Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2000-05-30
2003-05-20
Carlson, Karen Cochrane (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S330000, C530S331000, C548S311100
Reexamination Certificate
active
06566336
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a compound capable of alkylating a gene, an alkylating agent using the compound, a method for controlling the expression of the gene by using the alkylating agent, and a pharmaceutical composition containing the compound.
BACKGROUND ART
The rapid progress in molecular biology sequentially elucidates that the etiology of various diseases including cancer lies in DNA mutation. Furthermore, it is believed that the human genome project will complete the determination of the whole nucleotide sequence of human DNA within two or three years. Accordingly, it is increasingly expected the development of therapeutic methods of these diseases based on molecular-level findings. However, the practical application of these approaches is barred by such a serious barrier that not any general technology has been established for regulating gene expression extracellularly. In recent years, molecules binding to specific nucleotide sequences have been designed on the basis Of the recognition of DNA molecules by antibiotics such as distamycin. Hence, it can be said that gene expression is now possibly under regulation.
X-ray crystallography and NMR have elucidated that crescent-shape antibiotics such as distamycin and netropsin bind to the minor groove of DNA via hydrogen bonds between adenine (sometimes abbreviated as A hereinbelow) and thymine (sometimes abbreviated as T hereinbelow) in a DNA sequence with abundant A-T base pairs. Various molecules have been synthetically produced by utilizing the recognition. It has been speculated so far that the antibiotics will acquire an ability to recognize guanine-cytosine base pairs (G-C; guanine is sometimes abbreviated as G hereinbelow; cytosine is sometimes abbreviated as C hereinbelow), when the pyrrole ring is modified into imidazole [M. L. Kopla, C. Yoon, D. Goodsell, P. Pjura, R. E. Dickerson, Proc. Natl. Acad. Sci. USA, 82, 1376 (1985); J. W. Lown, K. Krowickl, U. G. Bhat, A. Skorobogaty, B. Ward, J. C. Dabrowiak, Biochemistry, 25, 7408 (1986)]. Practically, it never has been so simple. Hence, numerous intriguing experimental results have been reported.
Wemmer et al. examined distamycin bound DNA in detail by NMR. Consequently, they verified that two disks of distamycin can be stacked in the DNA miner group, where it has been believed that only one disk thereof can be placed [J. G. Pelton, D. E. Wemmer, Proc. Natl. Acad. Sci. USA, 86, 5723 (1989)]. Focusing attention to the binding mode by which the intriguing results hitherto can be explained, Dervan et al. demonstrated that the nucleotide sequence of a double-stranded DNA can be recognized by a polyamide comprising a pair of methylpyrrole (sometimes abbreviated as Py hereinbelow) and methylimidazole (sometimes abbreviated as Im hereinbelow) in an anti-parallel orientation.
More specifically, they introduced a general rule that Py-Im recognizes C-G base pair; Im-Py recognizes G-C base pair; and Py-Py recognizes A-T or T-A base pair [S. White, E. E. Baird, P. B. Dervan, Chemistry & Biology, 4, 569 (1997)]. Various polyamides in hair-pin structures or cyclic polyamides have been synthetically produced, so as to introduce a covalent bond in such pairs to prevent the entropy loss during binding, thereby generating stronger binding and higher recognition potency. It is also elucidated that a hair-pin structure with a &ggr;-linker represented by the formula —NHCH
2
CH
2
CH
2
CO— particularly exerts excellent binding and recognition potencies. The structure of a complex thereof with DNA is also determined [R. P. L. de Clairac, B. H. Geierstanger, M. Marksich, P. B. Dervan, D. E. Wemmer, J. Am. Chem. Soc., 119, 7909 (1997)].
Hair-pin polyamides just composed of pyrrole and imidazole can recognize base pairs up to 7 [J. M. Turner, E. F. Baird, P. B. Dervan, ibid., 119, 7636 (1997)]. When a &bgr;-alanine pair recognizing A-T sequence is introduced into a homo-dimer system, 11 base pairs can be recognized [S. E. Swalley, E. E. Baird, D. B. Dervan, Chem. Eur. J., 3, 1600 (1997)].
