Azoline compound and azole compound library and method for producing same

Patent metadata

Field	Value
Publication number	US-10197567-B2
Application number	US-201214003506-A
Country	US
Kind code	B2
Filing date	Mar 9, 2012
Priority date	Mar 9, 2011
Publication date	Feb 5, 2019
Grant date	Feb 5, 2019

Abstract

An object of the present invention is to provide a method of efficiently constructing a library abundant in diversity and also usable for screening of a compound that binds to a target substance having protease activity.
The present invention provides a method of constructing an azoline compound library containing two or more azoline compounds having an azoline backbone introduced into at least one of Cys, Ser, Thr, and 2,3-diamino acid, and analogs thereof of Xaa 0 of a peptide represented by the following formula (I):

A-(Xaa 0 ) n -B  (I)

[wherein, m numbers of Xaa 0 s respectively represent arbitrary amino acids, at least one of which is an amino acid selected from the group consisting of Cys, Ser, Thr, and 2,3-diamino acid, and analogs thereof, m represents an inter selected from 2 to 40, and A and B each independently represent a peptide composed of from 0 to 100 amino acids].

First claim

The invention claimed is:

1. A method of constructing an azoline compound library containing two or more azoline compounds having an azoline backbone introduced into at least one of Cys, Ser, Thr, and 2,3-diamino acid, and analogs thereof of Xaa 0 of a peptide comprising a sequence represented by -(Xaa 0 ) m -, wherein m is an integer selected from 2 to 40, and each Xaa 0 is an arbitrary amino acid, at least one of which is an amino acid that forms an azoline ring in the presence of heterocyclase and comprises the structural formula (II):

wherein R represents a hydrogen atom, a substituted or unsubstituted alkyl group having from 1 to 10 carbon atoms, or a substituted or unsubstituted aromatic group, X is O, S, or NH, comprising:
constructing an mRNA library encoding a plurality of precursor peptides, each precursor peptide independently comprising -(Xaa 0 ) m -, which is a patellamide C cassette domain modified to have one or more amino acid substitutions, deletions, and/or additions, and each precursor peptide independently and optionally comprises a recognition sequence 1 at its N-terminus, a recognition sequence 2 at its C-terminus, or both, and wherein the recognition sequences are recognized by the azoline backbone introducing enzyme;
expressing the plurality of precursor peptides in a cell-free translation system by using the mRNA library and thereby constructing a peptide library; and
reacting the azoline backbone-introducing enzyme and the peptide library, optionally in the presence a leader sequence of a substrate of the azoline backbone-introducing enzyme, and thereby introducing the azoline backbone into (Xaa 0 ) m ,
wherein the leader sequence is present where the recognition sequences are absent.

2. The method of constructing an azoline compound library according to claim 1 , wherein in the step of expressing the plurality of precursor peptides, each mRNA is bound to puromycin at its 3′ end prior to expressing each precursor peptide and thereby constructing a peptide library comprising peptide-mRNA complexes.

3. The method of constructing an azoline compound library according to claim 1 , wherein:
(Xaa 0 ) m - includes -(Xaa 1 -Xaa 2 ) n - wherein n represents an integer selected from 1 to 20, and Xaa 2 comprises the structural formula (II).

4. The method according to claim 1 , wherein at least one of the peptides of the peptide library is a peptide represented by any of SEQ ID NOS: 10 to 57.

5. The method according to claim 1 , wherein the leader sequence is provided as a fusion peptide, wherein the leader sequence is fused to a precursor peptide of the plurality of precursor peptides, said precursor peptide comprising a recognition sequence 1, -(Xaa 0 ) m -, and a recognition sequence 2.

6. The method according to claim 1 , wherein the leader sequence is not fused to the substrate of the azoline backbone-introducing enzyme.

7. The method according to claim 1 , further comprising macrocyclizing the azoline compound.

8. A method of constructing an azole compound library, comprising, after the step of introducing an azoline backbone in the method of constructing an azoline compound library according to claim 1 ,
reacting the library having an azoline backbone introduced therein with an azole backbone-introducing enzyme in the presence or absence of a peptide comprising a leader sequence of a substrate of the azole backbone-introducing enzyme and converting at least one of the azoline backbones into an azole backbone.

