LIGATION OF STAPLED POLYPEPTIDES

• US 20110144306A1
• Filed: 07/23/2009
• Published: 06/16/2011
• Est. Priority Date: 07/23/2008
• Status: Abandoned Application

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Verdine, Gregory L, Kennedy, Eileen Jeanne
• Application Number:
  US13/055,279
• Publication Number:
  US 20110144306A1
• Time in Patent Office: Days
• US Class Current: 530/351
• 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: A61K 38/1709 from mammals
• CPC Class Codes: A61K 38/2086 IL-13 to IL-16
• CPC Class Codes: C07K 1/107 by chemical modification of precursor peptides
• CPC Class Codes: C07K 14/4702 Regulators; Modulating activity
• CPC Class Codes: C07K 14/5437 IL-13

Abstract

The present invention provides technology for making large (e.g., greater than 50 amino acids), semi-synthetic, stapled or stitched proteins. The method essentially involves ligating a synthetically produced stapled or stitched peptide to a larger protein. Modified version of IL-13 and MYC are provided as illustrative examples.

Claims

1. A method of preparing a stitched or stapled protein, the method comprising steps of:

providing a stitched or stapled peptide;

providing a second protein or peptide to which the stitched or stapled peptide is to be ligated; and

ligating the stitched or stapled peptide to the second protein or peptide.

2. The method of claim 1, wherein the stitched or stapled peptide comprises an alpha-helix.

3. The method of claim 1, wherein the stitched or stapled peptide is less than 30 amino acids in length.

6. The method of claim 1, wherein the stapled peptide is of formula:

9. The method of claim 1, wherein the stitched peptide is of formula:

12. The method of claim 1, wherein the step of providing a stitched or stapled peptide comprises steps of:

synthetically producing an unstitched or unstapled peptide, wherein the peptide comprises unnatural amino acid residues suitable for stitching or stapling; and

stitching or stapling the unstitched or unstapled peptide.

13. The method of claim 1, wherein the step of providing a stapled peptide comprises steps of:

providing a peptide with at least two α

-methyl,α

-alkenylglycine or α

-hydro,α

-alkenylglycine residues; and

stapling the peptide using a Grubbs catalyst to form a macrocyclic hydrocarbon staple.

14. The method of claim 1, wherein the step of providing a stitched peptide comprises steps of:

providing a peptide with at least two α

-methyl,α

-alkenylglycine or α

-hydro,α

-alkenylglycine residues and at least one dialkenylglycine residue; and

stitching the peptide using a Grubbs catalyst to form at least two macrocyclic hydrocarbon staples.

15. The method of claim 13, wherein the two α

-methyl,α

-alkenylglycine or α

-hydro,α

-alkenylglycine residues are at positions i and i+3, at positions i and i+4, and/or at positions i and i+7.

18. The method of claim 13, wherein the α

-methyl,α

-alkenylglycine or α

-hydro,α

-alkenylglycine residue is of the formula;

20. The method of claim 1, wherein the second protein or peptide is prepared recombinantly.

21. The method of claim 1, wherein the second protein or peptide is prepared synthetically.

22. The method of claim 1, wherein the second protein or peptide is treated with a protease to yield an N-terminal cysteine.

27. The method of claim 1, wherein the second protein is produced by having cysteine as the second residue in an expression construct following an N-terminal methionine;

wherein the methionine is processed during protein expression to yield an N-terminal cysteine.

28. The method of claim 1, wherein the step of ligating comprises ligating using Expressed Protein Ligation (EPL).

29. The method of claim 1, wherein the step of ligating produces a scarless protein.

4. (canceled)

5. (canceled)

7-8. -8. (canceled)

10-11. -11. (canceled)

16-17. -17. (canceled)

19. (canceled)

23-26. -26. (canceled)

30-36. -36. (canceled)

37. A method of preparing a stitched or stapled protein, the method comprising steps of:

providing an unstitched or unstapled peptide, wherein the peptide comprises unnatural amino acid residues suitable for stitching or stapling;

providing a second protein or peptide to which the unstitched or unstapled peptide is to be ligated;

ligating the unstitched or unstapled peptide to the second protein or peptide; and

stitching or stapling the unstitched or unstapled peptide.

38-39. -39. (canceled)

40. A stapled IL-13, wherein Helix A or Helix D includes a hydrocarbon staple.

41-50. -50. (canceled)

51. A stapled cMyc, wherein the leucine zipper includes a hydrocarbon staple.

52. A stapled Max, wherein the DNA binding basic region of Max includes a hydrocarbon staple.

53-55. -55. (canceled)