Stabilized alpha helical peptides and uses thereof

• US 8,796,418 B2
• Filed: 09/18/2008
• Issued: 08/05/2014
• Est. Priority Date: 11/05/2003
• Status: Active Grant

• Current Assignee: Dana-Farber Cancer Institute, President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: Dana-Farber Cancer Institute, President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Walensky, Loren D., Korsmeyer, Stanley J., Verdine, Gregory L.
• Primary Examiner(s): Lukton, David
• Application Number:
  US12/233,555
• Publication Number:
  US 20090149630A1
• Time in Patent Office: 2,147 Days
• Field of Search: 530/333, 556/136
• US Class Current: 530/333
• 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 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/14 for treating abnormal movements, e.g. chorea, dyskinesia
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• 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 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 35/04 specific for metastasis
• 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 5/06 of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
• CPC Class Codes: A61P 5/14 : of the thyroid hormones, e.g. T3, T4
• CPC Class Codes: A61P 5/24 : of the sex hormones
• CPC Class Codes: A61P 7/00 : Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/02 : Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• CPC Class Codes: A61P 7/06 : Antianaemics
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/04 : Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• CPC Class Codes: A61P 9/08 : Vasodilators for multiple indications
• 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: A61P 9/12 : Antihypertensives
• CPC Class Codes: A61P 9/14 : Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
• CPC Class Codes: C07K 1/113 : without change of the primary structure
• CPC Class Codes: C07K 1/1136 : by reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating or stabilising agents
• CPC Class Codes: C07K 14/001 : by chemical synthesis
• CPC Class Codes: C07K 14/435 : from animals; from humans
• CPC Class Codes: C07K 14/4747 : Apoptosis related proteins
• CPC Class Codes: C07K 7/08 : having 12 to 20 amino acids gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68
• CPC Class Codes: C07K 7/64 : Cyclic peptides containing only normal peptide links

Abstract

Novel polypeptides and methods of making and using the same are described herein. The polypeptides include cross-linking (“hydrocarbon stapling”) moieties to provide a tether between two amino acid moieties, which constrains the secondary structure of the polypeptide. The polypeptides described herein can be used to treat diseases characterized by excessive or inadequate cellular death.

Claims

1. A method of producing a modified cell-permeable α

-helical polypeptide, the method comprising;

(a) selecting a precursor polypeptide comprising at least two reactive residues; and

(b) reacting the precursor polypeptide in conditions sufficient to promote formation of a crosslink spanning at least one turn of an alpha helix between two of the at least two reactive residues, thereby forming the modified cell-permeable α

-helical polypeptide;

wherein the modified cell-permeable α

-helical polypeptide is cell-permeable as determined by a cell permeability study; and

wherein at least one of the at least two reactive residues is an α

,α

-disubstituted amino acid.

4. The method of claim 1, 2, or 3, wherein the crosslink stabilizes an α

-helical structure.

5. The method of claim 1, 2, or 3, wherein the at least two reactive residues comprise at least one amino acid functionalized at its alpha carbon.

10. The method of claim 1, 2, or 3, wherein the modified cell-permeable α

-helical polypeptide is a proapoptotic or antiapoptotic polypeptide.

11. The method of claim 1 or 2, wherein the crosslink is formed by olefin metathesis.

12. The method of claim 1 or 2, wherein the modified cell-permeable α

-helical polypeptide has increased energy-dependent transport across a cell membrane relative to an unmodified polypeptide.

13. The method of claim 1 or 2, wherein the modified cell-permeable α

-helical polypeptide has increased endocytosis relative to an unmodified polypeptide.

21. The method of claim 1, 2, or 3, wherein the modified cell-permeable α

-helical polypeptide further comprises a fluorescent moiety or radioisotope.

22. The method of claim 1, 2, or 3, wherein the modified cell-permeable α

-helical polypeptide further comprises an affinity label.

23. The method of claim 1, 2, or 3, wherein the modified cell-permeable α

-helical polypeptide further comprises a targeting moiety.

24. The method of claim 1, 2, or 3, wherein the modified cell-permeable α

-helical polypeptide binds to a BCL-2 family polypeptide.

25. The method of claim 1, 2, or 3, wherein the modified cell-permeable α

-helical polypeptide comprises a BH3 domain.

