Compositions and methods for capture of cellular targets of bioactive agents

Patent metadata

Field	Value
Publication number	US-10976312-B2
Application number	US-201715402879-A
Country	US
Kind code	B2
Filing date	Jan 10, 2017
Priority date	Dec 12, 2012
Publication date	Apr 13, 2021
Grant date	Apr 13, 2021

Abstract

The present invention provides compositions and methods for capture and identification of the cellular targets of a bioactive agent. In particular, provided herein are bioactive agents tethered to capture ligand, cellular targets (optionally tagged with a reporter), capture proteins (optionally present as capture fusions), surfaces (e.g., displaying, capture ligands, capture proteins, or capture fusions), and methods of capturing and identifying the cellular targets of a bioactive agent therewith.

First claim

We claim:

1. A composition comprising:
(a) lysed cells comprising a cellular target of a small molecule bioactive agent;
(b) the small molecule bioactive agent tethered to a chloroalkane capture ligand, wherein the small molecule bioactive agent is capable of non-covalently binding to the cellular target; and
(c) a solid surface displaying a dehalogenase capture protein, wherein the dehalogenase capture protein is capable of forming a covalent bond with the chloroalkane capture ligand upon interaction thereof.

2. The composition of claim 1 , wherein the cellular target is a fusion with a reporter protein.

3. The composition of claim 2 , wherein the reporter protein is a bioluminescent reporter.

4. The composition of claim 3 , wherein the bioluminescent reporter comprises a polypeptide with at least 70% sequence identity with SEQ ID NO.: 3.

5. The composition of claim 1 , wherein the dehalogenase capture protein comprises at least 70% sequence identity with SEQ ID NO.: 1.

6. The composition of claim 1 , wherein the solid surface is selected from the group consisting of well, tube, slide, plate, matrix, resin and bead.

7. The composition of claim 6 , wherein the solid surface is magnetic or paramagnetic.

8. The composition of claim 1 , wherein the cellular target is bound to the small molecule bioactive agent, and the capture protein is bound to the capture ligand.

Assignees

• Promega Corp

Inventors

• Hitko Carolyn W
• Hurst Robin
• Kirkland Thomas
• Klaubert Dieter
• Meisenheimer Poncho
• Nath Nidhi
• Ohana Rachel Friedman
• Otto Paul
• Urh Marjeta
• Uyeda Harry Tetsuo
• Wood Keith
• Zhu Ji

Classifications

• G01N33/54306

  Solid-phase reaction mechanisms · CPC title

• G01N33/54326 Primary

  Magnetic particles · CPC title

• G01N33/54333 Primary

  Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction · CPC title