Extended tethering approach for rapid identification of ligands
First Claim
Patent Images
1. A process comprising the steps of:
- (i) contacting a Target Biological Molecule (TBM) having a first and a second site of interest, and containing or modified to contain a nucleophile, selected from the group consisting of thiol, protected thiol, reversible disulfide, hydroxyl, protected hydroxyl, amino, protected amino, carboxyl and protected carboxyl groups, at or near the first site of interest, with a plurality of first organic ligand candidates, said candidates having a functional group reactive with the nucleophile, under conditions such that a reversible covalent bond is formed between the nucleophile and a candidate that has affinity for the first site of interest, to form a TMB-first ligand complex;
(ii) identifying the first ligand from the TBM-first ligand complex;
(iii) designing a derivative of the first ligand identified in (ii) to provide a small molecule extender (SME) having a first functional group reactive with the nucleophile on the TBM and a second functional group reactive with a second ligand having affinity for the second site of interest, wherein said first functional group is capable of forming an irreversible covalent group with the thiol, protected thiol, reversible disulfide bond, hydroxyl, protected hydroxyl, amino, protected amino, carboxyl or protected carboxyl group on said TBM;
(iv) contacting the SME with the TBM to form a TBM-SME complex, and (v) contacting the TBM-SME complex with a plurality of second small organic ligand candidates, said candidates having a functional group reactive with the SME in said TBM-SME complex, wherein a candidate that has affinity for said second site of interest on said TBM forms a reversible covalent bond with said TBM-SME complex, whereby a second ligand is identified.
1 Assignment
0 Petitions
Accused Products
Abstract
The invention concerns a method for rapid identification and characterization of binding partners for a target molecule, and for providing binding partners with improved binding affinity. More specifically, the invention concerns an improved tethering method for the rapid identification of at least two binding partners that bind near one another to a target molecule.
20 Citations
27 Claims
-
1. A process comprising the steps of:
- (i) contacting a Target Biological Molecule (TBM) having a first and a second site of interest, and containing or modified to contain a nucleophile, selected from the group consisting of thiol, protected thiol, reversible disulfide, hydroxyl, protected hydroxyl, amino, protected amino, carboxyl and protected carboxyl groups, at or near the first site of interest, with a plurality of first organic ligand candidates, said candidates having a functional group reactive with the nucleophile, under conditions such that a reversible covalent bond is formed between the nucleophile and a candidate that has affinity for the first site of interest, to form a TMB-first ligand complex;
(ii) identifying the first ligand from the TBM-first ligand complex;
(iii) designing a derivative of the first ligand identified in (ii) to provide a small molecule extender (SME) having a first functional group reactive with the nucleophile on the TBM and a second functional group reactive with a second ligand having affinity for the second site of interest, wherein said first functional group is capable of forming an irreversible covalent group with the thiol, protected thiol, reversible disulfide bond, hydroxyl, protected hydroxyl, amino, protected amino, carboxyl or protected carboxyl group on said TBM;
(iv) contacting the SME with the TBM to form a TBM-SME complex, and (v) contacting the TBM-SME complex with a plurality of second small organic ligand candidates, said candidates having a functional group reactive with the SME in said TBM-SME complex, wherein a candidate that has affinity for said second site of interest on said TBM forms a reversible covalent bond with said TBM-SME complex, whereby a second ligand is identified. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
- (i) contacting a Target Biological Molecule (TBM) having a first and a second site of interest, and containing or modified to contain a nucleophile, selected from the group consisting of thiol, protected thiol, reversible disulfide, hydroxyl, protected hydroxyl, amino, protected amino, carboxyl and protected carboxyl groups, at or near the first site of interest, with a plurality of first organic ligand candidates, said candidates having a functional group reactive with the nucleophile, under conditions such that a reversible covalent bond is formed between the nucleophile and a candidate that has affinity for the first site of interest, to form a TMB-first ligand complex;
-
15. A process comprising:
-
(i) providing a Target Biological Molecule (TBM) containing or modified to contain a reactive nucleophile near a first site of interest on the TBM;
(ii) contacting the TBM from (i) with a small molecule extender having a group reactive with the nucleophile on the TBM and having a free thiol or protected thiol;
(iii) adjusting the conditions of contacting in step (ii) to cause a covalent bond to be formed between the nucleophile on the TBM and the group on the small molecule extender thereby forming a covalent complex comprising the TBM and the small molecule extender, the complex displaying a free thiol or protected thiol near a second site of interest on the TBM;
(iv) contacting the complex from (iii) with a library of small organic molecules each molecule having a free thiol or exchangeable disulfide linking group, under conditions of thiol exchange wherein the library member having the highest affinity for the second site of interest on the TBM forms a disulfide bond with the complex; and
(v) identifying the library member from (iv). - View Dependent Claims (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)
-
Specification