26. The method of claim 1, 2, or 3, wherein the crosslink is placed primarily on a single face of an alpha helix.

27. The method of claim 1, 2, or 3, wherein the at least two reactive residues are amino acids which are separated by three or six amino acids.

2. A method of producing a modified cell-permeable α

-helical polypeptide, the method comprising;

(a) selecting a precursor polypeptide comprising at least two reactive residues, each of the at least two reactive residues comprising a terminal olefin group; and

(b) reacting the precursor polypeptide in conditions sufficient to promote formation of a crosslink between the terminal olefin groups of two of the at least two reactive residues, thereby forming the modified cell-permeable α

-helical polypeptide, wherein the crosslink spans at least one turn of an alpha helix;

wherein the modified cell-permeable α

-helical polypeptide is cell-permeable as determined by a cell permeability study.

6. The method of claim 2, or 3, wherein at least one of the at least two reactive residues is a non-natural amino acid.

7. The method of claim 2, wherein each of the at least two reactive residues comprises an amino acid functionalized at its alpha carbon with the terminal olefin group.

8. The method of claim 7, wherein each amino acid functionalized at its alpha carbon with the terminal olefin group is further substituted at its alpha carbon with an α

-alkyl group.

9. The method of claim 8, wherein the α

-alkyl group is methyl.

3. A method of producing a modified cell-permeable α

-helical polypeptide, the method comprising

(a) selecting a precursor polypeptide comprising at least two reactive residues; and

(b) reacting the precursor polypeptide in conditions sufficient to promote formation of a crosslink between two of the at least two reactive residues, thereby forming the modified cell-permeable α

-helical polypeptide;

wherein the modified cell-permeable α

-helical polypeptide is cell-permeable as determined by a cell permeability study; and

wherein the modified cell-permeable α

-helical polypeptide has the formula;

14. The method of claim 3, wherein R₃ is alkyl, alkenyl, or alkynyl.

15. The method of claim 3, wherein x is 3.

16. The method of claim 3, wherein x is 6.

17. The method of claim 3, wherein R₃ comprises at least nine consecutive carbon-carbon bonds.

18. The method of claim 3, wherein at least one of R₁ and R₂ is methyl.

19. The method of claim 18, wherein R₁ and R₂ are methyl.

20. The method of claim 3, wherein R₃ is [R₄—

K—

R₄]_n.

28. A method of producing a pharmaceutical composition comprising a modified cell-permeable α

-helical polypeptide, the method comprising;

(a) selecting a precursor polypeptide comprising at least two reactive residues;

(b) reacting the precursor polypeptide in conditions sufficient to promote formation of a crosslink spanning at least one turn of an alpha helix between two of the at least two reactive residues, thereby forming the modified cell-permeable α

-helical polypeptide;

wherein the modified cell-permeable α

-helical polypeptide is cell-permeable as determined by a cell permeability study;

wherein at least one of the at least two reactive residues is an α

,α

-disubstituted amino acid; and

(c) combining the modified cell-permeable α

-helical polypeptide with a pharmaceutically acceptable carrier or adjuvant.

29. A method of producing a pharmaceutical composition comprising a modified cell-permeable α

-helical polypeptide, the method comprising;

(a) selecting a precursor polypeptide comprising at least two reactive residues, each of the at least two reactive residues comprising a terminal olefin group;

(b) reacting the precursor polypeptide in conditions sufficient to promote formation of a crosslink between the terminal olefin groups of two of the at least two reactive residues, thereby forming the modified cell-permeable α

-helical polypeptide;

wherein the crosslink spans at least one turn of an alpha helix;

wherein the modified cell-permeable α

-helical polypeptide is cell-permeable as determined by a cell permeability study; and

(c) combining the modified cell-permeable α

-helical polypeptide with a pharmaceutically acceptable carrier or adjuvant.

30. A method of producing a pharmaceutical composition comprising a modified cell-permeable α

-helical polypeptide, the method comprising

(a) selecting a precursor polypeptide comprising at least two reactive residues;

(b) reacting the precursor polypeptide in conditions sufficient to promote formation of a crosslink between two of the at least two reactive residues, thereby forming the modified cell-permeable α

-helical polypeptide;

wherein the modified cell-permeable α

-helical polypeptide is cell-permeable as determined by a cell permeability study; and

wherein the modified cell-permeable α

-helical polypeptide has the formula;