Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Having -c- – wherein x is chalcogen – bonded directly to...
Reexamination Certificate
2000-12-20
2003-09-02
McKane, Joseph K. (Department: 1626)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Having -c-, wherein x is chalcogen, bonded directly to...
C548S304700, C549S472000
Reexamination Certificate
active
06613787
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to novel compounds that recognize mixed sequences (i.e., GC as well as AT base pairs), and specifically bind the DNA minor groove through dimer formation.
BACKGROUND OF THE INVENTION
Design and discovery of molecules that can regulate gene expression in cells in a desirable and predictable manner is a central goal of research at the interface of chemistry and biology. See, e.g., Schreiber, S. L.,
Bioorg. Med. Chem.
6, 1127-1152 (1998); C. Denison and T. Kodadek,
Chem. Biol.
5, R129-R145 (1998); A. G. Papavassiliou,
Molecular Medicine Today
358-366 (1998); R. E. Bremer, et al.,
Chem. Biol.
5, 119-133 (1998); J. Gottesfeld et al.,
Nature
387, 202-205 (1997); H. Iida,
Current Opinion Biotechnology
10, 29-33 (1999). The developing field of “chemical genetics” requires molecules that have the necessary selectivity to recognize target genes. See, e.g., S. Schreiber, supra, and Schreiber, S.,
FASEB J.
11, p.M1 (1997).
A number of aromatic diamidines have been shown to bind to the minor-groove of DNA, and to exhibit useful antimicrobial activity. Various hypotheses of the mode of antimicrobial action of the aryl amidines have been proposed. However, evidence is growing that these compounds function by complex formation with DNA and subsequent selective inhibition of DNA dependent microbial enzymes. Intervention in transcription control has been demonstrated and seems to be a plausible mode of action for structurally diverse minor groove binders. B. P. Das, et al.,
J. Med. Chem.
20, 531-536 (1977); D. W. Boykin, et al.,
J. Med. Chem.
36, 912-916 (1995); A. Kumar et al.,
Eur. J. Med. Chem.
31, 767-773 (1996); R. J. Lombardy, et al.,
J. Med. Chem.
31, 912-916 (1996); R R. Tidwell. et al.,
Antimicrob. Agents Chemother.
37, 1713-1716 (1993); R. R. Tidwell, R. R. and C. A. Bell, “Pentamidine and Related Compounds in Treatment of
Pneumocystis carinii
Infection,” in
Pneumocystis carinii
, (Marcel Decker; New York, 561-583 (1993)); D. Henderson, and L. H. Hurley,
Nature Med.
1, 525-527 (1995); J. Mote, Jr., et al.,
J. Mol. Biol.
226, 725-737 (1994); and D. W. Boykin, et al.,
J. Med. Chem.
41, 124-129 (1998).
Organic cations that bind in the DNA minor groove also have biological activities that range from anti-opportunistic infection to anticancer properties. See e.g., C. Bailly, in
Advances in DNA Sequence
-
Specific Agents,
Vol. 3, pp. 97-156 (L. H. Hurley, Ed. JAI Press Inc., London, UK, 1998); J. A. Mountzouris and L. H. Hurley, in
Bioorganic Chemistry: Nucleic Acids,
pp. 288-323, (S. M. Hecht, Ed., Oxford Univ. Press, New York, 1996); E. Hildebrant, et al.,
J. Euk. Microbiol.
45, 112 (1998); and K. Hopkins et al.,
J. Med. Chem.
41, 3872 (1998). Such compounds have provided a wealth of fundamental information about nucleic acid recognition properties, and they continue to be important models in the study of nucleic acid complexes.
The DNA minor-groove and AT sequence recognition properties of molecules of this series have been probed extensively for more than 30 years. See, e.g., C. Zimmer and U. Wahnert,
Prog. Biophys. Mol. Biol.
47, 31 (1986); B. H. Geierstanger and D. E. Wemmer,
Annu. Rev. Biophys. Biomol. Struct.
24, 463 (1995); W. D. Wilson, in
Nucleic Acids in Chemistry and Biology,
Chapter 8 (G. M. Blackburn and M. J. Gait, Eds., IRL Press, Oxford, U.K., 1996). The compound netropsin (see
FIG. 1
) was the first minor groove-binding compound crystallized with a B-form DNA, and the structure of the complex provided clear suggestions about the molecular basis for AT base pair sequence-specific recognition. M. L. Kopka, et al.,
Proc. Natl. Acad. Sci.
