Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2001-01-26
2003-05-06
Riley, Jezia (Department: 1655)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S002600, C514S04400A, C435S006120, C536S023100, C536S024300, C536S025300
Reexamination Certificate
active
06559125
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to polyamide-alkylator conjugates and related products, including methods of using such conjugates as well as methods relating to their synthesis.
BACKGROUND OF THE INVENTION
None of the following discussion of the background of the invention, which is provided solely to aid the reader in understanding the invention, is admitted to be or to describe prior art to the invention.
The design of synthetic ligands capable of reading information stored in the DNA double helix has been a long-standing goal of chemistry and molecular biology. Cell-permeable small molecules, which target predetermined nucleotide sequences in double-stranded nucleic acids, particularly double-stranded DNA (dsDNA), are useful in regulating, or modulating, gene-expression. Small molecules specifically targeted to any predetermined DNA sequence would be useful tools in molecular biology and potentially in human medicine.
Oligodeoxynucleotides that recognize the major groove of double-helical DNA via triple-helix formation bind to a broad range of sequences with high affinity and specificity. See Moser, et al.,
Science, vol.
238:645-650 (1987), Duvaivalentin, et al.,
Proc. Nat'l Acad. Sci. USA, vol.
89:504-508 (1992), Maher, et al.,
Biochemistry, vol.
31:70-81 (1992). Although oligonucleotides and their analogs have been shown to interfere with gene expression, the triple helix approach so far has been limited to purine tracks and suffers from poor cellular uptake.
Another recent approach to targeting specific nucleotide sequences in dsDNA has involved molecules known as “polyamides.” See U.S. Ser. No. 08/607,078, PCT/US97/03332, U.S. Ser. Nos. 08/837,524, 08/853,525, PCT/US97/12733, U.S. Ser. No. 08/853,522, PCT/US97/12722, PCT/US98/06997, PCT/US98/02444, PCT/US98/02684, PCT/US98/01006, PCT/US98/03829, and PCT/US98/0714 all of which are incorporated herein by reference in their entirety, including any drawings. As described in the foregoing references, polyamides comprise polymers of amino acids covalently linked by amide bonds. Preferably, the amino acids used to form these polymers include N-methylpyrrole (Py) and N-methylimidazole (Im). Polyamides containing pyrrole (Py), and imidazole (Im) amino acids are synthetic ligands that have an affinity and specificity for DNA comparable to naturally occurring DNA binding proteins Trauger, J. W., Baird, E. E. & Dervan, P. B. (1996),
Nature
382, 559-561; Swalley, S. E., Baird, E. E. & Dervan, P. B. (1997),
J. Am. Chem. Soc.
119, 6953-6961; Turner, J. M., Baird, E. E. & Dervan, P. B. (1997),
J. Am. Chem. Soc.
119, 7636-7644; Trauger, J. W., Baird, E. E. & Dervan, P. B. (1998),
Angewandte Chemie
-
International Edition
37, 1421-1423; and Dervan, P. B. & Burli, R. W. (1999),
Current Opinion in Chemical Biology
3, 688-693.
The particular order of amino acids in such polyamides, and their pairing in dimeric, antiparallel complexes formed by association of two polyamide polymers, determines the sequence of nucleotides in dsDNA with which the polymers preferably associate. The development of pairing rules for minor groove binding polyamides derived from N-methylpyrrole (Py) and N-methylimidazole (Im) amino acids provided a useful code to control target nucleotide base pair sequence specificity. Specifically, an Im/Py pair in adjacent polymers was found to distinguish G•C from C•G and both of these from A•T or T•A base pairs. A Py/Py pair was found to specify A•T from G•C but could not distinguish A•T from T•A. More recently, it has been discovered that inclusion of a new aromatic amino acid, 3-hydroxy-N-methylpyrrole (Hp) (made by replacing a single hydrogen atom in Py with a hydroxy group), in a polyamide and paired opposite Py enables A•T to be discriminated from T•A by an order of magnitude. Utilizing Hp together with Py and Im in polyamides provides a code to distinguish all four Watson-Crick base pairs (i.e.. A•T, T•A, G•C, and C•G) in the minor groove of dsDNA, as described in Table 1.
