Targeted polypeptide degradation

• US 20050152888A1
• Filed: 12/03/2004
• Published: 07/14/2005
• Est. Priority Date: 12/03/2003
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Church, George M., Janse, Daniel M.
• Granted Patent: US 7,371,539 B2
• Time in Patent Office: Days
• US Class Current: 424/94.630
• CPC Class Codes: C07K 2319/20 containing a tag with affinity for a non-protein ligand
• CPC Class Codes: C07K 2319/95 containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: G01N 2500/02 Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Abstract

This invention pertains to compositions, methods, cells and organisms useful for selectively localizing polypeptides to the proteasome for degradation. Therapeutic methods and pharmaceutical compositions for treating disorders associated with the expression and/or activity of a polypeptide by targeting these polypeptides for degradation, as well as methods for targeting therapeutic polypeptides for degradation and/or activating therapeutic polypeptides by degradation are provided. The invention provides methods for identifying compounds that mediate proteasome localization and/or polypeptide degradation. The invention also provides research tools for the study of protein function.

Claims

1. A method of degrading a polypeptide in a ubiquitin-independent manner comprising:

a) providing a proteasome-localizing agent;

b) allowing the proteasome-localizing agent to bind the polypeptide to form a complex;

c) allowing the complex to bind the proteasome; and

d) allowing the proteasome to degrade the polypeptide.

2. The method of claim 1 further comprising:

e) allowing the proteasome-localizing agent to release from the proteasome;

f) allowing the proteasome-localizing agent to bind a second polypeptide to produce a second complex;

g) allowing the second complex to bind the proteasome; and

h) allowing the proteasome to degrade the second polypeptide.

3. A method of altering a biological activity of a compound comprising:

a) providing a compound including a polypeptide;

b) providing a proteasome-localizing agent;

c) allowing the proteasome-localizing agent to bind the polypeptide to form a complex;

d) allowing the complex to bind a proteasome;

e) allowing the polypeptide to be degraded by the proteasome in a ubiquitin-independent manner to alter the biological activity of the compound; and

f) allowing the compound to be released from the proteasome.

4. The method of claim 3 wherein the biological activity is a therapeutic activity.

5. The method of claim 3 wherein the biological activity of the compound is activated.

6. The method of claim 3 wherein the biological activity of the compound is inhibited.

7. A method of altering a therapeutic activity of a pharmaceutical compound in an individual in need thereof comprising:

a) providing the pharmaceutical compound to the individual;

b) providing a proteasome-localizing agent to the individual;

c) allowing the proteasome-localizing agent to bind the pharmaceutical compound to form a complex;

d) allowing the complex to bind a proteasome;

e) allowing at least a portion of the pharmaceutical compound to be degraded by the proteasome in a ubiquitin-independent manner to alter the therapeutic activity of the pharmaceutical compound.

8. The method of claim 7 wherein the therapeutic activity of the pharmaceutical compound is decreased.

9. The method of claim 8 wherein the pharmaceutical compound is a polypeptide.

10. The method of claim 8 wherein the pharmaceutical compound is a protein.

11. The method of claim 7 wherein the therapeutic activity of the pharmaceutical compound is increased.

12. A method of treating a disorder associated with a polypeptide in an individual in need thereof comprising:

a) providing the individual with a therapeutically effective amount of a proteasome-localizing agent;

b) allowing the proteasome-localizing agent to bind the polypeptide to form a complex;

c) allowing the complex to bind a proteasome; and

d) allowing the proteasome to degrade the polypeptide in a ubiquitin-independent manner to reduce an amount of the polypeptide in the individual.

13. The method of claim 12, further comprising:

e) allowing the proteasome-localizing agent to release from the proteasome;

f) allowing the proteasome-localizing agent to bind a second polypeptide to form a second complex;

g) allowing the second complex to bind a proteasome; and

h) allowing the proteasome to degrade the second polypeptide in a ubiquitin-independent manner to further reduce the amount of the polypeptide in the individual.

14. A compound comprising a complex including a proteasome-localizing agent associated with a polypeptide, wherein the proteasome-localizing agent is capable of binding the proteasome in a ubiquitin-independent manner.

15. The compound of claim 14 wherein the compound is a pharmaceutical compound.

16. A proteasome-localizing agent capable of binding a polypeptide and a proteasome in a ubiquitin-independent manner.

17. A compound capable of localizing a polypeptide to a proteasome for degradation in a ubiquitin-independent manner, the compound comprising a proteasome-localizing agent having a first module that is capable of binding the polypeptide and a second module that is capable of binding the proteasome.

18. A host cell expressing a proteasome associated with a first module, wherein the first module is capable of dimerizing with a second module to form a complex and is further capable of localizing the complex to the proteasome in a ubiquitin-independent manner.

19. The host cell of claim 18 wherein the second module is associated with a polypeptide.

20. The host cell of claim 18 further expressing a polypeptide associated with the second module.

21. The host cell of claim 18 wherein dimerization is altered by a compound that stimulates or inhibits binding of the first module to the second module.

22. The host cell of claim 18 wherein the host cell is Saccharomyces cerevisiae.

23. A transgenic animal expressing a proteasome associated with a first module, wherein the first module is capable of dimerizing with a second module to form a complex and is further capable of localizing the complex to the proteasome in a ubiquitin-independent manner.

24. The transgenic animal of claim 23 wherein the second module is associated with a polypeptide.

25. The transgenic animal of claim 23 further expressing a polypeptide associated with the second module.

26. The transgenic animal of claim 23 wherein dimerization is altered by a compound that stimulates or inhibits binding of the first module to the second module.

27. The transgenic animal of claim 23 wherein the transgenic animal is a mouse.

28. An in vitro method for targeted polypeptide degradation in a ubiquitin-independent manner comprising:

a) providing a proteasome;

b) providing a target polypeptide;

c) providing a proteasome-localizing agent;

d) allowing the proteasome-localizing agent to bind the target polypeptide to form a complex;

e) allowing the complex to bind the proteasome; and

f) allowing the proteasome to degrade the target polypeptide.

29. The in vitro method of claim 28 further comprising providing a compound that stimulates or inhibits the proteasome-localizing agent binding to the target polypeptide.

30. The in vitro method of claim 28 further comprising providing a compound that stimulates or inhibits the complex binding to the proteasome.

31. A method for identifying a test compound that targets a polypeptide to a proteasome in a ubiquitin-independent manner comprising:

a) providing a proteasome;

b) providing a polypeptide;

c) providing a test compound;

d) incubating the proteasome, the polypeptide and the test compound together; and

e) detecting degradation of the polypeptide, wherein the polypeptide is degraded if the test compound targets the polypeptide to the proteasome.