Cell-permeable, cell-compatible, and cleavable linkers for covalent tethering of functional elements

• US 10,618,907 B2
• Filed: 06/03/2016
• Issued: 04/14/2020
• Est. Priority Date: 06/05/2015
• Status: Active Grant

• Current Assignee: Promega Corporation
• Original Assignee: Promega Corporation
• Inventors: Levin, Sergiy, Ohana, Rachel Friedman, Kirkland, Thomas, Wood, Keith V.
• Primary Examiner(s): Haq, Shafiqul
• Application Number:
  US15/172,860
• Publication Number:
  US 20160355523A1
• Time in Patent Office: 1,411 Days
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: C07D 231/40 Acylated on said nitrogen atom
• CPC Class Codes: C07D 487/04 Ortho-condensed systems
• CPC Class Codes: C07D 491/22 in which the condensed system contains four or more hetero rings

Abstract

Provided herein are cell-permeable, cell-compatible, and chemoselectively-cleavable linkers for tethering (e.g., covalently) functional elements (e.g., a cellular interaction element and a capture element), and methods of use (e.g., intracellular capture and extracellular release of cellular targets) therewith.

Claims

1. A dual-function, cell-permeable, cell-compatible, chemoselectively-cleavable composition, comprising:

(a) a cellular interaction element, wherein the cellular interaction element is capable of non-covalently binding to a cellular target protein;

(b) a capture element comprising a haloalkane group; and

(c) a linker covalently tethering the cellular interaction element to the capture element, wherein said linker comprises a chemoselectively cleavable moiety selected from the group consisting of an allyl-heteroatom group, a propargyl-heteroatom group.

2. The composition of claim 1, wherein the cellular interaction element is a small molecule or peptide.

3. The composition of claim 1, wherein the capture element is a covalent substrate for an enzyme.

4. The composition of claim 1, wherein the capture element is an affinity element.

5. The composition of claim 1, wherein the chemoselectively cleavable moiety comprises an allyl-heteroatom group selected from the group consisting of:

allyl ether, allyl amine, allyl ester, allyl amide, allyl urea, allyl carbonate, and allyl carbamate.

6. The composition of claim 5, wherein the chemoselectively-cleavable moiety comprises an allyl carbamate group.

7. The composition of claim 1, wherein the chemoselectively-cleavable moiety comprises a propargyl-heteroatom group selected from the group consisting of:

propargyl ether, propargyl amine, propargyl ester, propargyl amide, propargyl urea, propargyl carbonate, and propargyl carbamate.

8. A method comprising:

(a) administering the dual-function, cell-permeable, cell compatible, chemoselectively-cleavable composition of claim 1 to a cell;

(b) allowing binding of the chemoselectively-cleavable composition via the cellular interaction element with a corresponding intracellular cellular target to provide chemoselectively-cleavable composition bound intracellular target;

(c) lysing said cell to produce a cell lysate; and

(d) providing surface immobilized intracellular target by contacting the cell lysate with a surface immobilized capture agent specific for the capture element of the chemoselectively-cleavable composition to immobilize the chemoselectively-cleavable composition bound intracelluar target on the surface.

9. The method of claim 8, further comprising:

(e) contacting the surface immobilized intracellular target with a chemoselective agent capable of cleaving the chemoselectivly-cleavable linker, thereby releasing the cellular interaction element and the bound intracellular target from the surface.

10. A system comprising:

(a) the dual-function, cell-permeable, cell-compatible, chemoselectively-cleavable composition of claim 1; and

(b) one or more of;

(i) a cell,

(ii) a cellular target,

(iii) a surface-displayed capture agent, and

(iv) a chemoselective agent.