Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
2001-08-01
2003-04-29
McKane, Joseph K. (Department: 1626)
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
C548S518000
Reexamination Certificate
active
06555692
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the fields of molecular biology, biochemistry, and drug design. More particularly, the present invention provides synthetic polyamides containing pyrrole and imidazole amino acids which bind specific base pair sequences of double helical DNA with affinities and specificities comparable to DNA binding proteins such as the transcription factors. A series of molecular templates are described which allow for rational targeting of any predetermined DNA sequence of therapeutic potential. This non-biological approach to DNA recognition provides an underpinning for the design of synthetic cell-permeable ligands for the control of gene-expression.
BACKGROUND OF THE INVENTION
In every human cell, genetic information is stored on a string-like DNA polymer which is approximately 1 meter in length and contains 3×10
9
units of information in the form of base pairs, within which is encoded approximately 80,000 to 100,000 genes or sets of instructions. (Watson, J. D.
Gene,
135, 309-315 (1993).) The specific interaction of proteins such as transcription factors with DNA controls the regulation of genes and hence cellular processes. (Roeder, R. G.
TIBS,
9, 327-335 (1996).) A wide variety of human conditions ranging from cancer to viral infection arise from malfunctions in the biochemical machinery that regulates gene-expression. (R. Tjian,
Sci. Am.,
2, 54-61 (1995).) Designed small, molecules which target specific DNA sequences offer a potentially general approach for gene-specific regulation. (Gottesfeld, et al.
Nature Accepted. (
1997). Such molecules could be powerful therapeutics for combating life threatening diseases which result from misregulation in transcription.
Designed bifunctional small molecules which target specific DNA sequences offer a potentially general approach for gene-specific, sequence-specific, or organism specific modification, detection or capture of plasmids, genes, cDNA, cosmids, or chromosomes. More specifically, a life threatening disease may result from a single error within the 3×10
9
units of information stored within the double helix. Sequence-specific polyamides may discriminate such small errors, hence bifunctional polyamides could have broad diagnostic applications which range from determining the molecular basis of life threatening diseases to sequence-specific visualization of disease genes in living organisms.
The genetic information is in fact, stored on two stands of DNA (in antiparallel orientation) in a structure termed the double helix. The DNA double helix consists of A,T and G,C base pairs held together by specific Watson-Crick hydrogen bonds like rungs on a twisted ladder. (Dickerson, et al.
Science,
216, 475 (1982). The common B-form of DNA is characterized by a wide (12 Å) and shallow major groove and a deep and narrow (4-6 Å) minor. Individual sequences may be distinguished by the pattern of hydrogen bond donors and acceptors displayed on the edges of the base pairs. (
Principles of Nucleic Acid Structure
Sanger, W.; Springer-verlag, N.Y., 1984.) In the minor groove, the A,T base pair presents two symmetrically placed hydrogen bond acceptors in the minor groove, the purine N3 and the pyrimidine O2 atoms. The G,C base pair presents these two acceptors, but in addition presents a hydrogen bond donor, the 2-amino group of guanine (Steitz, T. A.
Quart. Rev. Biophys.
23, 205).
Small molecules isolated from natural sources which bind DNA are found to be a structurally diverse class, as evidenced by consideration of four representative molecules, chromomycin, distamycin, actinomycin D, and calicheamicin. (Gao, et al.
J. Mol. Biol.
223, 259-279. (1992); Kamitori, et al.
J. Mol. Biol.
225, 445-456 (1992); Paloma, et al.
J. Am. Chem. Soc.
116, 3697-3708 (1994); Coll, et al.
Proc. Natl. Acad. Sci. U.S.A.
84, 8385-8389 (1987.)). There is no simple natural recognition code for the readout of specific sequences of DNA.
The structures of four small molecules isolated from natural sources are shown in FIG.
1
. Among these DNA-binding molecules, distamycin is distinguished by its structural simplicity, having no chiral centers and an oligopyrrolecarboxamide core structure. (Zimmer, C.
Prog. Nucleic Acid Res. Mol. Biol.
(1975) 15, 285; Baguley, B. C.
Molecular and Cellular Biochemistry
(1982) 43, 167-181; Zimmer, et al.,
Prog. Biophy. Mol. Biol.
