Method for identifying apoptosis modulating compounds

• US 6,165,732 A
• Filed: 07/31/1998
• Issued: 12/26/2000
• Est. Priority Date: 10/14/1997
• Status: Expired due to Term

• Current Assignee: Washington University In St Louis
• Original Assignee: Washington University In St Louis
• Inventors: Schlesinger, Paul H., Korsmeyer, Stanley J.
• Primary Examiner(s): Wortman, Donna C.
• Assistant Examiner(s): Brumback, Brenda G.
• Application Number:
  US09/127,048
• Time in Patent Office: 879 Days
• Field of Search: 424/9.1, 424/9.321, 435/4, 435/7.2, 435/7.21, 435/7.23, 530/350, 536/23.1, 536/23.5
• US Class Current: 435/7.2
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C07K 14/4747 Apoptosis related proteins

Abstract

Methods for identifying apoptosis-modulating compounds using lipid bilayers are provided. One method involves contacting a compound of interest with a lipid bilayer which contains an ion-channel formed by an anti-apoptotic or pro-apoptotic polypeptide of the BCL-2 family and assaying for changes in the ion conductance properties of the channel, including ion selectivity, single channel conductance and rectification. A second method identifies compounds which can form ion channels in planar lipid bilayers and determines the ion selectivity and pH dependence of such channel formation, where apoptosis modulating activity is predicted based on comparing these channel forming characteristics with those of Bcl-2 family members.

Claims

1. A method for identifying apoptosis-modulating compounds which comprises:

(a) providing a lipid bilayer that includes a K⁺ - or an Cl^- -selective channel wherein the covel comprises anti-apoptotic or pro-apoptotic polypeptide of the BCL-2 family, respectively, or fragment thereof which is capable of forming an ion selective channel in vitro;

(b) contacting a compound of interest with the bilayer; and

(c) determining ion-selectivity of the channel

wherein a change from K⁺ -selective to Cl^- -selective indicates the compound is a death agonist and a change from Cl^- -selective to K⁺ -selective indicates the compound is a death antagonist.

2. The method of claim 1, wherein the channel comprises an anti-apoptotic polypeptide of the BCL-2 family.

3. The method of claim 2, wherein the anti-apoptotic polypeptide is Bcl-2Δ

TM.

4. The method of claim 2, wherein the lipid bilayer comprises a proteoliposome loaded with a mixture of K⁺ and Cl^- in known amounts and wherein determining ion selectivity comprises measuring the relative rates of K⁺ and Cl^- efflux from the proteoliposome.

5. The method of claim 4, wherein the anti-apoptotic polypeptide is Bcl-2Δ

TM.

6. The method of claim 2, wherein the lipid bilayer comprises a planar bilayer in the presence of an ion concentration gradient and the determining ion selectivity step comprises measuring the reversal potential of the channel.

7. The method of claim 1, wherein the channel comprises a pro-apoptotic polypeptide of the BCL-2 family.

8. The method of claim 7, wherein the pro-apoptotic polypeptide is BaxΔ

TM.

9. The method of claim 7, wherein the lipid bilayer comprises a planar bilayer in the presence of an ion concentration gradient and the determining ion selectivity step comprises measuring the reversal potential of the channel.

10. The method of claim 7, wherein the lipid bilayer comprises a proteoliposome loaded with a mixture of K⁺ and Cl^- in known amounts and wherein determining ion selectivity comprises measuring the relative rates of K⁺ and Cl^- efflux from the proteoliposome.