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

Patent metadata

Field	Value
Publication number	US-10435439-B2
Application number	US-201715489618-A
Country	US
Kind code	B2
Filing date	Apr 17, 2017
Priority date	Dec 3, 2010
Publication date	Oct 8, 2019
Grant date	Oct 8, 2019

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 (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 carbon-carbon double bond, a group containing at least an aromatic ring, or a ketone group, (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.

2. The peptide according to claim 1 , wherein in the formula (I),
(A) is selected from the class consisting of a single bond, alkylene groups having from 1 to 10 carbon atoms, alkenylene groups having from 2 to 10 carbon atoms, and alkynylene groups having from 2 to 10 carbon atoms, which may be substituted with a substituent; and alkylene groups, alkenylene groups, and alkynylene groups having, in the main chain thereof, 10 or less atoms, in which one or two carbon atoms in the main chain have been substituted with an oxygen atom, a nitrogen atom, or a sulfur atom and which may be substituted with a substituent,
(B) is selected from the class consisting of —C═C—, —Ar—, —Ar—Ar—, —Ar—C≡C—, —Ar—C═C—, —Ar—NHC(O)—, and —Ar—C(O)—,
(C) is selected from the class consisting of hydrogen, a methyl group, an ethyl group, a propyl group, a butyl group, and a pentyl group, and
X is selected from the class consisting of Cl, Br, I, —OSO 2 Me, a tosyl group, a nosyl group, and —OSO 2 —Ar—R, wherein, R represents a group selected from the class consisting of CH 3 , NO 2 , CF 3 , and H.

3. The peptide according to claim 1 , wherein the special amino acid of the formula (I) is represented by formula (III) or formula (IV):

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

4. 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):

5. 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.

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

7. The peptide library according to claim 6 , wherein each of the peptides is linked to an mRNA encoding the peptide.

8. A method of constructing the peptide library as claimed in claim 6 , 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 (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 carbon-carbon double bond, a group containing at least an aromatic ring, or a ketone group, (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, 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 (I) 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 (I).

9. A method of constructing the peptide library as claimed in claim 8 , comprising:
in 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).

10. The method according to claim 8 , wherein an altered codon encoding the special amino acid of the formula (I) 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 represents any one base of A, U, G, and C.

11. A method of selecting a peptide that binds to a target protein from the peptide library as claimed in claim 6 , 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.

12. A method of selecting, from the peptide library as claimed in claim 11 , a peptide having inhibitory activity against the intermolecular interaction of a target protein, comprising:
(i) primary screening, including the steps (i) and (ii), 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.

13. A process of preparing a peptide that binds to a target protein:
(i) bringing the peptide library as claimed in claim 7 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.

14. The method according to claim 11 , wherein the target protein is a molecule that suppresses apoptosis.

15. A special amino acid represented by the following 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 carbon-carbon double bond, or a group containing at least an aromatic ring, or a ketone group; (C) represents a hydrogen atom or an a

Assignees

• Univ Tokyo

Inventors

• Suga Hiroaki
• Higuchi Takashi

Classifications

• A61P43/00

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

• A61P35/00

  Antineoplastic agents · CPC title

• G01N33/6845

  Methods of identifying protein-protein interactions in protein mixtures · CPC title

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C12N15/1062

  mRNA-Display, e.g. polypeptide and encoding template are connected covalently · CPC title