Methods for ligand discovery
The present invention provides novel methods for ligand discovery. The inventive methods rely on a process termed “tethering” where potential ligands are covalently bonded or “tethered” to a target and subsequently identified.
1. A method comprising:
a) providing a target having a reactive nucleophile at or near a site of interest;
b) contacting the target with an extender thereby forming a target-extender complex wherein the extender comprises a first functionality that reacts with the nucleophile in the target to form a covalent bond and a second functionality that is capable of forming a disulfide bond, wherein the extender is of the formula:
where R is unsubstituted C 1 –C 20 aliphatic, substituted C 1 –C 20 aliphatic, unsubstituted aryl, and substituted aryl; R′ is H, —SR 1 wherein R 1 is unsubstituted C 1 –C 10 aliphatic, substituted C 1 –C 10 aliphatic, unsubstituted aryl, and substituted aryl; X is a leaving group, and the boxes in each formula represent a binding determinant;
c) contacting the target-extender complex with a candidate ligand that is capable of forming a disulfide bond with said target-extender complex;
d) forming a disulfide bond between the target-extender complex and the candidate ligand thereby forming a target-extender-ligand conjugate; and,
e) identifying the candidate ligand present in the target-extender-ligand conjugate.
2. The method of claim 1 , wherein the candidate ligand present in the target-extender-ligand conjugate is identified by mass spectrometry.
3. The method of claim 1 , wherein the candidate ligand present in the target-extender-ligand conjugate is identified using a labeled probe.
4. The method of claim 1 , wherein the candidate ligand present in the target-extender-ligand conjugate is identified using a functional assay.
5. The method of claim 1 , wherein the candidate ligand present in the target-extender-ligand conjugate is identified using chromatography.
6. The method of claim 1 , wherein the candidate ligand present in the target-extender-ligand conjugate is identified using surface plasmon resonance.
7. A method comprising:
a) providing a target having a reactive nucleophile at or near a site of interest;
b) contacting the target with an extender thereby forming a target-extender complex wherein the extender comprises a first functionality that reacts with the nucleophile in the target to form a covalent bond and a second functionality that is capable of forming a disulfide bond, wherein the extender is of the formula:
where R is unsubstituted C 1 –C 20 aliphatic, substituted C 1 –C 20 aliphatic, unsubstituted aryl, and substituted aryl; R′ is H, —SR 1 wherein R 1 is unsubstituted C 1 –C 10 aliphatic, substituted C 1 –C 10 aliphatic, unsubstituted aryl, and substituted aryl; X is a leaving group, and the box in the formula represents a binding determinant;
c) contacting the target-extender complex with a candidate ligand that is capable of forming a disulfide bond with said target-extender complex;
d) forming a disulfide bond between the target-extender complex and the candidate ligand thereby forming a target-extender-ligand conjugate; and
e) identifying the candidate ligand present in the target-extender-ligand conjugate.
8. The method of claim 7 , wherein the candidate ligand present in the target-extender-ligand conjugate is identified by mass spectrometry.
9. The method of claim 7 , wherein the candidate ligand present in the target-extender-ligand conjugate is identified using a labeled probe.
10. The method of claim 7 , wherein the candidate ligand present in the target-extender-ligand conjugate is identified using a functional assay.
11. The method of claim 7 , wherein the candidate ligand present in the target-extender-ligand conjugate is identified using chromatography.
12. The method of claim 7 , wherein the candidate ligand present in the target-extender-ligand conjugate is identified using surface plasmon resonance.