Organic compounds -- part of the class 532-570 series – Organic compounds – Unsubstituted hydrocarbyl chain between the ring and the -c-...
Reexamination Certificate
2000-03-24
2002-12-17
Raymond, Richard L. (Department: 1642)
Organic compounds -- part of the class 532-570 series
Organic compounds
Unsubstituted hydrocarbyl chain between the ring and the -c-...
C560S139000, C560S153000, C560S158000, C530S317000, C530S321000, C528S271000
Reexamination Certificate
active
06495680
ABSTRACT:
BACKGROUND OF THE INVENTION
Naturally occurring membrane channels and pores are formed from a large family of diverse proteins, peptides and organic secondary metabolites whose vital biological functions include control of ion flow, signal transduction, molecular transport and production of cellular toxins. (Eisenberg, B. (1998) Ionic channels in biological membranes: natural nanotubes. Acc. Chem. Res. 31:117; Hill, B. (1992) Ionic Channels of Excitable Membranes 2
nd
edn. (Sinauer Associates, Sunderland); Gennis, R. B. (1989) Biomembranes, Molecular Structure and Function, Springer, New York). Many pore-forming peptides, such as gramicidin and alamethicin, function by creating pores within the plasma membrane of a target cell (Marsh, D. (1996) Peptide models for membrane channels. Biochem. J. 315(pt2):345; Smart, O. S.; Goodfellow, J. M.; Wallace, B. A. (1993) The pore dimensions of gramicidin A. Biophys. J. 65(6):2455; Ritov, V. B.: Tverdislova, I. L.; Avakyan, T. Yu; Menshikova, E. V.; Leikin, Yu N.; Bratkovskaya, L. B.; Shimon, R. G. (1992) Alamethicin-induced pore formation in biological membranes. Gen. Physiol. Biophys. 11(1):49). Pore-forming protein toxins, such as the
Staphylococcus aureus
&agr;-hemolysin and
Streptococcus streptolysin
-O, act similarly by boring holes into the cell membranes. (Bayley, H. (1997) Toxin structure: part of a hole? Curr. Biol. 7(12):R763; Ikigai, H.; Nakae, T. (1987) Assembly of the alpha-toxin-hexame of Staphylococcus aureus in the liposome membrane. J. Biol. Chem 262:2156; Palmer, M; Vulicevic I.; Saweljew, P.; Valeva, A.; Kehoe, M.; Bhakdi, S. (1998) Biochem. 37(8):2378. Streptolysin-O: a proposed model of allosteric interaction between a pore-forming protein and its target lipid bilayer. &agr;-Hemolysin has received intense interest as a prototype for artificial molecular gatekeepers that can be used for the design of drugs, (Bayley, H. (1997) Building doors into cells. Sci. Am. 277 (September):62); (Panchal, R. G.; Cusak, E.; Cheley, S.; Bayley, H. (1996) Turnor-protease-activated, pore-forming toxins from a combinatorial library, Nature Biotechnol 14:852) drug delivery agents (Fernandex, T.; Bayley, H. (1998) Ferrying proteins to the other side. Nature Biotechnol. 16(5):418) or highly sensitive and selective biosensors. (Braha, O.; Walker, B.; Cheley, S.; Kasianowicz, J. J.; Song, L.: Gouaux, J. E.; Bayley, H. (1997) Designed protein pores as components for biosensors. Chem. Biol. 4:497). Difficulties associated with using protein molecules in these designs include heat and mechanical instability, immunogenicity in biotherapeutics, and the like.
Lying at the center of the pore assembled from seven molecules of &agr;-hemolysin (and many other pore-forming proteins) is a nanosize channel. (Song, L; Hobaugh, M. R.; Shustak, C.; Cheley, S.; Bayley, H.; Gouaux, J. E. (1996) Structure of Staphylococcal &agr;-hemolysin, a heptameric transmembrane pore. Science 274:1859). The transmembrane segment, a &bgr;-barrel, of the channel ranges from 14 Å to 26 Å in diameter and 52 Å in length. The interior of the &bgr;-barrel was found to be primarily hydrophilic, while the exterior has a hydrophobic belt.
Despite the existence of numerous chemical models as artificial transmembrane channels, (Alkerfeldt, K. S.; Lear, J. D.; Wasserman, Z. R.; Chung, L. A.; DeGrado, W. F. (1993) Acc. Chem. Res. 26:191; Gokel, G. W.; Murillo, O. (1996) Acc. Chem. Res. 29:425) the design and synthesis of artificial systems that can mimic the biological function of natural compounds is still a formidable task. A successful model rivaling the structural robustness and versatility as observed in the natural systems has not been seen until the present invention. Such a model requires a tube-or barrel-like structure with a nanosized, hydrophilic internal cavity and a hydrophilic internal cavity and a hydrophobic outside surface.