TABLE 1
Pairing Code for Minor Groove Recognition
Pair
G · C
C · G
T · A
A · T
Im/Py
+
−
−
−
Py/Im
−
+
−
−
Hp/Py
−
−
+
−
Py/Hp
−
−
−
+
Favored (+), disfavored (−)
As discussed above, a number of different polyamide motifs have been reported in the literature, including “hairpins,” “H-pins,” “overlapped,” “slipped,” and “extended” polyamide motifs. Specifically, hairpin polyamides are those wherein the carboxy terminus of one amino acid polymer is linked via a linker molecule, typically aminobutyric acid or a derivative thereof to the amino terminus of the second polymer portion of the polyamide. Indeed, the linker amino acid &ggr;-aminobutyric acid (&ggr;), when used to connect first and second polyamide polymer portions, or polyamide subunits, C→N in a “hairpin motif,” enables construction of polyamides that bind to predetermined target sites in dsDNA with more than 100-fold enhanced affinity relative to unlinked polyamide subunits. See, for example, Turner, et al.,
J. Am. Chem. Soc., vol.
119:7636-7644 (1997), Trauger, et al.,
Angew. Chemie. Int. Ed. Eng., vol.
37: 1421-1423 (1997), Turner, et al.,
J. Am. Chem. Soc., vol.
120:6219-6226 (1998), and Trauger, et al.,
J. Am. Chem. Soc., vol.
120:3534-3535 (1998). Paired &bgr;-alanine residues (&bgr;/&bgr;), restore the curvature of the dimer for recognition of larger binding sites and in addition, code for AT/TA base pairs Trauger, J. W., Baird, E. E., Mrksich, M. & Dervan, P. B. (1996),
J. Am. Chem. Soc.
118, 6160-6166; Swalley, S. E., Baird, E. E. & Dervan, P. B. (1997),
Chem.
-
Eur. J.
3, 1600-1607; and Trauger, J. W., Baird, E. E. & Dervan, P. B. (1998),
J. Am. Chem. Soc.
120, 3534-3535. Eight ring hairpin polyamides can bind a 6 base pair match sequence at subnanomolar concentrations with good sensitivity to mismatch sequences. Dervan, P. B. et al.
Curr. Opin. Chem. Biol.
1999, 3,688-693. Moreover, eight-ring hairpin polyamides (comprised of two four amino acid polymer portions linked C→N) have been found to regulate transcription and permeate a variety of cell types in culture. See Gottesfield, J. M.; et al.,
Nature, vol.
387:202-205 (1997).
An H-pin polyamide motif, i.e., wherein two paired, antiparallel polyamide subunits are linked by a linker covalently attached to an internal polyamide pair, have also been reported. Another polyamide motif that can be formed between linked or unlinked polyamide subunits is an “extended” motif, wherein one of the polyamide subunits comprises more amino acids than the other, and thus has a single-stranded region. See U.S. Ser. No. 08/607,078. In contrast, an “overlapped” polyamide is one wherein the antiparallel polyamide subunits completely overlap, whereas in a “slipped” binding motif, the two subunits overlap only partially, with the C-terminal portions not associating with the N-terminal regions of the other subunit. See U.S. Ser. No. 08/607,078.
DNA alkylation by Duocarmycin A segment—hairpin polyamide conjugates is described in Tao et al., J. Am. Chem. Soc. 1999, 121:4961-4967. Alkylation by one such conjugate was reported to proceed with efficiency at nanomolar concentration against a particular DNA sequence. Other Duocarmycin A experiments we reported in Fujiwara, et al. J. Am. Chem. Soc. 1999, 121:7706-7702 and Tao, et al. Agnew. Chem. Int. Ed. 1999, 38; No. 5, pages 650-653.
SUMMARY OF THE INVENTION
The present invention is based on the surprising and unexpected discovery of new and useful polyamide-alkylator conjugates. As a result of their DNA binding properties, polyamides deliver reactive moieties for covalent reaction at specific DNA sequences and thereby inhibit DNA-protein interactions. This site specific alkylation of DNA is a useful tool to regulate gene expression. In addition to competing with transcription factors or promoters, the conjugates of the present invention will be used to target a gene's co
Chang Aileen
Dervan Peter B.
Wurtz Nicholas
California Institute of Technology
Foley & Lardner
Riley Jezia
Whittaker Michael A.
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