Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1998-09-09
2000-12-12
Riley, Jezia
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
435 6, 536 221, 536 64, C12Q 168, C07H 1900, C07H 2100, C07H 2102, C07H 2104, C07H 1524
Patent
active
061601020
DESCRIPTION:
BRIEF SUMMARY
1. FIELD OF THE INVENTION
The present invention refers to conjugates wherein a natural or modified oligonucleotide, capable of forming a specific triple-helix with a DNA-chain, is linked, via an appropriated linker, to the aglycone moiety of an anthracycline or to an anthracyclinone and to their use for the specific control of gene expression.
2. STATE OF THE ART
It is known [Claude Helene et al. Biochimica et Biophysica Acta 1049, 99-125 (1990); Uhlmann E. et al., Chemical Reviews, 90, 543-584 (1990)] that synthetic oligonucleotides can be used for selectively inhibiting the expression of a gene by binding to the mRNA (antisense mechanism) or to the DNA (antigene mechanism). In the first case the oligonucleotide binds, via hydrogen bonds, to a Watson-Crick complementary sequence, present on the mRNA-target and interfers with the synthesis of the corresponding protein. In the second case the oligonucleotide recognises, and binds only to a polypurine:polypyrimidine region of double-stranded DNA. The synthetic oligomer, which places itself in the major groove of the DNA target, leads to the formation of a triple-helix segment via Hoogsteen or reverse-Hoogsteen hydrogen bonds with the purine strand. The presence of the triple-helix structure can interfere with the replication and transcription of the gene through various mechanisms. In order to have a permanent effect it is necessary that the structure of the triple-helix be thermodinamically stable. Such stability can be increased by an appropriate molecule (intercalator) added to one or both ends of the oligonucleotide [Nguyen T. Thuong et al. Angew. Chem. Int. Ed. Engl., 32, 666-690 (1993)]. Among the intercalating molecules daunorubicin was already investigated [V. F. Zarytova et al. Nucleosides and Nucleotides, 10, 575-577 (1991); U. Asseline et al. Tetrahedron, 48, 1233-1254 (1992)] but the results obtained are considered not satisfactory, since the increase in stability of the so obtained triple-helix complex is similar to that shown by other conjugates already studied [T.Montaney-Garestier et al. in Molecular Basis of Specificity in Nucleic Acid-Drug Interaction, Kluver Academic Publishers, p. 275-290, Nederlands (1990)]. It is therefore clear the interest of new conjugates with molecular residue that impart a higher stability to the triple-helix structures they form.
DETAILED DESCRIPTION OF THE INVENTION
The present invention refers to new conjugates wherein a natural or synthetically modified oligonucleotide, capable of forming a stable triple-helix with specific DNA-regions, is covalently bound through an appropriated linker, to the aglycone moiety of an anthracycline or to an anthracyclinone. Surprisingly, this particular way of connecting the oligonucleotide to the intercalator is very efficient for obtaining the desired results, i.e. a higher stability compared to that of conjugates described in the art.
Oligonucleotides capable of forming a triple-helix with a DNA-chain are known and are described, for example, in: Nguyen T. Thuong et al. Angew, Chem. Int. Ed. Engl., 32, 666-690 (1993).
Among the oligonucleotides, according to the present invention the oligodeoxynucleotides (ODN) are preferred. In particular according to the invention the following ODN can be used: NO.:4 commonly used in this field [see, for example: Nguyen T. Thuong et al. Angew. Chem. Int. Ed. Engl., 32, 666-690 (1993); D. J. Kessler et al. Nucleic Acids Res., 21, 4810-4815 (1993)].
In particular the following linkers were used: --CH.sub.2 --CH.sub.2 --O).sub.p --, [(CH.sub.2).sub.2-5 --NH].sub.q --, [(CH.sub.2).sub.2-5 --S(O).sub.2 ].sub.q wherein n is an integer from 4 to 30, p is an integer from 1 to 6, m and q, same or different, are an integer from 2 to 9. Among useful anthracyclines, according to the invention, are, for example, the natural or synthetic anthracyclines having a free OH-group in position 4 and/or 6. In particular: daunorubicin, doxorubicin, carminomycin (or the corresponding aglycones), the corresponding 5-imino derivatives and the corresponding 3'
REFERENCES:
Biochimica et Biophysica Acta, 1049 (1990) 99-125, "Specific Regulation of Gene Expression by Antisense, Sense and Antigene Nucleic Acids", Claude Helene and Jean-Jacques Toulme.
Chemical Reviews, 1990, vol. 90, No. 4, "Antisense Oligonucleotides: A New Therapeutic Principle", Eugen Uhlmann and Anusch Peyman.
Angew. Chem. Int. Ed. Engl. 1993, 32, 666-690, "Sequence-Specific Recognition and Modification of Double-Helical DNA by Oligonucleotides", Nguyen T. Thuong and Claude Helene.
Nucleodies & Nucleotides, 10(103), 575-577 (1991), "Synthesis and Properties of Daunomycin Mono- and Oligonucleotide Derivatives", Zarytova V.F., Godovikova T.S., Maltseva T.V. Sergeyev D.S.
Tetrahedron, vol. 48, No. 7, pp. 1233-1254, 1992, "Solid-Phase Preparation of 5',3'-Heterobifunctional Oligodoxyribonucleotides Using Modified Solid Supports", Ulysse Asseline et al.
Molecular Basis of Specificity in Nucleic Acid-Drug Interactions, 275-290, 1990, "Design of Bifuctional Nucleic Acid Ligands", T. Montenay-Garestier et al.
Arcamone Federico
Bonazzi Stefania
Capobianco Massimo Luigi
Garbesi Anna Maria
Giannini Giuseppe
Consiglio Nazionale Delle Ricerche
Riley Jezia
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