The self-assembly of cyclic peptides (Ghadiri, M. R.; Granja, J. R.; Milligan, R. A.; McRee, D. E.; Khazanovich, H. (1993) Self-assembling organic nanotubes based on a cyclic peptide architecture. Nature 366:324) provides an example of self-assembling nanotubes. However, Ghadiri's nanotubes do not suit the above purpose since these tubes consist of stacked peptide rings. Due to the structure of these cyclic peptides and the way the tubes form, it is difficult to imagine how different molecular switches could be put site-specifically into these nanotubes.
Other approaches have been described (Bryson, J. W.; Betz, S. F; Lu, H. S.; Suich, D. J.; Zhou, H. X.; O'Neil, K. T.; DeGrado, W. F. (1995) Protein design: a hierarchic approach. Science 270:935) toward manipulating nanoscale structures by designing oligomeric bundles of &agr;-helices. Models for transmembrane helical oligomers may lead to simplified systems for designing pore-forming agents. (Dieckmann, G. R.; DeGrado, W. F. (1997) Modeling transmembrane helical oligomers Curr. Opin. Struct. Biol. 7(4):486). The advantage of these helix bundles is that they allow precise control over the positions to be modified, enabling site-specific engineering of the nanostructures with both natural and unnatural amino acids. However, one disadvantage of these designed helix bundles is that they may have the same instability and immunogenicity problems associated with natural peptides and proteins.
Until the present invention, a question that still remained was whether unnatural systems provide nanosize, tube-like structures. Various prior art approaches to folding structures have been taken, involving primarily the use of intramolecular hydrogen bonding, or donor-acceptor interactions. (Gellman, S. H. (1998) Foldamers: a manifesto. Acc. Chem. Res. 31:173) Examples of folded, potentially functionalizable structures include &bgr;-peptides and peptoid oligomers and many others involving unnatural backbones. (Appella, D. H; Christianson, L. A.; Karle, I. L; Powell, D. R.; Gellman, S. H. (1996) Beta-Peptide foldamers: robust helix formation in a new family of beta-amino acid oligomers. J. Am. Chem. Soc. 118:13071-13072; Seebach, D.; Overhand, M.; Kuhnle, F. N. M.; Martinoni, B.; Oberer, L.; Hommel, U.; Widmer, H. (1996) Beta-Peptides: synthesis by Arndt-Eistert homologation with concomitant peptide coupling. Structure determination by NMR and CD spectroscopy and by X-ray crystallography. Helical secondary structure of a &bgr;-hexapeptide in solution and its stability towards pepsin. Helv. Chim. Acta 79:913; Armand, P.; Kirshenbaum, K.; Falicov, A.; Dunbrack, R. L. Jr.; Dill, K. A.; Zuckermann, R. N.; Cohen, F. E. (1997) Chiral N-substituted glycines can form stable helical conformations. Fold. Des. 2(6):369; and others such as Cho, C. Y.; Moran, E. J.; Cherry, S. R.; Stephans, J. C.; Fodor, S. P.; Adams, C. L.; Sundaram, A.; Jacobs, J. W.; Schultz, P. G. (1993) An unnatural biopolymer, Science 261:1303). However, few examples have shown folded structures with cavities similar to those seen in protein molecules.
One approach toward building nanotubes involved designing oligomers that undergo polar solvent-driven folding (Nelson, J. C.; Saven, J. G.; Moore, J. S.; Wolynes, P. G. (1997) Solvophobically driven folding of nonbiological oligomers. Science 277:1793). While a helical conformation with nanosized tubular cavity was proposed for the folded structure, this system was, however, unsuitable for designing pore-forming agents since the interior is quite hydrophobic.
The assembly of well-defined protein secondary structures, such as &agr;-helix, &bgr;-sheet, and turns, leads to a bewildering array of tertiary structures. (Branden, C.; Tooze, J.,
Introduction to Protein Structure
, 2nd ed.; Garland Publishing: New York, 1998). As the first step toward developing artificial oligomers and polymers that fold like biomacromelecules, there is currently an intense interest in designing unnatural building blocks that adopt well-defined secondary structures. (Gellman, S. H., Acc. Chem. Res., 1998, 31, 173; Appella, D. H.; Christianson, L. A.; Karle, I. L.; Powell, D. R.; Gellman, S. H.
Emch, Schaffer, Schaub & Porcello & Co., L.P.A.
Raymond Richard L.
The University of Toledo
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