Serine substituted mutants of BCL-X.sub.L /BCL-2 associated cell death regulator

• US 5,856,445 A
• Filed: 10/18/1996
• Issued: 01/05/1999
• Est. Priority Date: 10/18/1996
• Status: Expired due to Term

• Current Assignee: Washington University In St Louis
• Original Assignee: Washington University In St Louis
• Inventors: Korsmeyer, Stanley J.
• Primary Examiner(s): Huff, Sheela
• Assistant Examiner(s): BURKE, JULIE ELAINE
• Application Number:
  US08/733,505
• Time in Patent Office: 809 Days
• Field of Search: 530/350, 435/69.1
• US Class Current: 530/350
• 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: C07K 14/4747 Apoptosis related proteins
• CPC Class Codes: C07K 2319/00 Fusion polypeptide

Abstract

Novel forms of mutant BAD polypeptides or fragments thereof having an amino acid substitution for serine-112 and/or serine-136 are provided along with their encoding polynucleotides. Also disclosed are methods for preparation of the mutant BAD polypeptides and methods for treating disease conditions involving decreased apoptosis.

Claims

1. An isolated polypeptide comprising an amino acid sequence of a mutant BCL-X_L /BCL-2 associated cell death regulator (BAD) polypeptide, wherein said amino acid sequence is identical to that of a naturally-occurring mammalian BAD polypeptide except for having an amino acid other than serine at one or both of first and second positions corresponding to position 112 and position 136 of SEQ ID NO:

1, said first and second positions being identified by alignment of said amino acid sequence to SEQ ID NO;

1, wherein said naturally-occurring mammalian BAD polypeptide (a) contains a BH1 domain comprising residues 142 to 152 of SEQ ID NO;

1 and (b) contains a BH2 domain, and wherein said mutant BAD polypeptide has cell death agonist activity.

2. The polypeptide of claim 1, wherein said naturally occurring mammalian BAD polypeptide comprises SEQ ID NO:

1 or SEQ ID NO;

55.

3. The polypeptide of claim 2, wherein the amino acid other than serine at one or both of said first and second positions is alanine.

4. The polypeptide of claim 3, wherein said amino acid sequence is SEQ ID NO:

12, SEQ ID NO;

13, or SEQ ID NO;

14.

5. The polypeptide of claim 3, wherein said amino acid sequence comprises SEQ ID NO:

56, SEQ ID NO;

57, or SEQ ID NO;

58.

6. An isolated polypeptide comprising a fragment of said mutant BAD polypeptide of claim 1, wherein said fragment comprises the BH1 domain, the BH2 domain and at least one of said first and second positions which is an amino acid other than serine, wherein said fragment has cell death agonist activity.

7. The fragment of claim 6 which comprises both of said first and second positions with each position being an amino acid other than serine.

8. The fragment of claim 6, wherein said mutant BAD polypeptide consists of SEQ ID NO:

12, SEQ ID NO;

13, or SEQ ID NO;

14.

9. The fragment of claim 6, wherein said mutant BAD polypeptide comprises SEQ ID NO:

56, SEQ ID NO;

57, or SEQ ID NO;

58.

10. A method for making a mutant BCL-X_L /BCL-2 associated cell death regulator (BAD) polypeptide which has cell death agonist activity, the method comprising

(a) selecting an amino acid sequence of a naturally occurring mammalian BAD polypeptide which (1) contains a BH1 domain comprising residues 142 to 152 of SEQ ID NO:

1, (2) contains a BH2 domain, and (3) contains serine residues at first and second positions corresponding to positions 112 and 136 of SEQ ID NO;

1, said first and second positions being identified by alignment of said amino acid sequence to SEQ ID NO;

1,

(b) preparing said mutant BAD polypeptide, said mutant BAD polypeptide containing said amino acid sequence except for an amino acid other than serine at one or both of said first and second positions; and

(c) testing said mutant BAD polypeptide for cell death agonist activity.

11. The method of claim 10, wherein said amino acid other than serine is alanine.

12. The method of claim 11, wherein said amino acid sequence is SEQ ID NO:

1.

13. The method of claim 12, wherein the mutant BAD polypeptide consists of SEQ ID NO:

12, SEQ ID NO;

13 or SEQ ID NO;

14.

14. The method of claim 11, wherein said amino acid sequence comprises SEQ ID NO:

55.

15. The method of claim 14, wherein the mutant BAD polypeptide comprises SEQ ID NO:

56, SEQ ID NO;

57 or SEQ ID NO;

58.

16. The method of claim 10, wherein said preparing step comprises expressing the mutant BAD polypeptide in a host cell transformed with a polynucleotide encoding the mutant BAD polypeptide.

17. The method of claim 16, wherein said polynucleotide comprises SEQ ID NO:

16, SEQ ID NO;

17 or SEQ ID NO;

18.

18. The method of claim 16, wherein said polynucleotide comprises SEQ ID NO:

52, SEQ ID NO;

53 or SEQ ID NO;

54.