Methods for screening compound libraries

Abstract

Disclosed are methods for screening compound libraries using frontal chromatography in combination with mass spectrometry to identify and rank those members of the library that bind to a target receptor. Methods are also disclosed which permit a compound library to be rapidly screened to determine if any member of the library has an affinity for the target receptor as measured by a pre-selected indicator compound.

1 . A method for screening a compound library to determine the relative or absolute affinity of a plurality of putative ligands to a target receptor or a plurality of target receptors, which method comprises: 
 (a) providing a compound library comprising a plurality of putative ligands,    (b) applying the compound library to a column comprising a target receptor or a plurality of target receptors, each target receptor optionally bound to a solid phase support, under frontal chromatography conditions to provide an effluent;    (c) continuously or intermittently applying the effluent to a mass spectrometer to provide mass spectra of the constituent putative ligands present in the effluent; and    (d) evaluating the mass spectra to determine a break through time for the putative ligands.    
   
   
       2 . The method of  claim 1 , wherein said method further comprises the step of: 
 (e) determining an affinity to the target receptor for a putative ligand in the compound library relative to another putative ligand in the library by comparing the break through time for the putative ligand to the break through time for the other putative ligand.    
   
   
       3 . The method of  claim 1 , wherein said method further comprises the step of: 
 (f) determining a dissociation constant, K d , for a putative ligand in the compound library and the target receptor.    
   
   
       4 . The method of  claim 1 , wherein said method further comprises the steps of: 
 (g) collecting the effluent from step (c) to provide a plurality of effluent fractions; and    (h) optionally desalting each effluent fraction prior to step (d).    
   
   
       5 - 6 . (canceled)  
   
   
       7 . The method of  claim 1 , wherein the compound library comprises putative ligands selected from the group consisting of carbohydrates, monosaccharides, oligosaccharides, polysaccharides, amino acids, peptides, oligopeptides, polypeptides, proteins, nucleosides, nucleotides, oligonucleotides, polynucleotides, lipids, retinoids, steroids, glycopeptides, glycoproteins, glycolipids, proteoglycans, and synthetic analogs or derivatives thereof, synthetic small molecule organic compounds, and natural products.  
   
   
       8 - 9 . (canceled)  
   
   
       10 . The method of  claim 1 , wherein each target receptor is independently selected from the group consisting of proteins, glycoproteins, glycosaminoglycans, proteoglycans, integrins, enzymes, lectins, selectins, cell-adhesion molecules, toxins, bacterial pili, transport proteins, receptors involved in signal transduction or hormone-binding, hormones, antibodies, major histocompatability complexes, immunoglobulin superfamilies, cadherins, DNA or DNA fragments, RNA and RNA fragments, whole cells, cell fragments, tissues, bacteria, fungi, viruses, parasites, preons, and synthetic analogs or derivatives thereof.  
   
   
       11 . The method of  claim 1 , wherein each target receptor is bound to a solid phase support.  
   
   
       12 - 13 . (canceled)  
   
   
       14 . The method of  claim 1 , wherein the column contains from about 1 fmol to about 10 nmol of target receptor active sites.  
   
   
       15 . The method of  claim 1 , wherein the effluent from the column is diluted with a supplemental diluent before step (c).  
   
   
       16 - 17 . (canceled)  
   
   
       18 . The method of  claim 1 , wherein the mass spectrometer is an electrospray mass spectrometer.  
   
   
       19 . The method of  claim 1  wherein a plurality of compound libraries are screened to determine the relative affinity of a plurality of putative ligands in each library to a target receptor or a plurality of target receptors.  
   
   
       20 - 31 . (canceled)  
   
   
       32 . A method for screening a compound library to determine the relative affinity of a plurality of putative ligands to a target receptor or a plurality of target receptors relative to an indicator compound or a plurality of indicator compounds, which method comprises: 
 (a) providing a compound library comprising a plurality of putative ligands;    (b) providing at least one void marker compound;    (c) providing an indicator compound or a plurality of indicator compounds for each target receptor, each indicator compound having a pre-determined affinity for the target receptor and having a pre-determined break through time on the column in the absence of the compound library relative to a void marker compound;    (d) applying the compound library to a column comprising a target receptor or a plurality of target receptors, each target receptor optionally bound to a solid phase support, under frontal chromatography conditions to equilibrate or partially equilibrate the column with the compound library;    (e) applying (i) a mixture comprising the compound library, the void marker compound and the indicator compound or compounds, or (ii) the void marker compound and the indicator compound or compounds, to the column under frontal chromatography to provide an effluent;    (f) analyzing the effluent to determine a break through time for the indicator compound or compounds.    
   
   
       33 . The method of  claim 32 , wherein said method further comprises the step of: 
 (g) determining whether any putative ligands of the compound library have an affinity for a target receptor greater than the indicator compound by comparing the break through time for the indicator compound from step (f) with the pre-determined break through time for the indicator compound in the absence of the compound library.    
   
   
       34 . The method of  claim 32 , wherein the analysis of the effluent is conducted using a mass spectrometer.  
   
   
       35 - 44 . (canceled)  
   
   
       45 . The A method of  claim 32  wherein a plurality of compound libraries are screened to determine the relative affinity of a plurality of putative ligands to a target receptor or a plurality of target receptors relative to an indicator compound or a plurality of indicator compounds.  
   
   
       46 - 56 . (canceled)  
   
   
       57 . The method of  claim 32  wherein the indicator compound has a pre-determined affinity for the target receptor and 
 a pre-determined signal intensity in the presence of the compound library.    
   
   
       58 - 69 . (canceled)  
   
   
       70 . A method for screening a compound library to identify inhibitors of a target receptor, which method comprises: 
 (a) providing a compound library comprising a plurality of putative ligands,    (b) applying the compound library to a column comprising a target receptor optionally bound to a solid phase support under frontal chromatography conditions to equilibrate or partially equilibrate the column with the compound library;    (c) providing a first indicator compound which is capable of being chemically modified by the target receptor to form a second indicator compound;    (d) applying (i) a mixture comprising the compound library and the first indicator compound, or (ii) the first indicator compound, to the column under frontal chromatography conditions to provide an effluent;    (e) analyzing the effluent to determine the presence and/or concentration of the second indicator compound.    
   
   
       71 . The method of  claim 70 , wherein the analysis of the effluent is conducted using a mass spectrometer.  
   
   
       72 - 74 . (canceled)  
   
   
       75 . The method of  claim 70  wherein the target receptor is an enzyme.  
   
   
       76 . The method of  claim 75  wherein the first indicator compound is a substrate for the enzyme.  
   
   
       77 - 78 . (canceled)