substrat pour la détection de 5-C-ADN méthyltransférase de mammifères
WO1997044346A2 (fr) * 1996-05-22 1997-11-27 Mcgill University Inhibiteurs specifiques d'adn metyltransferase
US6268137B1 (en) 1996-05-22 2001-07-31 Methylgene, Inc. Specific inhibitors of DNA methyl transferase
WO1997044346A3 (fr) * 1996-05-22 1998-02-05 Univ Mcgill Inhibiteurs specifiques d'adn metyltransferase
US7125857B2 (en) 1997-08-29 2006-10-24 The Regents Of The University Of California Modulators of DNA cytosine-5 methyltransferase and methods for use thereof
US7138384B1 (en) 1997-08-29 2006-11-21 The Regents Of The University Of California Modulators of DNA cytosine-5 methyltransferase and methods for use thereof
US7342108B2 (en) 1998-06-25 2008-03-11 The General Hospital Corporation De novo DNA cytosine methyltransferase genes, polypeptides and uses thereof
US7368551B2 (en) 1998-06-25 2008-05-06 The General Hospital Corporation De novo DNA cytosine methyltransferase genes, polypeptides and uses thereof
WO2002103052A1 (fr) * 2001-06-15 2002-12-27 Quiatech Ab Sonde amplifiable
US7118864B2 (en) 2001-06-15 2006-10-10 Quiatech Ab Amplifiable probe
WO2003031648A2 (fr) * 2001-10-05 2003-04-17 Epigenomics Ag Procede pour detecter la methylation de l'adn au moyen d'analogues marques de la s-adenosylmethionine
WO2003031648A3 (fr) * 2001-10-05 2003-10-30 Epigenomics Ag Procede pour detecter la methylation de l'adn au moyen d'analogues marques de la s-adenosylmethionine
US7670777B2 (en) 2001-10-05 2010-03-02 Epigenomics Ag Method for detecting DNA methylation using labelled S-adenosylmethionine analogs

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AU8953891A (en) 1992-05-20
CA2072083A1 (fr) 1992-04-24
EP0506944A1 (fr) 1992-10-07
AU648851B2 (en) 1994-05-05
EP0506944A4 (en) 1994-08-17

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