Specific inhibitors of DNA methyl transferase

Details Specific inhibitors of DNA methyl transferase Specific inhibitors of DNA methyl transferase

1998-12-08

2001-07-31

Schwartzman, Robert A. (Department: 1635)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S375000, C435S455000, C536S023100, C536S024100, C536S024500

Reexamination Certificate

active

06268137

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to modulation of gene expression. In particular, the invention relates to modulation of gene expression of the gene encoding DNA methyltransferase, and to modulation of gene expression that is regulated by the enzyme DNA methyltransferase.
2. Summary of the Related Art
Modulation of gene expression has become an increasingly important approach to understanding various cellular processes and their underlying biochemical pathways. Such understanding enriches scientific knowledge and helps lead to new discoveries of how aberrancies in such pathways can lead to serious disease states. Ultimately, such discoveries can lead to the development of effective therapeutic treatments for these diseases.
One type of cellular process that is of particular interest is how the cell regulates the expression of its genes. Aberrant gene expression appears to be responsible for a wide variety of inherited genetic disorders, and has also been implicated in numerous cancers and other diseases. Regulation of gene expression is a complex process, and many aspects of this process remain to be understood. One of the mysteries of this process resides in the fact that while the genetic information is the same in all tissues that constitute a multicellular organism, the expression of functions encoded by the genome varies significantly in different tissues.
In some cases, tissue-specific transcription factors are known to play a role in this phenomenon. (See Maniatis et al., Science 236: 1237-1245 (1987); Ingarham et al., Annual Review of Physiology 52: 773-791 (1990). However, several important cases exist that cannot be readily explained by the action of transcription factors alone. For example, Midgeon, Trends Genet. 10: 230-235 (1994), teaches that X-inactivation involves the inactivation of an allele of a gene that resides on the inactive X-chromosome, while the allele on the active X-chromosome continues to be expressed. In addition, Peterson and Sapienza, Annu. Rev. Genet. 27: 7-31 (1993), describes “parental imprinting”, where an allele of a gene that is inherited from one parent is active and the other allele inherited from the other parent is inactive. In both of these cases, both alleles exist in an environment containing the same transcription factors, yet one allele is expressed and the other is silent. Thus, something other than transcription factors must be involved in these phenomena.
Investigators have been probing what type of “epigenetic information” may be involved in this additional control of the expression pattern of the genome. Holliday, Philos. Trans. R. Soc. Lond. B. Biol. Sci. 326: 329-338 (1990) discusses the possible role for DNA methylation in such epigenetic inheritance. DNA contains a set of modifications that is not encoded in the genetic sequence, but is added covalently to DNA using a different enzymatic machinery. These modifications take the form of methylation at the 5 position of cytosine bases in CG dinucleotides. Numerous studies have suggested that such methylation may well be involved in regulating gene expression, but its precise role has remained elusive. For example, Lock et al., Cell 48: 39-46 (1987), raises questions about whether the timing of hypermethylation and X-inactivation is consistent with a causal role for methylation. Similarly, Bartolomei et al., Genes Dev. 7: 1663-1673 (1993) and Brandeis et al., EMBO J. 12: 3669-3677 (1993), disclose timing/causation questions for the role of methylation in parental imprinting.
Some of the shortcomings of existing studies of the role of DNA methylation in gene expression reside in the tools that are currently available for conducting the studies. Many studies have employed 5-azaC to inhibit DNA methylation. However, 5-azaC is a nucleoside analog that has multiple effects on cellular mechanisms other than DNA methylation, thus making it difficult to interpret data obtained from these studies. Similarly, 5-azadC forms a mechanism based inhibitor upon integration into DNA, but it can cause trapping of DNA methyltransferase (MTase) molecules on the DNA, resulting in toxicities that may obscure data interpretation.
More recently, Szyf et al., J. Biol. Chem. 267: 12831-12836 (1995), discloses a more promising approach using expression of antisense RNA complementary to the DNA MTase gene to study the effect of methylation on cancer cells. Szyf and Von Hofe, PCT/US94/13685 (1994), discloses the use of antisense oligonucleotides complementary to the DNA MTase gene to inhibit tumorigenicity. These developments have provided powerful new tools for probing the role of methylation in numerous cellular processes. In addition, they have provided promising new approaches for developing therapeutic compounds that can modulate DNA methylation. One limitation to these approaches is that their effect is not immediate, due to the half life of DNA MTase enzyme. Thus, although the expression of DNA MTase is modulated, residual DNA MTase enzyme can continue to methylate DNA until such residual enzyme is degraded. There is, therefore, a need for new inhibitors of DNA MTase enzyme which are effective at inhibiting methylation, but without the toxic side effects of the earlier mechanism-based inhibitors.
BRIEF SUMMARY OF THE INVENTION
The invention provides novel inhibitors of DNA MTase enzyme and methods for using such inhibitors as analytical and diagnostic tools, as potentiators of transgenic plant and animal studies and gene therapy approaches, and as potential therapeutic agents.
In a first aspect, the invention provides novel hairpin oligonucleotide inhibitors of DNA methyltransferase (DNA MTase) enzyme. The normal substrate for DNA MTase is a hemimethylated double stranded DNA molecule having a CG dinucleotide opposite a 5-methyl CG dinucleotide, e.g., in a hairpin forming oligonucleotide. Methylation occurs at the 5-position of the cytosine base in the CG dinucleotide. The present inventors have discovered that substitution of the CG dinucleotide with a phosphorothioate IG, UG, 5-bromocytosineG, 5-fluorocytosineG, abasicG or CG dinucleotide in a hairpin forming oligonucleotide results in a powerful mechanism-based inhibitor of DNA MTase. Thus, inhibitors according to this aspect of the invention have the general structure:
wherein each N is independently any nucleotide, n is a number from 0-20, C is 5-methylcytidine, G is guanidine, y is a number from 0-20, L is a linker, each D is a nucleotide that is complementary to an N such that Watson-Crick base pairing takes place between that D and the N such that the N
n
-C-G-N
y
and the D
n
-G-B-D
y
form a double helix, B is cytosine, inosine, uridine, 5-bromocytosine, abasic deoxyribose, or 5-fluorocytosine, dotted lines between nucleotides represent hydrogen bonding between the nucleotides, the linkage between G and B is a phosphorothioate or phosphorodithioate linkage and the total number of nucleotides ranges from about 10 to about 50. In one preferred embodiment, L is an oligonucleotide region having from 1 to about 10 nucleotides. DNA MTase inhibitors according to this aspect of the invention bind DNA MTase enzyme avidly in a noncovalent manner and inhibit DNA MTase in an S-adenosylmethionine (SAM)-independent manner.
In a second aspect, the invention provides inhibitors of DNA MTase enzyme which also inhibit-the expression of the DNA MTase gene. Inhibitors according to this aspect of the invention have the general structure:
wherein the substituents are the same as for inhibitors according to the first aspect of the invention, except that X is an antisense oligonucleotide of from about 10 to about 50 nucleotides in length, which is complementary to a portion of an RNA encoding DNA MTase enzyme, and L can optionally be X.
In a third aspect, the invention provides a diagnostic method for determining whether a particular sample of cells is cancerous. The method according to this aspect of the invention comprises preparing an extract from the cells in the cell sample, adding labeled inhibitor according t

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