Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Chemical aftertreatment – e.g. – acylation – methylation – etc.
Reexamination Certificate
1998-08-13
2003-07-29
Kunz, Gary L. (Department: 1647)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Chemical aftertreatment, e.g., acylation, methylation, etc.
C530S300000
Reexamination Certificate
active
06600017
ABSTRACT:
BACKGROUND OF THE INVENTION
In general, the invention features fluorescent A&bgr; peptides and methods for their use.
The extracellular deposition of &bgr;-amyloid in senile plaques is one of the neuropathological hallmarks of Alzheimer disease (AD). The major constitutive component of amyloid plaques is the A&bgr; peptide, a 39 to 43 residue polypeptide (Selkoe et al., (1986)
J. Neurochem.
46:1820-1834) that is proteolytically derived from the amyloid precursor protein (APP), a much larger type I transmembrane protein. A&bgr; is folded into the &bgr;-sheet structure that is characteristic of amyloid fibrils.
Amyloid plaque formation likely involves two basic steps: the initial formation of a seeding aggregate that establishes the amyloid fibril lattice (Kirschner et al., (1987)
Proc. Natl. Acad. Sci. USA
84:6953-6957), followed by the elongation of the fibril by the sequential addition of subunits (Maggio et al., (1992)
Proc. Natl. Acad. Sci. USA
89:5462-5466). Some of the key parameters that promote the assembly of amyloid fibril include high peptide concentration, long incubation time, low pH (pH 5-6) (Barrow et al., (1991)
Science
253:179-182; Burdick et al., (1992)
J. Biol. Chem.
267:546-554; and Fraser et al., (1991)
Biophys. J.
60:1190-1201), solvent composition (Shen and Murphy, (1995)
Biophisical J.
69:640-651), and salt concentration (Hilbich et al., (1991)
J. Mol. Biol.
218:149-163). Assembly of A&bgr; into the fibrils may also be promoted by molecules that interact with A&bgr; and increase its rate of aggregation in vitro including ApoE (Strittmatter et al., (1993)
Proc. Natl. Acad. Sci. USA
90:8098-8102; and Wisniewski and Frangione, (1992)
Neurosci. Lett.
135:235-238), &agr;1-antichymotrypsin (Abraham et al., (1988)
Cell
52:487-501), complement C1q (Rogers et al., (1992)
Proc. Natl. Acad. Sci. U.S.A.
89:10016-10020), heparin sulfate proteoglycan (Snow et al., (1988)
Am. J. Pathol.
133:456-463), and zinc ions (Bush et al., (1994)
J. Biol. Chem.
269:2152-12158; and Bush et al., (1994)
Science
265:1464-1467).
Although many of the parameters influencing fibril assembly have been elucidated, relatively little is known about the structure of soluble A&bgr;. Gel filtration analysis of A&bgr; in solution reveals the presence of multiple, discrete structures that have variously been interpreted as monomer, dimer, trimer, and higher order aggregates (Barrow et al., (1992)
J. Mol. Biol.
225:1075-1093; Bush et al., (1994)
J. Biol. Chem.
269:2152-12158; Hilbich et al., (1991)
J. Mol. Biol.
218:149-163; Soreghan et al., (1994)
J. Biol. Chem.
269:28551-28554; and Zagorski and Barrow, (1992)
Biochemistry
31:5621-5631). The oligomeric structure depends on the concentration of the peptide, time of incubation, and the length of its carboxyl terminus (Soreghan et al., (1994)
J. Biol. Chem.
269:28551-28554).
SUMMARY OF THE INVENTION
In general, the invention features a composition that includes an aggregating amyloid A&bgr; peptide to which is covalently bonded a fluorescent label. In one preferred embodiment, the fluorescent label is covalently bonded to a cysteine amino acid. The invention also features a method for generating such a preferred aggregating amyloid A&bgr; peptide. The method involves (a) generating an amyloid A&bgr; peptide including a cysteine amino acid substitution; (b) covalently bonding a fluorescent label to the peptide at the cysteine amino acid; and (c) determining whether the peptide is capable of aggregating with another A&bgr; peptide.
