Stabilized compounds having secondary structure motifs

• US 9,951,099 B2
• Filed: 10/06/2016
• Issued: 04/24/2018
• Est. Priority Date: 05/18/1999
• Status: Expired due to Term

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Verdine, Gregory L., Schafmeister, Christian E.
• Primary Examiner(s): Russel, Jeffrey E.
• Application Number:
  US15/287,513
• Publication Number:
  US 20170088581A1
• Time in Patent Office: 565 Days
• 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 38/02 Peptides of undefined number of amino acids; Derivatives thereof
• CPC Class Codes: C07K 1/00 General methods for the preparation of peptides , i.e. processes for the organic chemical preparation of peptides or proteins of any length
• CPC Class Codes: C07K 1/04 on carriers C07K1/003, C07K1/006 take precedence
• CPC Class Codes: C07K 1/047 Simultaneous synthesis of different peptide species; Peptide libraries
• CPC Class Codes: C07K 1/10 using coupling agents C07K1/006 takes precedence
• CPC Class Codes: C07K 1/113 without change of the primary structure
• CPC Class Codes: C07K 14/001 by chemical synthesis
• CPC Class Codes: C07K 14/4746 p53
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups

Abstract

The present invention provides novel stabilized crosslinked compounds having secondary structure motifs, libraries of these novel compounds, and methods for the synthesis of these compounds libraries thereof. The synthesis of these novel stabilized compounds involves (1) synthesizing a peptide from a selected number of natural or non-natural amino acids, wherein the peptide comprises at least two moieties capable of undergoing reaction to promote carbon-carbon bond formation; and (2) contacting the peptide with a reagent to generate at least one crosslinker and to effect stabilization of a secondary structure motif. The present invention, in a preferred embodiment, provides stabilized p53 donor helical peptides. Additionally, the present invention provides methods for disrupting the p53/MDM2 binding interaction comprising (1) providing a crosslinked stabilized α-helical structure; and (2) contacting the crosslinked stabilized α-helical structure with MDM2.

Claims

1. A method for synthesizing a peptide comprising a cross-link, the method comprising:

(a) synthesizing an amino acid sequence, wherein the amino acid sequence comprises a first amino acid comprising a first moiety and a second amino acid comprising a second moiety, and wherein the first and second moieties are reactive toward one another in the presence of a catalyst; and

(b) reacting the amino acid sequence under conditions sufficient to promote a reaction between the first and second moieties toward each other in the presence of the catalyst, thereby resulting in formation of the cross-link in the peptide;

wherein the cross-link spans one or two turns of an α

-helix;

the cross-link comprises a carbon-carbon double bond; and

the peptide disrupts binding of p53 to MDM2.

2. The method of claim 1, wherein the peptide is stabilized in comparison to a corresponding uncross-linked peptide.

3. The method of claim 1, wherein the peptide comprises an MDM2 binding site of a p53 donor helix.

4. The method of claim 1, wherein at least one amino acid in the amino acid sequence is a non-natural amino acid.

5. The method of claim 1, wherein at least one amino acid in the amino acid sequence comprises an olefin.

6. The method of claim 1, wherein at least one amino acid in the amino acid sequence is an α

-allyl amino acid.

7. The method of claim 1, wherein at least one amino acid in the amino acid sequence has a 1-penten-5-yl side chain.

14. The method of claim 1, wherein the reaction between the first and second moieties is a ring-closing metathesis (RCM) reaction.

15. The method of claim 1, wherein the catalyst is a ruthenium catalyst.

8. A method for synthesizing a library of stabilized α

-helix peptides, the method comprising;

(a) providing a collection of resin-bound amino acids;

(b) deprotecting each of said resin-bound amino acids;

(c) separating said collection of deprotected resin-bound amino acids into N equal portions, wherein N represents the number of different types of reacting amino acids to be coupled;

(d) coupling of each N types of reacting amino acids to each portion of the deprotected resin-bound amino acids;

(e) combining each of the N portions together;

(f) repeating steps (b)-(e) until a desired peptide product is obtained,

     1) wherein at least two of the amino acids within the desired peptide product comprise substituted or unsubstituted vinyl side chains capable of undergoing ring closing metathesis reaction to generate a first cross-linker, or

     2) wherein a first amino acid within the desired peptide product comprises a substituted or unsubstituted divinyl side chain, and wherein a second and a third amino acid within the desired peptide comprise substituted or unsubstituted olefins, whereby said divinyl side chain and said olefins are reactive in ring closing metathesis reactions to generate a second and a third cross-linker originating from the first amino acid; and

(g) contacting said desired peptide product with a ring closing metathesis catalyst to generate a library of cross-linked stabilized α

-helix peptide structures.

9. The method of claim 8, further comprising step h) diversifying the library of stabilized α

-helix peptides at a specific functional moiety of the cross-linked stabilized α

-helix peptide structures after the cross-linker is formed.

10. The method of claim 9, wherein hydroxyl moieties are introduced into members of the library of stabilized α

-helix peptides.

11. The method of claim 9, wherein diversifying comprises introducing functionalities of hydrogen, alkyl, aryl, phenoxy, methoxy, halide, benzene, heteroaryl, carboxyl, carboxylalkyl, carboxyaryl, and arylalkyl, thio, or hydroxyl into the cross-linked stabilized α

-helix peptide structures.

12. The method of claim 9, further comprising step i) screening the library of cross-linked stabilized a-helix peptide structures to identify those that promote or inhibit cell functions.

13. The method of claim 12, wherein inhibiting cell functions comprises disrupting binding of p53 to MDM2.