9. A screening method for identifying an azoline compound that binds to a target substance, comprising:
constructing an azoline compound library containing two or more azoline compounds having an azoline backbone introduced into at least one of Cys, Ser, Thr, and 2,3-diamino acid, and analogs thereof of Xaa 0 of a peptide comprising a sequence represented by -(Xaa 0 ) m -, wherein m is an integer selected from 2 to 40, and each Xaa 0 is an arbitrary amino acid, at least one of which is an amino acid that forms an azoline ring in the presence of heterocyclase and comprises the structural formula (II):

wherein R represents a hydrogen atom, a substituted or unsubstituted alkyl group having from 1 to 10 carbon atoms, or a substituted or unsubstituted aromatic group, X is O, S, or NH, by:
constructing an mRNA library encoding a plurality of precursor peptides, each precursor peptide independently comprising -(Xaa 0 ) m -, which is a patellamide C cassette domain modified to have one or more amino acid substitutions, deletions, and/or additions, and each precursor peptide independently and optionally comprises a recognition sequence 1 at its N-terminus, a recognition sequence 2 at its C-terminus, or both, and wherein the recognition sequences are recognized by the azoline backbone introducing enzyme;
expressing the plurality of precursor peptides in a cell-free translation system by using the mRNA library and thereby constructing a peptide library; and
reacting the azoline backbone-introducing enzyme and the peptide library, optionally in the presence a leader sequence of a substrate of the azoline backbone-introducing enzyme, and thereby introducing the azoline backbone into (Xaa 0 ) m ,
wherein the leader sequence is present where the recognition sequences are absent;

bringing the azoline compound library into contact with a target substance, followed by incubation, and
selecting the azoline compound that has bound to the target substance.

10. The screening method according to claim 9 , wherein in the step of expressing the plurality of precursor peptides, each mRNA is bound to puromycin at its 3′ end prior to expressing each precursor peptide and thereby constructing a peptide library comprising peptide-mRNA complexes, and further comprising:
analyzing the base sequence of the mRNA of the azoline compound thus selected.

11. A method of preparing an azoline compound having an azoline backbone introduced into at least one of Cys, Ser, Thr, and 2,3-diamino acid, and analogs thereof of Xaa 0 of a peptide comprising a sequence represented by -(Xaa 0 ) m -, wherein m is an integer selected from 2 to 40, and each Xaa 0 is an arbitrary amino acid, at least one of which is an amino acid that forms an azoline ring in the presence of azoline backbone-introducing enzyme and comprises the structural formula (II):

wherein R represents a hydrogen atom, a substituted or unsubstituted alkyl group having from 1 to 10 carbon atoms, or a substituted or unsubstituted aromatic group, X is O, S, or NH, comprising:
preparing an mRNA encoding a precursor peptide comprising -(Xaa 0 ) m -, which is a patellamide C cassette domain modified to have one or more amino acid substitutions, deletions, and/or additions, said precursor polypeptide optionally comprising a recognition sequence 1 at its N-terminus, a recognition sequence 2 at its C-terminus, or both, and wherein the recognition sequences are recognized by the azoline backbone introducing enzyme, and wherein at least one Xaa 0 of the precursor peptide is a noncanonical amino acid;
expressing the precursor peptide in a cell-free translation system by using the mRNA; and
reacting the azoline backbone-introducing enzyme and the precursor peptide in the presence of a leader sequence of a substrate of the azoline backbone-introducing enzyme and thereby introducing an azoline backbone into (Xa

Assignees

• Suga Hiroaki
• Goto Yuki
• Ito Yumi
• Univ Tokyo

Inventors

• Suga Hiroaki
• Goto Yuki
• Ito Yumi

Classifications

• G01N33/54393 Primary

  Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding · CPC title

• C07K14/47

  from mammals · CPC title

• C12N15/1062

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

• C12P17/16

  containing two or more hetero rings {(thiamine open chain analogs C12P17/167, i.e. not condensed among themselves or through a common carbocyclic ring system)} · CPC title

• C12Q1/37 Primary

  involving peptidase or proteinase · CPC title