Translocation of non-natural chemical entities through anthrax protective antigen pore

Patent metadata

Field	Value
Publication number	US-9498538-B2
Application number	US-201314402856-A
Country	US
Kind code	B2
Filing date	May 21, 2013
Priority date	May 21, 2012
Publication date	Nov 22, 2016
Grant date	Nov 22, 2016

Abstract

Disclosed is a new approach for delivering compounds and drugs to the cytosol of living cells through the use of engineered protein transporters. The engineered protein transporters include a pore and a pore specific delivery protein, wherein a reagent such as a drug is attached to one or more of the engineered protein transporters.

First claim

The invention claimed is:

1. A method of disrupting a molecular interaction in a living cell, comprising
contacting the living cell with a pore forming protein and a fusion molecule comprising a pore specific delivery protein linked to a reagent, wherein the reagent is delivered to the cytosol of the living cell in an effective amount for disrupting a molecular interaction in the living cell.

2. The method of claim 1 , wherein the reagent is a labeled compound, a halogenated compound, a morpholino, a therapeutic RNA, a protein mimic, antibody mimic, a minor image biomolecule or a monobody, or an engineered protein scaffold.

3. The method of claim 2 , wherein the labeled compound is a peptide labeled with a biotin or a click chemistry reagent.

4. The method of claim 2 , wherein the halogenated compound is a fluorinated peptide.

5. The method of claim 2 , wherein the protein mimic is an antibody mimic.

6. The method of claim 1 , wherein the molecular interaction is a protein-protein binding interaction and the reagent inhibits the protein-protein binding.

7. The method of claim 1 , wherein the molecular interaction is a nucleic acid-protein binding interaction and the reagent inhibits the nucleic acid-protein binding.

8. The method of claim 1 , wherein the molecular interaction is a nucleic acid function and the reagent inhibits the nucleic acid function.

9. A fusion molecule, comprising a pore specific delivery protein linked to a reagent, wherein the reagent is a labeled compound, a halogenated compound, a morpholino, a therapeutic RNA, a protein mimic, antibody mimic, a minor image biomolecule or a monobody, or an engineered protein scaffold.

10. The fusion protein of claim 9 , wherein the labeled compound is a peptide labeled with a biotin or a click chemistry reagent.

11. The fusion protein of claim 9 , wherein the halogenated compound is a fluorinated peptide.

12. A kit comprising:
a container housing together or in separate compartments a pore forming protein , a pore specific delivery protein, a peptide thioester and instructions for preparing a fusion protein and delivering the fusion protein to a living cell.

13. The kit of claim 12 , further comprising a SrtA enzyme, which is optionally SrtA*.

14. The kit of claim 12 , wherein the peptide thioester is G n -Xaa-COSR, wherein n is 1-6 and wherein Xaa is an amino acid (SEQ ID NOs 1-4).

15. The kit of claim 12 , wherein the peptide thioester is G n -Xaa-COSR, wherein n is 3-5 and, wherein Xaa is Gly, Phe, Ser or Leu (SEQ ID NOs 5-7).

16. The kit of claim 12 , wherein the peptide thioester is GGGGG-Xaa-COSR, wherein Xaa is Gly, Phe, Ser or Leu (SEQ ID NO: 7).

17. The kit of claim 12 , wherein the peptide thioester is G n -X m -COSR (SEQ ID NO: 8), wherein n is 1-6, m is 1-6, and wherein X is an amino acid, naturally occurring or non-naturally occurring.

18. The kit of claim 17 , wherein X is a D-amino acid.

19. The kit of claim 12 , wherein the peptide thioester is G n -Y-COSR (SEQ ID NO: 9-11), wherein n is 1-6 and wherein Y is a non-amino acid chemical entity.

20. The kit of claim 19 , wherein in Y is a PEG unit.

21. A method for delivering a reagent to the cytosol of a living cell, comprising
contacting the living cell with a pore forming protein and a fusion molecule comprising a pore specific delivery protein linked to a reagent, wherein the reagent is delivered to the cytosol of the targeted living cell, and wherein the fusion molecule is prepared using a continuous flow enzymatic ligation reaction by flowing a N-terminal pore specific delivery protein and a peptide thioester comprising the reagent over a stationary phase containing a cysteine transpeptidase enzyme, wherein a N-terminal protein -COSR product is formed, and flowing a C-terminal protein over the stationary phase, wherein the C-terminal protein domain has a cysteine at the N-termini, to produce a modified protein having a chemical entity linking the N-terminal pore specific delivery protein and the C-terminal protein domain and wherein the linked molecule is the fusion molecule.

22. The method of claim 21 , wherein the cysteine transpeptidase enzyme is a sortase.

Assignees

• Liao Xiaoli
• Rabideau Amy E
• Pentelute Bradley L
• Ling Jingjing
• Akçay Gizem
• Collier John
• Massachusetts Inst Technology
• Harvard College

Inventors

• Liao Xiaoli
• Rabideau Amy E
• Pentelute Bradley L
• Ling Jingjing
• Akçay Gizem
• Collier John

Classifications

• C07K1/107 Primary

  by chemical modification of precursor peptides · CPC title

• A61K47/48261

  Human Necessities · mapped topic

• C12P21/06

  produced by the hydrolysis of a peptide bond, e.g. hydrolysate products (preparing foodstuffs by protein hydrolysis A23J3/00) · CPC title

• A61K47/48346 Primary

  Human Necessities · mapped topic

• C07K1/047

  Simultaneous synthesis of different peptide species; Peptide libraries · CPC title