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US 5,750,361 AGranted
Formation and use of prion protein (PRP) complexes
AssigneeThe Regents of the University of California
InventorsStanley B. Prusiner, Kivotoshi Kaneko, Fred E. Cohen
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Abstract
Prion protein (PrP) peptides having at least one .alpha.-helical domain and forming a random coil conformation in aqueous solutions bind cellular PrP (PrP.sup.C) to form a complex having characteristics of the scrapie isoform (PrP.sup.Sc). Methods for screening compounds able to inhibit or decrease the binding of PrP peptides to PrP.sup.C are disclosed, as well as methods for assaying PrP.sup.Sc.
Claims27
A method of screening for compounds which inhibits the binding of PrP.sup.C to a PrP peptide, comprising the steps of:
- (a) contacting in vitro a test compound with a purified naturally occurring first component PrP.sup.C in the presence of a purified second component PrP peptide, wherein said PrP peptide has a random coil or .alpha. helical confirmation, and wherein the first and second components form a prion protein complex with a PrP.sup.SC characteristic selected from the group consisting of (1) increased .beta.-sheet content, (2) diminished aqueous solubility, and (3) resistance to poroteolytic digestion, relative to PrP.sup.C ;
- (b) detecting formation of a prion protein complex;
- by determining (1) increased .beta.-sheet, (2) diminished aqueous solubility, or (3) resistance to proteolytic digestion, relative to PrPc and and
- (c) comparing levels of prion protein complex in the presence of said test compound with levels in the absence of the test compound, wherein reduced levels of said prion protein complex in the presence of said test compound is indicative that said test compound inhibits said binding of PrP.sup.C to a PrP peptide.
1 more element
A method for screening compounds which inhibit induction of a prion protein complex, comprising the steps of:
- (a) contacting in vitro a test compound with a purified naturally occurring first component PrP.sup.C in the presence of a second purified component PrP peptide wherein said PrP peptide has a random coil or .alpha. helical confirmation, and wherein the first and second components form a prion protein complex with a PrP.sup.SC characteristic selected from the group consisting of (1) increased .beta.-sheet context, (2) diminished aqueous solubility, and (3) resistance to poroteolytic digestion, relative to PrP.sup.C ;
- (b) detecting induction of the prion protein complex;
- by determining (1) increased .beta.-sheet content, (2) diminished aqueous solubility, or (3) resistance to proteolytic digestion, relative to PrPc and
- (c) comparing induction of the prion protein complex in the presence of said test compound with induction of the protein prion complex in the absence of the test compound, wherein reduced levels of said prion protein complex in the presence of said test compound is indicative that said test compound inhibits induction.
1 more element
A method for screening compounds which inhibit induction of a prion protein complex, comprising the steps of:
- (a) contacting in vitro a test compound with a first component PrPc having, the amino acid sequence of SEQ ID NO:
- 10 in the presence of a second purified component PrP peptide wherein said PrP peptide has a random coil or .alpha. helical confirmation and wherein the first and second components form a prion protein complex with a PrPsc characteristic selected from the group consisting of (1) increased .beta.-sheet content, (2) diminished aqueous solubility and (3) resistance to proteolytic digestion, relative to PrPc;
- (b) detecting induction of the prion protein complex by determining (1) increased .beta.-sheet content, (2) diminished aqueous solubility or (3) resistance to proteolytic digestion, relative to PrPc;
- and
- (c) comparing induction of the prion protein complex in the presence of said test compound with induction of the protein prion complex in the absence of the test compound, wherein reduced levels of said prion protein complex in the presence of said test compound is indicative that said test compound inhibits induction.
2 more elements
An assay methodology, comprising:
- providing a purified naturally occurring PrP.sup.C protein;
- contacting the PrP.sup.C protein with a test compound;
- combining a purified peptide with the PrP.sup.C protein which peptide is characterized by having a random coil or .alpha. helical confirmation and causing the PrP.sup.C protein to undergo conformational changes including the formation of .beta.-sheets in the absence of the test compound;
- and
- determining the effect of the test compound on preventing the conformational changes.
2 more elements
An assay for PrP.sup.Sc, said assay comprising the steps of:
- (a) mixing a first component of purified naturally occurring PrP.sup.C and a purified second component PrP peptide wherein said peptide has a random coil or .alpha. helical confirmation under conditions in which a prion protein complex is formed;
- (b) adding a test sample suspected of containing PrP.sup.Sc ;
- (c) measuring the amount of PrP peptide displaced from the prion protein complex by formation of PrP.sup.C /PrP.sup.Sc complexes,
- wherein the amount of PrP peptide displaced from the prion protein complex is proportional to the amount of PrP.sup.Sc present in the test sample.
1 more element
Dependencies are inferred from the claim text.