Methods for identifying modulators of apoptosis

• US 7,422,862 B2
• Filed: 11/27/2002
• Issued: 09/09/2008
• Est. Priority Date: 11/28/2001
• Status: Expired due to Fees

• Current Assignee: Burnham Institute For Medical Research
• Original Assignee: The Burnham Institute
• Inventors: Guo, Bin, Reed, John C
• Primary Examiner(s): Canella; Karen A.
• Assistant Examiner(s): Reddig; Peter J
• Application Number:
  US10/306,878
• Publication Number:
  US 20030175819A1
• Time in Patent Office: 2,113 Days
• Field of Search: 435/4, 530/387.1
• US Class Current: 435/7.8
• CPC Class Codes: G01N 2510/00 Detection of programmed cell death, i.e. apoptosis
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5011 for testing antineoplastic activity
• CPC Class Codes: G01N 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/5079 Mitochondria
• CPC Class Codes: G01N 33/5091 for testing the pathological state of an organism
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds

Abstract

The invention provides a method of identifying an effective compound that modulates the binding of Humanin to Bax or Bid. The invention also provides a method of identifying an effective compound that modulates an activity of Bax or Bid. In addition, the invention provides a method of identifying a Humanin-like compound that binds to Bax or Bid or modulates an activity of Bax or Bid, or inhibits the apoptotic activity of Bax or Bid. The invention further provides an isolated polypeptide containing a mitochondrial-derived form of Humanin (SEQ ID NO:3) or a functional fragment thereof where the fragment contains the methionine at position 16 of SEQ ID NO:3.

Claims

1. A method of identifying an effective compound that decreases the binding of Humanin to Bax, comprising the steps of:

(a) contacting said Humanin, wherein Humanin comprises the amino acid sequence SEQ ID NO;

2 or SEQ ID NO;

3, with said Bax, wherein Bax comprises the amino acid sequence SEQ ID NO;

5, in vitro under conditions suitable to form a Humanin-Bax complex;

(b) contacting said Humanin-Bax complex with a candidate compound; and

(c) determining the ability of said candidate compound to decrease the binding of said Humanin to said Bax, wherein decreasing the binding of said Humanin to said Bax indicates that said candidate compound is an effective compound that decreases the binding of said Humanin to said Bax.

2. The method of claim 1, wherein said candidate compound is selected from the group consisting of:

a polypeptide, peptidomimetic, non-peptidyl compound, carbohydrate, lipid, a synthetic compound, a natural product, an antibody or antibody fragment, a small organic molecules, a small inorganic molecule, and a nucleotide sequence.

3. The method of claim 1, wherein said candidate compound is a small organic molecule.

4. The method of claim 1, wherein said candidate compound is a polypeptide.

5. The method of claim 1, wherein said binding is determined using a method selected from the group consisting of:

a co-immunoprecipitation assay, scintillation proximity assay (SPA), UV or chemical cross-linking, biomolecular interaction analysis (BIA), mass spectrometry (MS), nuclear magnetic resonance (NMR), and fluorescence polarization assays (FPA).

10. The method of claim 1, wherein Humanin comprises the amino acid sequence SEQ ID NO:

2.

11. The method of claim 1, wherein Humanin comprises the amino acid sequence SEQ ID NO:

3.

6. A method of identifying an effective compound that decreases the binding of Humanin to Bax, comprising the steps of:

(a) contacting said Humanin, wherein Humanin comprises the amino acid sequence SEQ ID NO;

2 or SEQ ID NO;

3, with said Bax, wherein Bax comprises the amino acid sequence SEQ ID NO;

5, under conditions suitable to form a Humanin-Bax complex in a yeast two-hybrid assay;

(b) contacting said Humanin-Bax complex with a candidate compound; and

(c) determining the ability of said candidate compound to decrease the binding of said Humanin to said Bax, wherein decreasing the binding of said Humanin to said Bax indicates that said candidate compound is an effective compound that decreases the binding of said Humanin to said Bax.

7. The method of claim 6, wherein said candidate compound is selected from the group consisting of:

a polypeptide, peptidomimetic, non-peptidyl compound, carbohydrate, lipid, a synthetic compound, a natural product, an antibody or antibody fragment, a small organic molecules, a small inorganic molecule, and a nucleotide sequence.

8. The method of claim 6, wherein said candidate compound is a small organic molecule.

9. The method of claim 6, wherein said candidate compound is a polypeptide.

12. The method of claim 6, wherein Humanin comprises the amino acid sequence SEQ ID NO:

2.

13. The method of claim 6, wherein Humanin comprises the amino acid sequence SEQ ID NO:

3.