Peptide with safer secondary structure, peptide library, and production methods for same

Patent metadata

Field	Value
Publication number	US-9657289-B2
Application number	US-201113990123-A
Country	US
Kind code	B2
Filing date	Dec 5, 2011
Priority date	Dec 3, 2010
Publication date	May 23, 2017
Grant date	May 23, 2017

Abstract

An object of the invention is to provide a peptide having a stabilized secondary structure.
The present invention provides a peptide having a secondary structure stabilized by a crosslinked structure and containing at least one combination of a special amino acid of the formula (I):

(wherein, (A) represents a single bond or a linking group having, in the main chain thereof, from 1 to 10 atoms; (B) represents a group containing at least one π bond; (C) represents a hydrogen atom or an alkyl group which may be substituted with a substituent; and X represents a group substitutable by a substitution reaction with a sulfanyl group) and an amino acid having, in the side chain thereof, a sulfanyl group; and having the crosslinked structure formed through a thioether bond between the side chain of the special amino acid residue and the sulfanyl group.

First claim

The invention claimed is:

1. A peptide having a secondary structure stabilized by a crosslinked structure, comprising
at least one combination of a special amino acid represented by the following formula (II):

wherein m represents an integer selected from 1 to 10 and x is selected from the group consisting of Cl, Br, I, —OSO 2 Me, a tosyl group, a nosyl group, and —OSO 2 —Ar—R and wherein, R is CH 3 , NO 2 , CF 3 , or H, and
an amino acid having, in the side chain thereof, a sulfanyl group; and having the crosslinked structure formed through a thioether bond between the side chain of the special amino acid residue and the sulfanyl group.

2. The peptide according to claim 1 , wherein the amino acid having a sulfanyl group is selected from the group consisting of cysteine and cysteine analogues represented by the following formulas (V) and (VI):

3. The peptide according to claim 1 , wherein the special amino acid residue and the amino acid residue having, in the side chain thereof, a sulfanyl group are, in each combination, placed with 2, 3, 6, or 10 amino acid residues therebetween.

4. A peptide library comprising two or more kinds of the peptide as claimed in claim 1 .

5. The peptide library according to claim 4 , wherein each of the peptides is linked to an mRNA encoding the peptide.

6. A method of constructing the peptide library as claimed in claim 4 , comprising:
(i) producing an mRNA library which contains at least one combination of a codon encoding an amino acid having, in the side chain thereof, a sulfanyl group and a codon encoding the special amino acid represented by the formula (II) in an RNA encoding a random amino acid sequence; in each combination, the codon encoding an amino acid having, in the side chain thereof, a sulfanyl group and the codon encoding the special amino acid of the formula (II) being placed with 2, 3, 6, or 10 amino acid units therebetween; and
(ii) translating the mRNA by using a cell-free translation system containing a tRNA to which the special amino acid has been linked and thereby obtaining a group of peptides having the special amino acid placed in the random sequence; and
(iii) forming, in each of the peptides, a crosslinked structure by binding the sulfanyl group to the side chain of the special amino acid of the formula (II), wherein formula (II) is

wherein m represents an integer selected from 1 to 10 and x is selected from the group consisting of Cl, Br, I, —OSO 2 Me, a tosyl group, a nosyl group, and —OSO 2 —Ar—R, and wherein, R is CH 3 , NO 2 , CF 3 , or H.

7. A method of constructing the peptide library as claimed in claim 6 , comprising:
in the step (i), binding puromycin to the 3′-end of the mRNA to obtain a puromycin-bound mRNA library;
in the step (ii), causing the puromycin-bound mRNA library to express in a cell-free translation system to obtain a peptide-mRNA complex having the special amino acid placed in the random sequence; and
conducting the step (iii).

8. The method according to claim 6 , wherein an altered codon encoding the special amino acid of the formula (II) is AUG codon and the mRNA random sequence is composed of repetition of a triplet of either one of an NNC or NNU sequence, wherein N is A, U, G, is C.

9. A special amino acid represented by the following formula (II);

wherein m represents an integer selected from 1 to 10 and x is selected from the group consisting of Cl, Br, I, —OSO 2 Me, a tosyl group, a nosyl group, and —OSO 2 —Ar—R, and wherein, R is CH 3 , NO 2 , CF 3 , or H.

10. A method of selecting a peptide that binds to a target protein from the peptide library as claimed in claim 4 , comprising:
(i) bringing the peptide library into contact with the target protein, followed by incubation; and
(ii) selecting a peptide molecule that binds to the target protein.

11. The method according to claim 10 , wherein the peptide being selected has inhibitory activity against the intermolecular interaction of a target protein, comprising:
(i) primary screening, including the steps (i) and (ii) as claimed in claim 10 , for selecting a peptide that binds to the target protein; and
(ii) secondary screening for evaluating inhibitory activity of the peptide, which has been selected in the primary screening (i), against intermolecular interaction of the target protein and thereby determining that the peptide has inhibitory activity against the intermolecular interaction of the target protein.

12. A process of preparing a peptide that binds to a target protein:
(i) bringing the peptide library as claimed in claim 5 into contact with the target protein while incubating;
(ii) selecting a peptide-mRNA complex that binds to the target protein;
(iii) amplifying the mRNA of the selected peptide-mRNA complex to obtain a peptide-mRNA complex;
(iv) repeating the steps (i) to (iii) at least once to concentrate the peptide-mRNA complex having high-affinity; and
(v) causing the mRNA of the peptide-mRNA complex concentrated in the step (iv) to express the peptide.

13. The method according to claim 10 , wherein the target protein is a molecule that suppresses apoptosis.

Assignees

• Suga Hiroaki
• Higuchi Takashi
• Univ Tokyo

Inventors

• Suga Hiroaki
• Higuchi Takashi

Classifications

• A61P35/00

  Antineoplastic agents · CPC title

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• C40B40/10

  Libraries containing peptides or polypeptides, or derivatives thereof · CPC title

• C07K2/00

  Peptides of undefined number of amino acids; Derivatives thereof · CPC title

• C07K14/001 Primary

  by chemical synthesis · CPC title