Method for producing and screening mass-coded combinatorial libraries for drug discovery and target validation
View Patent ↗Methods for identifying a member of a mass-coded molecular library, which is a ligand for a biomolecule and binds to the biomolecule at the binding site of a known second ligand for the biomolecule are described. The methods includes contacting a mass-coded molecular library with a biomolecule; separating the biomolecule-ligand complexes from the unbound members of the mass-coded molecular library; contacting the biomolecule-ligand complexes with a second ligand to dissociate biomolecule-ligand complexes in which the ligand binds to the biomolecule at the binding site of the second ligand, thereby forming biomolecule-second ligand complexes and dissociated ligands; separating the dissociated ligands and biomolecule-second ligand complexes; and determining the molecular mass of each dissociated ligand.
1. A method for identifying a member of a mass-coded molecular library which is a ligand for a biomolecule and binds to the biomolecule at the binding site of a known second ligand for the biomolecule, said method comprising the steps of:
(a) contacting the biomolecule with a mass-coded molecular library, said mass-coded molecular library comprising compounds of the general formula XY n , wherein n is an integer from 2 to about 6, X is a scaffold and each Y is, independently, a peripheral moiety, wherein there exist at least about 250 distinct combinations of n peripheral moieties, and wherein each of at least about 90% of the combinations of n peripheral moieties has a molecular mass sum that is distinct from the molecular mass sum of all other combinations of n peripheral moieties, whereby members of the mass-coded molecular library which are ligands for the biomolecule bind to the biomolecule to form biomolecule-ligand complexes and members of the mass-coded library which are not ligands for the biomolecule remain unbound;
(b) separating the biomolecule-ligand complexes from the unbound members of the mass-coded molecular library;
(c) contacting the biomolecule-ligand complexes with the second ligand to dissociate biomolecule-ligand complexes in which the ligand binds to the biomolecule at the binding site of the second ligand, thereby forming biomolecule-second ligand complexes and dissociated ligands;
(d) separating the dissociated ligands and biomolecule-second ligand complexes; and
(e) determining the molecular mass of each dissociated ligand,
wherein the molecular mass of each dissociated ligand corresponds to a set of peripheral moieties present in that ligand, thereby identifying a member of the mass-coded molecular library which is a ligand for the biomolecule and binds to the biomolecule at the binding site of the known second ligand for the biomolecule.
2. The method of claim 1 wherein the second ligand is a polypeptide, a nucleic acid molecule or a cofactor.
3. The method of claim 1 wherein the biomolecule is immobilized on a solid support.
4. The method of claim 3 wherein the solid support is a water-insoluble matrix contained within a chromatographic column.
5. The method of claim 1 wherein the biomolecule is a protein or a nucleic acid molecule.
6. The method of claim 1 wherein a solution comprising the biomolecule is contacted with the mass-coded combinatorial library to form, if one or more members of the combinatorial library are ligands for the biomolecule, a solution comprising biomolecule-ligand complexes and unbound members of the mass-coded combinatorial library.
7. The method of claim 6 wherein the unbound members of the mass-coded combinatorial library are separated from the biomolecule-ligand complexes by directing the solution comprising biomolecule-ligand complexes and the unbound members of the mass-coded combinatorial library through a size exclusion chromatography column, whereby the unbound members of the mass-coded combinatorial library elute from said column after the biomolecule-ligand complexes.
8. The method of claim 6 wherein the unbound members of the mass-coded combinatorial library are separated from the biomolecule-ligand complexes by contacting the solution comprising biomolecule-ligand complexes and the unbound members of the mass-coded combinatorial library with a size-exclusion membrane, whereby the unbound compounds pass through said membrane and the biomolecule-ligand complexes do not pass through said membrane.
9. The method of claim 1 wherein a solution comprising the biomolecule-ligand complexes is contacted with the second ligand to form a solution comprising biomolecule-second ligand complexes and dissociated ligands.
10. The method of claim 9 wherein the dissociated ligands are separated from the biomolecule-second ligand complexes by directing the solution comprising biomolecule-second ligand complexes and dissociated ligands through a size exclusion chromatography column, whereby the dissociated ligands elute from said column after the biomolecule-second ligand complexes.
11. The method of claim 9 wherein the dissociated ligands are separated from the biomolecule-second ligand complexes by contacting the solution comprising biomolecule-second ligand complexes and dissociated ligands with a size-exclusion membrane, whereby the dissociated ligands pass through said membrane and the biomolecule-second ligand complexes do not pass through said membrane.