82, 1376 (1985). The structure of netropsin also led to the development of minor-groove binding netropsin analogs, the lexitropsins, that could specifically recognize GC base pairs and could thus have extended sequence recognition capability. See, J. W. Lown et al.,
Biochemistry
25, 7408 (1986); M. L. Kopka and T. A. Larsen, in
Nucleic Acid Targeted Drug Design,
pp. 303-374C (L. Probst and T. J. Perun, Eds., Marcel Dekker Inc., New York, 1992); and M. L. Kopka et al.,
Structure
5, 1033 (1997). Initial efforts in the design of such analogs did provide compounds with enhanced recognition of GC base pairs, but unfortunately, the specificity obtained was not significant. A breakthrough in this area occurred with the discovery that the monocationic compound distamycin (
FIG. 1
) could bind into the minor groove of some AT sequences of DNA as a stacked, antiparallel dimer. See J. G. Pelton and D. E. Wemmer,
Proc. Natl. Acad. Sci.
86, 5723 (1989), and J. G. Pelton and D. E. Wemmer,
J. Am. Chem. Soc.
112, 1393 (1990).
One of the early recognition principles for AT sequences was the fact that the minor groove is narrower in AT than in GC regions, and it is perhaps the most surprising feature of the dimer complex that the minor groove in B-form DNA can readily expand to the width required for dimer binding. The expansion of the groove not only allows the dimer to bind but also provides for recognition of both strands in the duplex through complementary strand recognition by the two molecules of the dimer. Replacement of pyrrole group in distamycin by imidazole provided improved GC recognition specificity with dimer complexes and current design efforts in this system have reached a high level of success. See e.g., C. L. Kielkopf, et al.,
Nature Struct. Biol.
5, 104 (1998); S. Whiteet al.,
Nature
391, 468 (1998); C. L. Kielkopf et al.,
Science
282, 111 (1998); S. E. Swalleyet al.,
J. Am. Chem. Soc
121, 1113 (1999); and D. M. Herman, et al.,
J. Am. Chem. Soc
121, 1121 (1999). With recent incorporation of hydroxypyrole groups as a recognition unit, AT and TA as well as GC and CG base pairs can now be effectively distinguished in DNA sequences by pyrrole-imidazole polyamides related to distamycin.
The pyrrole-imidazole polyamide system is the only one of the well-known minor-groove binding motifs that has been found to form the stacked-dimer recognition unit. Even netropsin, the first minor-groove binding agent to be structurally characterized in detail and a dicationic relative of the monocation distamycin (FIG.
1
), does not form a dimer recognition unit. A recent crystal structure of a 2:1 netrospin-DNA complex found that the two netropsin molecules in the complex bound in the minor groove as tandem monomer units instead of the side-by-side dimer observed with distamycin. See e.g., X. Chen, et al.,
J. Mol. Biol.
267, 1157 (1997); X. Chen, et al,
Nucleic Acids Res.
26, 5464 (1998); and X. Chen, et al,
Nature Struct. Biol.
1, 169 (1994). The two charges of netropsin as well as other minor groove agents, such as the furan derivatives shown in
FIG. 1
, have been postulated to prevent stacked-dimer formation.
Recent evidence suggests that some monocationic cyanine dyes can form an array of stacked dimers in the DNA minor groove. See J. L. Seifert, et al.,
J. Am. Chem. Soc.
(in press, 1999). There are, however, other monocationic minor-groove agents, such as Hoechst 33258 (see FIG.
1
and analogs, that apparently do not form dimer DNA recognition motifs. These results indicate that the electrostatic and stereochemical requirements for minor-groove recognition of DNA by dimers are very restrictive, and further suggest that stacked dimer formation by dications is unlikely.
SUMMARY OF THE INVENTION
The present invention is based on the inventors' surprising discovery of a new class of organic dications, based on unfused-aromatic systems, that selectively recognize mixed DNA sequences (i.e., AT as well as GC base pairs) in a manner that is very sensitive to compound structure. These are the first non-peptide compounds that have mixed-sequence recognition capability and the result is particularly promising, since similar compounds readily enter cells and have generally low toxicity. See K. Hopkins et al.,
J. Med. Chem.
41, 3872-3878 (1998). A surprising feature of this discovery is that recognition occurs through highly cooperative
Boykin David W.
Tidwell Richard R.
Wilson W. David
Jenkins & Wilson & Taylor, P.A.
McKane Joseph K.
Small Andrea D.
The University of North Carolina at Chapel Hill
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