Method for producing and screening mass-coded combinatorial libraries for drug discovery and target validation

Abstract

The present invention provides a method for producing a mass-coded combinatorial library comprising a set of compounds having the general formula X(Y) n , where X is a scaffold, each Y is, independently, a peripheral moiety, and n is an integer greater than 1. The method comprises selecting a peripheral moiety precursor subset from a peripheral moiety precursor set. The subset includes a sufficient number of peripheral moiety precursors that at least about 50 distinct combinations of n peripheral moieties derived from the peripheral moiety precursors in the subset exist. The subset of peripheral moiety precursors is selected so that at least about 90% of all possible combinations of n peripheral moieties derived from the subset have a molecular mass sum which is distinct from the molecular mass sums of all of the other combinations of n peripheral moieties. The method further comprises contacting the peripheral moiety precursor subset with a scaffold precursor which has n reactive groups. Methods of use of the mass-coded combinatorial library produced by this method for identifying a ligand to a particular biomolecule are also disclosed.

1 - 33 . (canceled)  
   
   
       34 . A method for identifying a member of a mass-coded combinatorial library which is a ligand for a first biomolecule but is not a ligand for a second biomolecule, 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 said mass-coded combinatorial library is produced by reacting a scaffold precursor with a sufficient number of distinct peripheral moiety precursors such that there exist at least about 250 distinct combinations of n peripheral moieties derived from said peripheral moiety precursors, said method comprising the steps of: 
 (a) contacting the first biomolecule with the mass-coded molecular library, whereby members of the mass-coded molecular library, which are ligands for the first biomolecule bind to the first biomolecule to form first biomolecule-ligand complexes and members of the mass-coded library which are not ligands for the first biomolecule remain unbound;    (b) separating the first biomolecule-ligand complexes from the unbound members of the mass-coded molecular library;    (c) dissociating the first biomolecule-ligand complexes;    (d) determining the molecular mass of each ligand for the first biomolecule;    (e) contacting the second biomolecule with the mass-coded molecular library, whereby members of the mass-coded molecular library which are ligands for the second biomolecule bind to the second biomolecule to form second biomolecule-ligand complexes and members of the mass-coded library which are not ligands for the second biomolecule remain unbound;    (f) separating the second biomolecule-ligand complexes from the unbound members of the mass-coded molecular library;    (g) dissociating the second biomolecule-ligand complexes;    (h) determining the molecular mass of each ligand for the second biomolecule; and    (i) determining which molecular mass or masses determined in step (d) are not determined in step (h), thereby providing the molecular masses of members of the mass-coded combinatorial library which are ligands for the second biomolecule,    wherein the each molecular mass determined in step (i) corresponds to a set of n peripheral moieties present in a ligand for the first biomolecule which is not a ligand for the second biomolecule, thereby identifying a member of the mass-coded combinatorial library which are ligands for the first biomolecule but are not ligands for the second biomolecule.    
   
   
       35 . The method of  claim 34  wherein the first and second biomlecules are each, independently, a protein or a nucleic acid molecule.  
   
   
       36 . The method of  claim 35  wherein the first and second biomolecules are each a protein and amino acid sequence of the second biomolecule is derived from the amino acid sequence of the first biomolecule by insertion, deletion or substitution of one or more amino acid residues.  
   
   
       37 . The method of  claim 35  wherein the first biomolecule is a first protein and the second biomolecule is a second protein, said first and second proteins having the same amino acid sequence, wherein said first and second proteins have different posttranslational modifications.  
   
   
       38 . The method of  claim 37  wherein the first protein differs from the second protein in extent of phosphorylation, glycosylation or ubiwuitination.  
   
   
       39 . The method of  claim 35  wherein the second biomolecule is a complex of the first biomolecule with a ligand.  
   
   
       40 . The method of  claim 35  wherein the first and second biomolecules are each immobilized on a solid support.  
   
   
       41 . The method of  claim 40  wherein the solid support is a water-insoluble matrix contained within a chromatographic column.  
   
   
       42 . The method of  claim 35  wherein a solution comprising the first biomolecule is contacted with the mass-coded molecular library to form a solution comprising first biomolecule-ligand complexes and unbound members of the mass-coded molecular library and a solution comprising the second biomolecular library to form a solution comprising second biomolecule-ligand complexes and unbound members of the mass-coded molecular library.  
   
   
       43 . The method of  claim 42  wherein the unbound members of the mass-coded molecular library are separated from the second biomolecule-ligand complexes by directing the solution comprising second biomolecule-ligand complexes and the unbound members of the mass-coded molecular library through a size exclusion chromatography column, whereby the unbound members of the mass-coded molecular library elute from second column after the second biomolecule-ligand complexes.  
   
   
       44 . The method of  claim 42  wherein the unbound members of the mass-coded molecular library are separated from the second biomolecule-ligand complexes by contacting the solution comprising second biomolecule-ligand complexes and the unbound members of the mass-coded molecular library with a size-exclusion membrane, whereby the unbound compounds pass through said membrane and the second biomolecule-ligand complexes do not pass through said membrane.  
   
   
       45 - 50 . (canceled)