Furthermore, Dervan, Gottesfeld et al. designed a polyamide capable of binding to the fourth finger in the recognition sequence of one of Zn finger proteins, namely TFIIIA, in a manner antagonistic against a minor group of sequences to potentially bind to the fourth finger and then revealed that the expression of 5S RNA could be regulated selectively at in vitro experiments [J. M. Gottesfeld, L. Neely, J. W. Trauger, E. E. Baird, P. B. Dervan, Nature, 387, 202 (1997)]. Additionally, they simultaneously demonstrated that the polyamide permeated into nuclei at in vitro experiments.
Recent attention has been drawn toward oligonucleotides and peptide nucleic acids (PNA), as tools for gene regulation. However, these molecules suffered lower cell permeability. Taking account of significant cell permeability of methylpyrrole-methylimidazole polyamides, these molecules are promising compounds as a molecule regulating gene expression, a powerful tool in molecular biology, and a human medicine.
Conventional polyamides of Py-Im series never bind to gene via a covalent bond but only recognize in the minor groove hydrogen bonds between base pairs.
DISCLOSURE OF THE INVENTION
The present invention provides a compound recognizing the minor group of hydrogen bonds between base pairs and being capable of forming a covalent bond with bases. The inventive compound can recognize a specific nucleotide sequence and can strongly bind to adjacent bases via covalent bonding, to regulate the expression of a DNA with the nucleotide sequence.
The invention relates to a compound represented by the general formula I:
wherein
R represents a lower amyl group or a polyamide group;
and X represents nitrogen or CH;
wherein R represents a lower alkyl group or a polyamide group;
and X represents nitrogen or CH.
The invention relates to an alkylating agent of gene, comprising a compound represented by the general formula I. More specifically, the inventive alkylating agent recognizes a specific nucleotide sequence in a gene, such as W-W-V wherein V represents A or G; and W represents A or T or U (abbreviation of uracil), or G-W-V wherein V represents A or G; and W represents A or T or U, thereby selectively alkylating the sequence.
Furthermore, the invention relates to a method for regulating the expression of a gene region including a specific nucleotide sequence, by using the alkylating agent.
Still furthermore, the invention relates to a pharmaceutical composition for the treatment and prophylaxis of various gene-inducible diseases, the composition containing a compound represented by the general formula I.
REFERENCES:
Gomi et al., Japanese Journal of Cancer Research (1992) 83:113-120.*
Lehninger, Principles of Biochemistry, 2d Ed., Worth Publishers, NY (1993) p. 974.*
Fujiwara et al., “Sequence Selective DNA Alkylation by Duocarmycin Derivatives Using Molecular Recognition of Pyrrole-imidazole Polyamide”, Nucleic Acids Symposium Series, 1998, No. 39, pp. 101-102.
Sugiyama et al., “Distamycin A Modulates the Sequence Specificity of DNA Alkylation by Duocarmycin A”, Proceedings of the National Academy of Science of the United States of America, 1996, vol. 93, No. 25, pp. 14405-14410.
Isomura et al., “Efficient Guanine Alkylation Through Cooperative Heterodimeric Formation of Duocarmycin A and Distamycin”, Nucliec Acids Symposium Series, 1995, No. 34, pp. 47-48.
Okamoto et al., “Differential Effect of Duocarmycin A and Its novel Derivative DU-86 on DNA Strand Breaks in HeLa S3 Cells”, Japanese Journal of Cancer Research, 1994, vol. 85, No. 12, p. 1304-1311.
Yamamoto et al., “Concerted DNA Recognition and Novel Site-Specific Alkylation by Duocarmycin A with Distamicin A”, Biochemistry, 1993, vol. 32, No. 4, pp. 1059-1066.
Sugiyama et al., “A Novel Guanine N3 Alkylation by Antitumor Antibiotic Duocarmycin A”, Tetrahedron Letters, 1993, vol. 34, No. 13, p. 2179-2182.
Asai et al., A Novel Property of Duocarmycin and Its Analogues for Co
Saito Isao
Sugiyama Hiroshi
Tao Zhi Fu
Carlson Karen Cochrane
Corless Peter F.
Edwards & Angell LLP
Japan Science and Technology Corporation
O'Day Christine C.
LandOfFree
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