In other preferred embodiments, the wild type amyloid A&bgr; peptide is a human A&bgr; peptide; the amyloid A&bgr; peptide has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; the cysteine amino acid of the peptide replaces an amino acid in a wild type A&bgr; peptide and, for example, replaces an internal amino acid or a hydrophobic amino acid of the peptide. Preferred aggregating A&bgr; peptides include, without limitation, those having the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, except that a cysteine replaces either a phenylalanine at position 4, an aspartic acid at position 7, a glycine at position 25, or a leucine at position 34. Preferred fluorescent labels include any thiol-reactive fluorescent dyes (for example, 5-(2-iodoacetyl)amino)ethyl)aminonapthylene-1-sulfonic acid (1,5-IEDANS) or fluorescein) or any of the light-emitting moieties chosen from the group consisting of dipyrromethene boron fluoride (Bodipy), fluorescein thiosemicarbazide (FTC), sulforhodamine 101 acid chloride (Texas Red), phycoerythrin, rhodamine, carboxytetramethylrhodamine, 4,6-diamidino-2-phenylindole (DAPI), an indopyras dye, pyrenyloxytrisulfonic acid (Cascade Blue), 514 carboxylic acid (Oregon Green), eosin, erythrosin, pyridyloxazole, benzoxadiazole, aminonapthalene, pyrene, maleimide, a coumarin, 4-fluoro-7-nitrobenofurazan (NBD), 4-amino-N-[3-(vinylsulfonyl)-phenyl]naphthalimide-3,6-disulfonate) (Lucifer Yellow), propidium iodide, a porphyrin, a cyanine dye (CY
3
, CY
5
, CY
9
), a lanthanide cryptate, a lanthanide chelate, or a derivative or analog thereof.
The aggregating A&bgr; peptides of the invention are useful in methods for detecting or monitoring A&bgr; production, accumulation, aggregation, or disaggregation. One particular method for the detection of an amyloid aggregate (for example, an amyloid plaque) involves (a) contacting the sample with an aggregating amyloid A&bgr; peptide to which is covalently bonded a fluorescent label; and (b) detecting the fluorescent label in association with the sample as an indication of an amyloid aggregate.
In preferred embodiments, this method is carried out to diagnose Alzheimer's disease or a predisposition thereto; the fluorescent label is covalently bonded to a cysteine amino acid; the cysteine amino acid replaces an amino acid in a wild type A&bgr; peptide; the cysteine amino acid replaces a hydrophobic amino acid or an internal amino acid in a wild type amyloid A&bgr; peptide; the aggregating A&bgr; peptide is chosen, without limitation, from peptides having the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, except that a cysteine replaces either a phenylalanine at position 4, an aspartic acid at position 7, a glycine at position 25, or a leucine at position 34; and the fluorescent label is a thiol-reactive fluorescent dye (for example, 5-(2-((iodoacetyl)amino)ethyl)aminoapthylene-1-sulfonic acid (1,5-IEDANS) or fluorescein) or is chosen from the light-emitting moieties, dipyrromethene boron fluoride (Bodipy), fluorescein thiosemicarbazide (FTC), sulforhodamine 101 acid chloride (Texas Red), phycoerythrin, rhodamine, carboxytetramethylrhodamine, 4,6-diamidino-2-phenylindole (DAPI), an indopyras dye, pyrenyloxytrisulfonic acid (Cascade Blue), 514 carboxylic acid (Oregon Green), eosin, erythrosin, pyridyloxazole, benzoxadiazole, aminonapthalene, pyrene, maleimide, a coumarin, 4-fluoro-7-nitrobenofurazan (NBD), 4-amino-N-[3-(vinylsulfonyl)-phenyl]naphthalimide-3,6-disulfonate) (Lucifer Yellow), propidium iodide, a porphyrin, a cyanine dye (CY
3
, CY
5
, CY
9
), a lanthanide cryptate, a lanthanide chelate, or a derivative or analog thereof.
The aggregating A&bgr; peptides of the invention also find use in screens for identifying compounds capable of affecting the aggregation of A&bgr; amyloid peptide. One particular method involves (a) providing a sample of A&bgr; amyloid peptide; (b) contacting the sample with (i) an aggregating amyloid A&bgr; peptide to which is covalently bonded a fluorescent label; and (ii) a candidate compound; and (c) measuring association of the fluorescent label with the sample, a change in the level of fluorescent label found in association with the sample relative to a control sample lacking the candidate compound being an indication that the compound is capable of affecting A&bgr; amyloid peptide aggregation. In a preferred embodiment of this method, the sample includes unlabeled A&bgr; amyloid peptide bound to a solid support, and the aggregation is measured by association of the fluorescen
Glabe Charles
Rodriguez William Garzon
Clark & Elbing LLP
Kunz Gary L.
The Regents of the University of California
Turner Sharon
LandOfFree
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