Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-11-12
2002-04-02
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
57, C530S350000
Reexamination Certificate
active
06365359
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to proteins, functionally equivalent pharmacophores and methods of creating and/or detecting inhibitors of prion formation. Specifically, the invention relates to small molecules, peptides and peptide analogs with the ability to either inhibit prion formation or replication and methods of treating a neuropathology such as a prion-mediated neuropathology.
BACKGROUND OF THE INVENTION
Prions are infectious pathogens that cause central nervous system spongiform encephalopathies in humans and animals. Prions are distinct from bacteria, viruses and viroids. The predominant hypothesis at present is that no nucleic acid component is necessary for infectivity of prion protein. Further, a prion which infects one species of animal (e.g., a human) will not efficiently infect another (e.g., a mouse).
From a clinical perspective, the prion diseases represent a variety of neurodegenerative states characterized at the neuropathologic level by the presence of spongiform degeneration and astrocytic gliosis in the central nervous system (DeArmond & Prusiner (1996)
Current Topics in MicroBiology and Immunology
, 207:125-146). Frequently, protein aggregates and amyloid plaques are seen that are often resistant to proteolytic degradation. The neuroanatomic distribution of the lesions varies with the specific types of prion disease. In humans, sporadic Creutzfeldt-Jakob Disease (CJD) accounts for 85% of all cases. The disease presents in the sixth decade of life with dementia and ataxia. Familial disease carries a variety of monikers such as Gertsmann-Straussler-Scheinker disease (GSS), familial CJD (fCJD) and Fatal Familial Insomnia (FFI) that relate the precise mutation in the PrP gene to a clinical syndrome (Prusiner & Hsaio (1994)
Annals of Neurology
, 35:385-395; Parchi, et al. (1996)
Annals of Neurology
, 39:767-778; Montagna, et al. (1998)
Brain Pathology
, 8:515-520). Disease typically presents in the fourth decade of life with an autosomal dominant pedigree. While the infectious prion diseases represents less than 1% of all cases, their link to mad cow disease in the U.K. (new variant CJD), growth hormone inoculations in the U.S. and France (iatrogenic CJD), and ritualistic cannibalism in the Fore tribespeople (Kuru) have raised the public awareness of this facet of the disease (Devillemeur, et al. (1996)
Neurology
, 47:690-695; Hill, et al. (1997)
Nature
, 389:448-450; Goodfield (1997)
Nature
, 387:841-841).
A critical advance in our understanding of prion diseases came with the partial purification of a proteinaceous material that retained the ability to reinfect laboratory rodents (McKinley, et al. (1983)
Cell
, 35:57-62). Micro-sequencing and molecular biologic tools led to the cloning of the prion gene, a normal component of mammalian and avian genomes (Prusiner, et al. (1984)
Cell
, 38:127-134). The gene contains a single open reading frame and codes for a protein that is proteolytically processed and glycosylated to form a macromolecule with 219 amino acids, a disulfide bridge, two N-linked sugars and a glycophosphotidyl inositol anchor that is exported to the cell surface and concentrated in an endocytic compartment known as the caveolar space (Endo, et al.
Biochemistry
, 28:8380-8 1989); Stahl, et al. Biochemistry 29:8879-84 (1990); Yost, et al.
Nature
, 343:669-72 (1990); DeFea, et al.
J. Biol. Chem
., 269:16810-16820 (1994); Hegde, et al.,
Science
279:827-34 (1998)). Biophysical characterization of the deglycosylated recombinant PrP refolded into a monomeric form resembling the normal cellular isoform (PrP
C
) reveals a two domain molecule with an N-terminal region (57-89) that binds 4 Cu
++
atoms per chain (Viles, et al. (1999)
Proc. Natl. Acad. Sci. USA
, 96:2042-2047) and a C-terminal region (124-231) that contains 3 substantial helices and 2-3 residue &bgr;-strands joined by 2-3 hydrogen bonds (see
FIG. 1
) (Riek, et al. (1996)
Nature
, 382:180-182; James, et al. (1997)
Proc. Natl. Acad. Sci. USA
, 94:10086-10091; Donne, et al. (1997)
Proc. Natl. Acad. Sci. USA
, 94:13452-13457). By contrast, the disease causing form of the prion protein (PrP
Sc
) is a multimeric assembly substantially enriched in &bgr;-sheet structure (40% &bgr;-sheet, 30% &agr;-helices as judged by FTIR spectroscopy) (Pan, et al. (1993)
Proc. Natl. Acad. Sci. USA
, 90:10962-10966). Immunologic studies of PrP
Sc
suggest that the conformational change is largely in the region from residues 90-145 or perhaps 175 (Peretz, et al. (1997)
J. Mol. Biol
. 273:614-622) These features have been codified in a model of PrP
Sc
(see
FIG. 2
) that emphasize the dramatic conformational distinction between PrP
C
and PrP
Sc
.