47, 31 (1986)). Structural studies of distamycin-DNA complexes reveal modular complexes in which adjacent pyrrolecarboxamides makes similar contacts with adjacent DNA base pairs. The relative simplicity of distamycin, with respect both to its chemical structure and its complexes with DNA, guided the initial decision to use distamycin as a basis for designed polyamides having novel DNA-binding sequence specificity. (Dervan, P. B.
Science
232, 464-471 (1986).)
A schematic representation of recognition of A,T rich sequences in the minor groove by Distamycin is shown below:
Two distinct DNA binding modes exist for Distamycin A. In the first binding mode, a single molecule of Distamycin binds in the middle of the minor groove of a 5 base pair A,T rich sequence. The amide hydrogens of the N-methylpyrrole-carboxamides form bifurcated hydrogen bonds with Adenine N3 and thymine O2 atoms on the floor of the minor groove.
10
In the second binding mode, 2 distamycin ligands form an antiparallel side-by-side dimer in the minor groove of a 5 base pair A,T rich site. (Pelton, J. G. & Wemmer, D. E. (1989)
Proc. Natl. Acad. Sci.
86, 5723-5727; Pelton, J. G. & Wemmer, D. E. (1990)
J. Am. Chem. Soc.
112, 1393-1399; Chen, et al. (1994).
Nature Struct. Biol.
1, 169-175) In the 2:1 model each polyamide subunit forms hydrogen bonds to a unique DNA strand in the minor groove.
Polyamides containing N-methylpyrrole (Py) and N-methylimidazole (Im) amino acids provide a model for the design of artificial molecules for recognition of double helical DNA. For side-by-side complexes of Py/Im-polyamides in the minor groove of DNA, the DNA binding sequence specificity depends on the sequence of side-by-side amino acid pairings. (Wade, et al. (1992).
J. Am. Chem. Soc.
114, 8783-8794; Mrksich, et al. (1992).
Proc. Natl. Acad. Sci. U.S.A.
89, 7586-7590; Wade, W. S., Mrksich, M. & Dervan, P. B. (1993); Biochemistry 32, 11385-11389 (1993)). A pairing of Im opposite Py targets a G&Circlesolid;C base pair while a pairing of Py opposite Im targets a C&Circlesolid;G base pair. A Py/Py combination, is degenerate targeting both A&Circlesolid;T and T&Circlesolid;A base pairs. Specificity for G,C base pairs results from the formation of a putative hydrogen bond between the imidazole N3 and the exocyclic amine group of guanine. Validity of the pairing rules is supported by a variety of footprinting and NMR structure studies. (Mrksich, et al.,
J. Am. Chem. Soc.,
115, 2572 (1993); Geierstanger, et al.
Science,
266, 646 (1994); Mrksich et al.,
J. Am. Chem. Soc.,
117, 3325 (1995).)
A schematic representation of the polyamide pairing rules is shown below:,
In parallel with the elucidation of the scope and limitations of the pairing rules, efforts have been made to increase the DNA-binding affinity and specificity of pyrrole-imidazole polyamides by covalently linking polyamide subunits. (Mrksich, M. & Dervan, P. B. (1993).
J. Am. Chem. Soc.
115, 9892-9899; Dwyer, et al. (1993).
J. Am. Chem. Soc.
115, 9900-9906; Mrksich, M. & Dervan, P. B. (1994).
J. Am. Chem. Soc.
116, 3663-3664; Chen, Y. H. and Lown, J. W. (1994)
J. Am. Chem. Soc.
116, 6995-7005. Chen, Y. H. and Lown, J. W.
Heterocycles
41, 1691-1707 (1995). Geierstanger, et al.,
Nature Structural Biology,
3, 321 (1996). Chen, et al.
J. Biomol. Struct. Dyn.
14, 341-355 (1996); Cho, et al.
Proc. Natl. Acad. Sci. USA,
92, 10389 (1995)). A simple hairpin polyamide motif with &ggr;-aminobutyric acid (&ggr;) serving as a turn-specific internal-guide-residue provides a synthetically accessible method of linking polyamide subunits within the 2:1 motif. The head-to-tail linked polyamide ImPyPy
California Institute of Technology
Foley & Lardner
McKane Joseph K.
Small Andrea D.
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