ATP-dependent protease and use of inhibitors for same in the treatment of cachexia and muscle wasting

• US 5,972,636 A
• Filed: 12/02/1997
• Issued: 10/26/1999
• Est. Priority Date: 05/13/1991
• Status: Expired due to Term

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Goldberg, Alfred L.
• Primary Examiner(s): Patterson, Jr., Charles L.
• Application Number:
  US08/982,295
• Time in Patent Office: 693 Days
• Field of Search: 435/23, 435/226, 514/12, 514/410, 514/423, 514/512
• US Class Current: 435/23
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: C07K 14/8139 Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin
• CPC Class Codes: C12N 9/6421 from mammals
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: G01N 2333/96466 Cysteine endopeptidases (3.4.22)
• CPC Class Codes: G01N 2500/20 cell-free systems

Abstract

The ATP-ubiquitin-dependent process has been shown to be responsible for the excessive protein degradation which occurs in conditions or disease states in which there is severe loss of body mass and negative nitrogen balance has been identified and key constituents in the process identified. A method of inhibiting the accelerated or enhanced proteolysis, a method of identifying inhibitors of the process, multipain and the proteasome inhibitor are the subject of the claimed invention.

Claims

1. A method of screening for an inhibitor of muscle protein degradation, said method comprising:

a) providing cultured cells in which a protein whose degradation is ubiquitin-dependent is produced;

b) contacting the cultured cells with a substance to be assessed for its ability to inhibit muscle protein degradation, under conditions appropriate for entry of the substance into the cultured cells;

c) determining the extent to which the protein is present in the cytosol of the cultured cells, wherein accumulation of the protein in the cytosol is indicative that the substance inhibits the ATP-ubiquitin-dependent degradative process; and

d) identifying a substance that inhibits the ATP-ubiquitin-dependent degradative process as an inhibitor of muscle protein degradation.

2. The method of claim 1, wherein the cultured cells are COS cells which produce a mutant form of an enzyme, which is short-lived.

3. The method of claim 2 wherein the enzyme is a mutant form of β

-galactosidase from E. coli.

4. The method of screening for an inhibitor of muscle protein degradation as claimed in claim 1, wherein said method further comprises measuring cell growth, ATP content, or protein synthesis to identify substances having toxic activity.

5. The method of screening for an inhibitor of muscle protein degradation as claimed in claim 1, wherein said protein is a short-lived protein.

6. The method of screening for an inhibitor of muscle protein degradation as claimed in claim 1, wherein said protein is a long-lived protein.

7. The method of screening for an inhibitor of muscle protein degradation as claimed in claim 6, wherein said protein is selected from the group consisting of the oncogene product myc and the oncogene product fos.

8. A method of identifying an inhibitor of the ATP-ubiquitin-dependent process, comprising:

a) incubating a muscle, obtained from an animal afflicted with a muscle-wasting condition with a compound to be tested as a potential inhibitor of the ATP-ubiquitin-dependent process, under conditions appropriate for degradation of ubiquinated proteins;

b) measuring the release of a product of breakdown of muscle protein in the presence of the compound;

c) comparing the measurement made in (b) with release of the product of breakdown of muscle protein, obtained from an animal exhibiting muscle wasting, in the absence of the compound; and

d) identifying said compound as an inhibitor of the ATP-ubiquitin-dependent process if the presence of said compound results in decreased release of the product of breakdown of muscle protein.

9. The method of claim 8 wherein the product of breakdown of muscle protein is 3-methylhistidine.

10. The method of identifying an inhibitor of the ATP-ubiquitin-dependent process as claimed in claim 8, wherein said muscle is a soleus or extensor digitorum longus muscle.

11. The method of identifying an inhibitor of the ATP-ubiquitin-dependent process as claimed in claim 8, wherein said product of breakdown of muscle protein is 3-methylhistidine.

12. The method of identifying an inhibitor of the ATP-ubiquitin-dependent process as claimed in claim 8, wherein the muscle wasting results from denervation, fasting, febrile infection, or metabolic acidosis.

13. A method of identifying an inhibitor of the ATP-ubiquitin-dependent process in an animal, comprising:

a) providing an animal afflicted with a muscle-wasting condition;

b) administering to the animal a compound to be tested as a potential inhibitor of the ATP-ubiquitin-dependent process;

c) measuring the excretion of 3-methylhistidine by the animal;

d) comparing the measurement made in (c) with excretion of 3-methylhistidine by an animal subjected to muscle wasting without administration of the compound; and

e) identifying said compound as an inhibitor of the ATP-ubiquitin-dependent process if the presence of said compound results in decreased excretion of 3-methylhistidine.

14. A method of screening for a potential pharmaceutical agent, said method comprising:

a) providing cultured cells in which a protein whose degradation is ubiquitin-dependent is produced;

b) contacting the cultured cells with a substance to be assessed for its ability to inhibit the ATP-ubiquitin-dependent degradative process, under conditions appropriate for entry of the substance into the cultured cells;

c) determining the extent to which the protein is present in the cytosol of the cultured cells, wherein accumulation of the protein in the cytosol is indicative of inhibition of the ATP-ubiquitin-dependent degradative process; and

d) identifying a substance that inhibits the ATP-ubiquitin-dependent degradative process as a potential pharmaceutical agent useful for treating a condition mediated by ATP-ubiquitin-dependent proteolysis.

15. The method of screening for a pharmaceutical agent as claimed in claim 14, wherein said condition is associated with a negative nitrogen balance.

16. The method of screening for a potential pharmaceutical agent as claimed in claim 14, wherein said condition is selected from the group consisting of cancer, AIDS, muscle wasting after surgery or injury, infection, cachexia, corticosteroid treatment, sepsis, burn, trauma, neuromotor degenerative disease, muscular dystrophy, acidosis, and spinal or nerve injury.

17. A method of screening for potential growth promoters, said method comprising:

a) providing cultured cells in which a protein whose degradation is ubiquitin-dependent is produced;

b) contacting the cultured cells with a substance to be assessed for its ability to inhibit the ATP-ubiquitin-dependent degradative process, under conditions appropriate for entry of the substance into the cultured cells;

c) determining the extent to which the protein is present in the cytosol of the cultured cells, wherein accumulation of the protein in the cytosol is indicative of inhibition of the ATP-ubiquitin-dependent degradative process; and

d) identifying a substance that inhibits the ATP-ubiquitin-dependent degradative process as a potential growth promoter.