A large number of genetic and transgenetic studies have helped to cement the role of the prion protein in the pathogenesis of this group of neurodegenerative diseases. First, a variety of genetic linkage studies of kindreds with familial prion diseases mapped the defect to the Prn-p locus. Subsequent studies identified specific point mutations that caused inherited disease (Hsiao, et al. (1989)
Nature
, 338:342-345; Dlouhy, et al. (1992)
Nat. Genet
., 1:64-67; Petersen, et al. (1992)
Neurology
, 42:1859-1863; Poulter, et al. Brain 115:675-85 (1992); Gabizon, et al. (1993)
Am. J. Hum. Genet
., 53:828-835). These loci are shown in FIG.
3
. Subsequently, the Prn-p gene was knocked out in mice with no obvious phenotypic sequelae (Büeler, et al. (1992)
Nature
, 356:577-582). While wild type mice will develop a prion disease ~180d after intracerebral inoculation, the hemizygous animals require ~400d to succumb to an infectious inoculum and the homozygous knockouts are resistant to prion infection (Prusiner, et al. (1993)
Proc. Natl. Acad. Sci. USA
, 90:10608-10612; Büeler, et al. (1994)
Molecular Medicine
, 1:19-30). Transgenic mice carrying a sufficiently high number of copies of mutant gene (the human GSS mutation P101L) on the knockout background develop a spontaneous neurodegenerative disease that is faithful to the neuropathologic expectations developed from a study of the human kindreds. Knockout mice carrying a redacted form of the PrP transgene (90-141; 175-231) also develop a prion disease upon inoculation with full length RML prions (Supattapone, et al. (1999)
Cell
, 96:869-878). The infection process is more efficient with the “mini” RML prion demonstrating that an artificial prion can be created and that replication efficiency demands fidelity at the amino acid sequence level.
While the predominantly helical PrP
C
and &bgr;-sheet rich PrP
Sc
have exceptionally different secondary and tertiary structures as judged by CD, FTIR, and NMR spectroscopy (Caughey, et al. (1991)
Biochemistry
, 30:7672-7680; Pan, et al. (1993)
Proc. Natl. Acad. Sci. USA
, 90:10962-10966; Riek, et al. (1996)
Nature
, 382:180-182; James, et al. (1997)
Proc. Natl. Acad. Sci. USA
, 94:10086-10091; Donne, et al. (1997)
Proc. Natl. Acad. Sci. USA
, 94:13452-13457), they appear to share a common amino acid sequence and disulfide bridge (Cohen & Prusiner (1998)
Annual Review of Biochemistry
, 67:793-819). Recent work has shown that a conformational change that is aided by an auxiliary molecule is an obligatory step in PrP
Sc
formation (Telling, et al. (1995)
Cell
, 83:79-90; Kaneko, et al. (1997)
J. Mol. Biol
. 270:574-586). The exceptional stability of PrP
Sc
and the marginal stability of PrP
C
together with a variety of transgenetic and cellular transfection studies have led to the conclusion that PrP
C
is a kinetically trapped intermediate in the folding of PrP
Sc
(Cohen & Prusiner (1998)
Annual Review of Biochemistry
, 67:793-819). This kinetic barrier can be reduced by exogenous administration of the PrP
Sc
template, mutations to the wild type (wt) PrP sequence, or stochastic processes resulting in infectious, inherited, or sporadic prion diseases. Epitope mapping and peptide studies suggest that much of this conformational plasticity is localized to the middle third of this 231 residue GPI anchored glycoprotei
Cohen Fred E.
James Thomas L.
Kaneko Kiyotoshi
Prusiner Stanley B.
Bozicevic Karl
Bozicevic Field & Francis LLP
Carlson Karen Cochrane
The Regents of the University of California
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