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Patent Application

App. No. 13/055,279

LIGATION OF STAPLED POLYPEPTIDES

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Patent No.: US None
App. No.: 13/055,279

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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 (76)

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.

4 . (canceled)

5 . (canceled)

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

wherein

each instance of L 1 and L 2 is, independently, a bond, cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkenylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkynylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkenylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkynylene; substituted or unsubstituted arylene; substituted or unsubstituted heteroarylene; or substituted or unsubstituted acylene;

each instance of R a is, independently, hydrogen; cyclic or acyclic, branched or unbranched, substituted or unsubstituted aliphatic; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; cyclic or acyclic, substituted or unsubstituted acyl; or R a is a suitable amino protecting group;

each instance of R b is, independently, a suitable amino acid side chain; hydrogen; cyclic or acyclic, branched or unbranched, substituted or unsubstituted aliphatic; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; cyclic or acyclic, substituted or unsubstituted acyl; substituted or unsubstituted hydroxyl; substituted or unsubstituted thiol; substituted or unsubstituted amino; cyano; isocyano; halo; or nitro;

each instance of R e is, independently, —R E , —OR E , —N(R E ) 2 , or —SR E , wherein each instance of R E is, independently, hydrogen, cyclic or acyclic, branched or unbranched, substituted or unsubstituted aliphatic; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; substituted or unsubstituted acyl; a resin; a suitable hydroxyl, amino, or thiol protecting group; or two R E groups together form a substituted or unsubstituted 5- to 6-membered heterocyclic or heteroaromatic ring;

each instance of R f is, independently, hydrogen; cyclic or acyclic, branched or unbranched, substituted or unsubstituted aliphatic; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; substituted or unsubstituted acyl; a resin; a suitable amino protecting group; a label optionally joined by a linker, wherein the linker is selected from cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkenylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkynylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkenylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkynylene; substituted or unsubstituted arylene; substituted or unsubstituted heteroarylene; or substituted or unsubstituted acylene; or R f and R a together form a substituted or unsubstituted 5- to 6-membered heterocyclic or heteroaromatic ring;

each instance of R LL is, independently, hydrogen; cyclic or acyclic, branched or unbranched, substituted or unsubstituted aliphatic; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; substituted or unsubstituted acyl; substituted or unsubstituted hydroxyl; substituted or unsubstituted thiol; substituted or unsubstituted amino; azido; cyano; isocyano; halo; nitro;

or two adjacent R LL groups are joined to form a substituted or unsubstituted 5- to 8-membered cycloaliphatic ring; substituted or unsubstituted 5- to 8-membered cycloheteroaliphatic ring; substituted or unsubstituted aryl ring; or substituted or unsubstituted heteroaryl ring;

each instance of X AA is, independently, a natural or unnatural amino acid;

each instance of z is, independently, an integer between 2 to 6;

each instance of j is, independently, an integer between 1 to 10;

each instance of s and t is, independently, an integer between 0 and 100;

each instance of q is, independently, an integer between 0 to 2;

and wherein

corresponds to a single or double bond.

7 - 8 . (canceled)

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

wherein

each instance of K, L 1 , L 2 , and M, is, independently, a bond, cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkenylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkynylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkenylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkynylene; substituted or unsubstituted arylene; substituted or unsubstituted heteroarylene; or substituted or unsubstituted acylene;

each instance of R e is, independently, —R E , —OR E , N(R E ) 2 , or —SR E , wherein each instance of R E is, independently, hydrogen, cyclic or acyclic, branched or unbranched, substituted or unsubstituted aliphatic; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; substituted or unsubstituted acyl; a resin; a suitable hydroxyl, amino, or thiol protecting group; or two R E groups together form a substituted or unsubstituted 5- to 6-membered heterocyclic or heteroaromatic ring;

each instance of R KL , R LL , and R LM , is, independently, hydrogen; cyclic or acyclic, branched or unbranched, substituted or unsubstituted aliphatic; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; substituted or unsubstituted acyl; substituted or unsubstituted hydroxyl; substituted or unsubstituted thiol; substituted or unsubstituted amino; azido; cyano; isocyano; halo; nitro;

or two adjacent R KL are joined to form a substituted or unsubstituted 5- to 8-membered cycloaliphatic ring; substituted or unsubstituted 5- to 8-membered cycloheteroaliphatic ring; substituted or unsubstituted aryl ring; or substituted or unsubstituted heteroaryl ring; two adjacent R KL are joined to form a substituted or unsubstituted 5- to 8-membered cycloaliphatic ring; substituted or unsubstituted 5- to 8-membered cycloheteroaliphatic ring; substituted or unsubstituted aryl ring; or substituted or unsubstituted heteroaryl ring; or two adjacent R LM groups are joined to form a substituted or unsubstituted 5- to 8-membered cycloaliphatic ring; substituted or unsubstituted 5- to 8-membered cycloheteroaliphatic ring; substituted or unsubstituted aryl ring; or substituted or unsubstituted heteroaryl ring;

each instance of X AA is, independently, a natural or unnatural amino acid;

each instance of y and z is, independently, an integer between 2 to 6;

each instance of j is, independently, an integer between 1 to 10;

each instance of p is, independently, an integer between 0 to 10;

each instance of s and t is, independently, an integer between 0 and 100;

each instance of u, v, and q, is, independently, an integer between 0 to 2;

and wherein

corresponds to a single or double bond.

10 - 11 . (canceled)

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.

16 - 17 . (canceled)

18 . The method of claim 13 , wherein the α-methyl,α-alkenylglycine or α-hydro,α-alkenylglycine residue is of the formula:

wherein n is an integer between 1 and 10, inclusive; and R b is H or methyl.

19 . (canceled)

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.

23 - 26 . (canceled)

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.

30 - 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 . (canceled)

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

41 - 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 . (canceled)

Assignments (1)

ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Mar 23, 2011

From: VERDINE, GREGORY L.; KENNEDY, EILEEN JEANNE

To: PRESIDENT AND FELLOWS OF HARVARD COLLEGE

Reel/Frame 026001/0951 →