Ligation of stapled polypeptides

Patent metadata

Field	Value
Publication number	US-9458189-B2
Application number	US-201514748287-A
Country	US
Kind code	B2
Filing date	Jun 24, 2015
Priority date	Jul 23, 2008
Publication date	Oct 4, 2016
Grant date	Oct 4, 2016

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.

First claim

What is claimed is:

1. A method of preparing a stabilized, non-immunogenic, and folded protein comprising a stitched α-helical peptide segment, the method comprising steps of:
providing a stitched α-helical peptide segment, wherein the stitched α-helical peptide segment 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 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 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 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; 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; 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;
providing a second protein to which the stitched α-helical peptide segment is to be ligated, wherein the second protein is produced recombinantly or purified from a natural source; and
ligating the stitched α-helical peptide segment to the second protein.

2. The method of claim 1 , wherein the stitched α-helical peptide segment is less than 30 amino acids in length.

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

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

5. The method of claim 1 , wherein the step of providing a stitched α-helical peptide segment comprises steps of:
synthetically producing an unstitched peptide segment, wherein the peptide segment comprises unnatural amino acid residues suitable for stitching; and
stitching the unstitched peptide.

6. The method of claim 1 , wherein the step of providing a stitched α-helical peptide segment 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.

7. The method of claim 6 , 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.

8. The method of claim 6 , 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.

9. The method of claim 1 , wherein the second protein is prepared recombinantly.

10. The method of claim 1 , wherein the second protein is treated with a protease to yield an N-terminal cystei

Assignees

• Harvard College

Inventors

• Verdine Gregory L
• Kennedy Eileen Jeanne

Classifications

• A61K38/1709

  from mammals · CPC title

• C07K1/107 Primary

  by chemical modification of precursor peptides · CPC title

• C07K14/4702 Primary

  Regulators; Modulating activity · CPC title

• A61K38/00

  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 title

• C07K14/5437

  IL-13 · CPC title