Formation and use of prion protein (PRP) complexes

• US 5,750,361 A
• Filed: 11/02/1995
• Issued: 05/12/1998
• Est. Priority Date: 11/02/1995
• Status: Expired due to Fees

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Kaneko, Kivotoshi, Cohen, Fred E., Prusiner, Stanley B.
• Primary Examiner(s): Hutzell, Paula K.
• Assistant Examiner(s): NAVARRO, ALBERT MARK
• Application Number:
  US08/556,823
• Time in Patent Office: 922 Days
• Field of Search: 530/350, 536/23.1, 435/23, 435/24, 435/6, 435/188, 435/240.2, 436/2, 436/164, 436/181
• US Class Current: 435/23
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: G01N 2800/2828 Prion diseases
• CPC Class Codes: G01N 33/6896 Neurological disorders, e.g. Alzheimer's disease
• CPC Class Codes: Y10T 436/25875 Gaseous sample or with change of physical state

Abstract

Prion protein (PrP) peptides having at least one α-helical domain and forming a random coil conformation in aqueous solutions bind cellular PrP (PrP^C) to form a complex having characteristics of the scrapie isoform (PrP^Sc). Methods for screening compounds able to inhibit or decrease the binding of PrP peptides to PrP^C are disclosed, as well as methods for assaying PrP^Sc.

Claims

1. A method of screening for compounds which inhibits the binding of PrP^C to a PrP peptide, comprising the steps of:

(a) contacting in vitro a test compound with a purified naturally occurring first component PrP^C in the presence of a purified second component PrP peptide, wherein said PrP peptide has a random coil or α

helical confirmation, and wherein the first and second components form a prion protein complex with a PrP^SC characteristic selected from the group consisting of (1) increased β

-sheet content, (2) diminished aqueous solubility, and (3) resistance to poroteolytic digestion, relative to PrP^C ;

(b) detecting formation of a prion protein complex;

by determining (1) increased β

-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^C to a PrP peptide.

2. The method of claim 1, wherein the prion protein complex has increased insolubility relative to PrP^C.

3. The method of claim 2, wherein said complex has increased β

-sheet conformation relative to the first component PrP^C.

4. The method of claim 1, wherein said first component PrP^C is selected from the group consisting of human PrP^C, hamster PrP^C, mouse PrP^C, bovine PrP^C, and ovine PrP^C.

5. The method of claim 4, wherein said PrP^C is human PrP^C.

6. The method of claim 1, wherein said first component PrP^C is the peptide having the amino acid sequence of SEQ ID NO:

10.

7. The method of claim 2, wherein said second component is selected from the group consisting of SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NQ;

8, SEQ ID NO;

9, and SEQ ID NO;

10.

24. The method of a claim 1, wherein the prion protein complex comprises a fibrous aggregate, of which at least 65% sediments at 100,000×

g for 1 hour at 20°

C.

25. The method of claim 1, wherein the prion protein complex is at least 20% protease resistant relative to said PrPc.

8. 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^C in the presence of a second purified component PrP peptide wherein said PrP peptide has a random coil or α

helical confirmation, and wherein the first and second components form a prion protein complex with a PrP^SC characteristic selected from the group consisting of (1) increased β

-sheet context, (2) diminished aqueous solubility, and (3) resistance to poroteolytic digestion, relative to PrP^C ;

(b) detecting induction of the prion protein complex;

by determining (1) increased β

-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.

9. The method of claim 8, wherein said prion protein complex has increased insolubility relative to PrP^C.

10. The method of claim 8, wherein said first component PrP^C is selected from the group consisting of human PrP^C, hamster PrP^C, mouse PrP^C, bovine PrP^C, and ovine PrP^C.

11. The method of claim 10, wherein said PrP^C is human PrP^C.

13. The method of claim 8, wherein said second component is selected from the group consisting of:

SEQ ID NO;

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, and SEQ ID NO;

10.

26. The method of a claim 8, wherein the prion protein complex comprises a fibrous aggregate, of which at least 65% sediments at 100,000×

g for 1 hour at 20°

C.

27. The method of claim 8, wherein the prion protein complex is at least 20% protease resistant relative to said PrPc.

12. 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 α

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 β

-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 β

-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.

14. An assay methodology, comprising:

providing a purified naturally occurring PrP^C protein;

contacting the PrP^C protein with a test compound;

combining a purified peptide with the PrP^C protein which peptide is characterized by having a random coil or α

helical confirmation and causing the PrP^C protein to undergo conformational changes including the formation of β

-sheets in the absence of the test compound; and

determining the effect of the test compound on preventing the conformational changes.

15. The assay methodology of claim 14, wherein conformational changes result in increased insolubility as compared to PrP^C.

16. The assay methodology of claim 14, wherein conformational changes result in increased protease resistance as compared to PrP^C.

17. The assay methodology of claim 14, wherein PrP^C is selected from the group consisting of human PrP^C, hamster PrP^C, mouse PrP^C, bovine PrP^C, and ovine PrP^C.

18. The assay methodology of claim 17, wherein PrP^C is human PrP^C.

19. The assay methodology of claim 14, wherein PrP^C is a human naturally occurring variant of PrP^C.

20. An assay for PrP^Sc, said assay comprising the steps of:

(a) mixing a first component of purified naturally occurring PrP^C and a purified second component PrP peptide wherein said peptide has a random coil or α

helical confirmation under conditions in which a prion protein complex is formed;

(b) adding a test sample suspected of containing PrP^Sc ;

(c) measuring the amount of PrP peptide displaced from the prion protein complex by formation of PrP^C /PrP^Sc complexes,

wherein the amount of PrP peptide displaced from the prion protein complex is proportional to the amount of PrP^Sc present in the test sample.

21. The method of claim 20, wherein the second component PrP peptide is labelled and the amount of PrP peptide displaced from the prion protein complex is measured by removing the PrP^C /PrP^Sc complex and determining the amount of label remaining.

22. The method of claim 20, wherein the first component PrP^C is immobilized by attachment to a solid phase, and the amount of displaced labelled PrP peptide is measured in the unbound phase.

23. The method of claim 21, wherein the PrP peptide is labeled with a reagent selected from the group consisting of a radioactive isotope, a fluorescent dye, and a chromophore.