Butelase-mediated peptide ligation

Patent metadata

Field	Value
Publication number	US-11091786-B2
Application number	US-201615765156-A
Country	US
Kind code	B2
Filing date	Sep 29, 2016
Priority date	Oct 1, 2015
Publication date	Aug 17, 2021
Grant date	Aug 17, 2021

Abstract

The present invention relates to a method of forming a peptide of Formula (I) (P 1 -Xaa 1 -Xaa 2 -P 2 ) by ligating a first peptide of Formula (II) (P 1 -Xaa 1 -X—R, wherein X is O or S) to a second peptide of Formula (III) (Xaa 1 -Xaa 2 -P 2 ) by enzymatically cleaving the bond between “Asx” and “X” in the first peptide of Formula (II) and ligating the fragment P 1 -Asx of the first peptide to the second peptide of Formula (III), wherein the enzymatic cleavage and ligation reaction is catalyzed by butelase 1 (SEQ ID NO: 1) and the peptide of Formula (I) is a depsipeptide, preferably a thiodepsipeptide. Further encompassed are peptides and dendrimeric peptide assemblies prepared using the presently disclosed method, as well as use of the dendrimeric peptide assemblies as a vaccine, medicament, or diagnostic agent, particularly as an antimicrobial agent.

First claim

What is claimed is:

1. A method of forming a peptide of Formula (I)
P 1 -Asx-Xaa 1 -Xaa 2 -P 2 (I), the method comprising:
enzymatically cleaving a bond between “Asx” and “X” in a first peptide of Formula (II)
P 1 -Asx-X—R  (II); and

ligating a P 1 -Asx fragment of the first peptide of Formula (II) to a second peptide of Formula (III)
Xaa 1 -Xaa 2 -P 2 (III) to form the peptide of Formula (I);
wherein P 1 and P 2 are each independently any peptide, modified or unmodified; Asx is Asp or Asn; X is S; R is a substituted or unsubstituted alkyl selected from the group consisting of
—(CH 2 ) n —CONH 2 , —(CH 2 ) n —COOH, —(CH 2 ) n —CO-AA 1 ,

wherein n is an integer of 1 to 10, R 1 is H, or any other side chain of a naturally occurring amino acid, and AA 1 is any amino acid or is absent; Xaa 1 is any naturally occurring amino acid with the exception of Pro; Xaa 2 is a naturally occurring amino acid;
wherein the enzymatic cleavage and ligation are catalyzed by a polypeptide having at least 90% of the ligase activity of butelase 1 (SEQ ID NO:1).

2. The method according to claim 1 , wherein Asx is Asn, R is —CH(R 1 )—CO-AA 1 , R 1 is H, and AA 1 is any amino acid or absent.

3. The method according to claim 1 , wherein Asx is Asn, R is —CH(R 1 )—CO-AA 1 , R 1 is H, and AA 1 is Val.

4. The method according to claim 1 , wherein the polypeptide having at least 90% of the ligase activity of butelase 1 (SEQ ID NO:1) comprises:
(a) the amino acid sequence as set forth in SEQ ID NO:1 (butelase 1);
(b) an amino acid sequence that shares at least 80% sequence identity with the amino acid sequence as set forth in SEQ ID NO:1; or
(c) a fragment of any one of (a)-(b).

5. The method according to claim 1 , wherein the polypeptide having the ligase activity of butelase 1 (SEQ ID NO:1) comprises the amino acid sequence as set forth in SEQ ID NO:2.

6. The method according to claim 1 , wherein the polypeptide having at least 90% of the ligase activity of butelase 1 (SEQ ID NO:1) comprises
(a) the amino acid residue Asn at the position corresponding to position 19 of SEQ ID NO:1; and/or
(b) the amino acid residue His at the position corresponding to position 124 of SEQ ID NO:1; and/or
(c) the amino acid residue Cys at the position corresponding to position 166 of SEQ ID NO:1.

7. The method according to claim 1 , wherein the polypeptide having at least 90% of the ligase activity of butelase 1 (SEQ ID NO:1) comprises
(a) any one of the amino acid sequences as set forth in SEQ ID Nos:3-109;
(b) an amino acid sequence that shares at least 80% sequence identity with any one of the amino acid sequences of (a) over its entire length;
(c) a fragment of any one of (a)-(b).

8. The method according to claim 1 , wherein P 1 or P 2 is modified by one or more of an affinity tag, a detectable label, a solid support material, a scaffold molecule, or any combination thereof.

9. The method according to claim 1 , wherein P 1 or P 2 is modified by one or more of a biotin, a fluorescent marker, a polymer resin, a dendrimer, or any combination thereof.

10. The method according to claim 9 , wherein P 1 or P 2 is modified by a dendrimer.

11. The method according to claim 10 , wherein each dendrimer is conjugated to 2 or more copies of the second peptide via P 2 , such that the ligation of the first peptide and the second peptide results in a dendrimeric peptide assembly comprising 2 or more copies of the ligated peptide P 1 -Asx-Xaa 1 -Xaa 2 -P 2 .

12. The method according to claim 10 , wherein each dendrimer is a lysyl dendrimer.

13. The method according to claim 1 , wherein the first peptide is P 1 -Asn-thioglc-Val, and P 1 is an antimicrobial peptide.

14. The method according to claim 13 , wherein P 1 is an antimicrobial peptide comprising a BHHB tetrapeptide motif.

15. The method according to claim 14 , wherein P 1 is an antimicrobial peptide comprising an Arg-Leu-Tyr-Arg (SEQ ID NO:121) tetrapeptide.

16. The method according to claim 15 , wherein the first peptide is Ac-Arg-Leu-Tyr-Arg-Asn-thioglc-Val (SEQ ID NO:120).

17. The method according to claim 1 , wherein the second peptide is Arg-Ile-βAla conjugated to a lysyl dendrimer via βAla.

18. The method according to claim 12 , wherein the first peptide is Ac-Arg-Leu-Tyr-Arg-Asn-thioglc-Val (SEQ ID NO:120), and each lysyl dendrimer is conjugated to 2 or more copies of the second peptide Arg-Ile-βAla via βAla, such that the ligation of the first peptide and the second peptide results in a dendrimeric peptide assembly comprising 2 or more copies of Ac-Arg-Leu-Tyr-Arg-Asn-Arg-Ile-βAla (SEQ ID NO:131).

19. A dendrimeric peptide assembly comprising 2 or more copies of Ac-Arg-Leu-Tyr-Arg-Asn-Arg-Ile-βAla (SEQ ID NO:131).

20. The method according to claim 1 , wherein Xaa 2 is a hydrophobic amino acid or Cys.

Assignees

• Univ Nanyang Tech

Inventors

• Tam James P
• Nguyen Kien Truc Giang
• Cao Yuan
• Liu Chuan Fa

Classifications

• C07K1/02

  in solution {(C07K1/003, C07K1/006 take precedence)} · CPC title

• C12P21/02 Primary

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

• C07K5/1019

  with the first amino acid being basic · CPC title

• C07K7/06

  having 5 to 11 amino acids · CPC title

• C12N9/93

  Ligases (6) · CPC title