Compositions and methods for capture of cellular targets of bioactive agents
View Patent ↗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.
1. A method of capturing a cellular target comprising the steps of:
(a) administering to a cell, a conjugate comprising a small molecule bioactive agent tethered by a carbamate linker to a chloroalkane capture ligand, the cell comprising a cellular target of the small molecule bioactive agent, under conditions such that the cellular target non-covalently binds the small molecule bioactive agent;
(b) lysing the cell to produce a cell lysate;
(c) contacting the cell lysate with a solid surface displaying a capture protein comprising a dehalogenase modified to covalently bind to the chloroalkane capture ligand under conditions in which the capture protein forms a covalent bond with the chloroalkane capture ligand; and
(d) separating the solid surface with the bound cellular target from the cell lysate.
2. The method of claim 1 , wherein the cellular target is endogenous to the cell.
3. The method of claim 1 , wherein the linker is a cleavable linker.
4. The method of claim 1 , wherein the small molecule bioactive agent tethered to the capture ligand is cell permeable.
5. The method of claim 1 , wherein the tethering of the small molecule bioactive agent to the capture ligand has minimal impact on the cell permeability and/or potency of the small molecule bioactive agent.
6. The method of claim 1 , further comprising
(e) detecting or analyzing the cellular target bound to the solid surface.
7. The method of claim 6 , wherein the detecting or analyzing comprises mass spectrometry (MS).
8. The method of claim 1 , further comprising:
(e) eluting the cellular target from the solid surface; and
(f) detecting, identifying, or analyzing the cellular target that was bound to the solid surface.
9. The method of claim 8 , wherein the detecting or analyzing comprises mass spectrometry (MS).
10. The method of claim 1 , wherein the cellular target is expressed intracellularly as a fusion with a reporter.
11. The method of claim 10 , wherein the reporter is a bioluminescent reporter.
12. The method of claim 11 , wherein the bioluminescent reporter comprises a polypeptide with at least 70% sequence identity with SEQ ID NO.: 3.
13. The method of claim 1 , wherein the capture protein comprises at least 70% sequence identity with SEQ ID NO.: 1.
14. The method of claim 1 , wherein the solid surface is selected from the group consisting of a well, a tube, a slide, a plate, a matrix, resin and a bead.
15. The method of claim 14 , wherein the solid surface is magnetic or paramagnetic.
16. The method of claim 1 , wherein the cell is a live cell.