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.

What is claimed is:  
     
         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 . The method of  claim 1 , wherein the compound library comprises less than about 10,000 putative ligands.  
     
     
         6 . The method of  claim 5 , wherein the compound library comprises about 5 to about 100 putative ligands.  
     
     
         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.  
     
     
         8 . The method of  claim 1 , wherein the compound library comprises putative ligands selected from the group consisting of synthetic small molecule organic compounds.  
     
     
         9 . The method of  claim 1 , wherein the compound library comprises putative ligands selected from the group consisting of natural products.  
     
     
         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 . The method of  claim 11 , wherein each target receptor is covalently bound to the solid phase support or bound via biotin-avidin or biotin-streptavidin binding.  
     
     
         13 . The method of  claim 11 , wherein the solid phase support is selected from the group consisting of polymeric beads, polymeric gels, glass beads, silica chips, silica capillaries, agarose, diatomaceous earths and pulp.  
     
     
         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 . The method of  claim 15 , wherein the supplement diluent comprises a major amount of an organic solvent and a minor amount of an aqueous buffer.  
     
     
         17 . The method of  claim 16 , wherein the organic solvent is selected from the group consisting of acetonitrile, methanol and isopropanol.  
     
     
         18 . The method of  claim 1 , wherein the mass spectrometer is an electrospray mass spectrometer.  
     
     
         19 . A method for screening a plurality of compound libraries to determine the relative affinity of a plurality of putative ligands in each library to a target receptor or a plurality of target receptors, which method comprises: 
 (a) providing a plurality of compound libraries, each library comprising a plurality of putative ligands,    (b) applying each compound library to a separate 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 from each column;    (c) intermittently applying the effluent from each column 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 in each compound library.    
     
     
         20 . The method of  claim 19 , wherein said method further comprises the step of: 
 (e) determining an affinity to the target receptor for a putative ligand in a compound library relative to another putative ligand in the same library by comparing the break through time for the putative ligand to the break through time for the other putative ligand.    
     
     
         21 . The method of  claim 19 , wherein said method further comprises the steps of: 
 (f) collecting each effluent from step (c) to provide a plurality of effluent fractions; and    (g) optionally desalting each effluent fraction prior to step (d).    
     
     
         22 . The method of  claim 19 , wherein said plurality of columns comprises from 2 to about 100 columns.  
     
     
         23 . The method of  claim 19 , wherein each compound library independently comprises less than about 10,000 putative ligands.  
     
     
         24 . The method of  claim 23 , wherein each compound library independently comprises about 5 to about 100 putative ligands.  
     
     
         25 . The method of  claim 19 , wherein each compound library independently 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.  
     
     
         26 . The method of  claim 19 , wherein the compound library comprises putative ligands selected from the group consisting of natural products.  
     
     
         27 . The method of  claim 19 , wherein each compound library independently comprises putative ligands selected from the group consisting of synthetic small molecule organic compounds.  
     
     
         28 . The method of  claim 19 , wherein each target receptor is independently selected from the group consisting of proteins, glycoproteins, glycosaminoglycans, proteoglycans, integrins, enzymes, lectins, selecting, 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.  
     
     
         29 . The method of  claim 19 , wherein each target receptor is bound to a solid phase support.  
     
     
         30 . The method of  claim 19 , wherein each column contains from about 1 fmol to about 10 nmol of target receptor active sites.  
     
     
         31 . The method of  claim 19 , wherein the mass spectrometer is an electrospray mass spectrometer.  
     
     
         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 predetermined affinity for the target receptor and having a predetermined 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 . The method of  claim 34 , wherein the mass spectrometer is an electrospray mass spectrometer.  
     
     
         36 . The method of  claim 32 , wherein the compound library comprises less than about 50,000 putative ligands.  
     
     
         37 . The method of  claim 36 , wherein the compound library comprises about 5 to about 100 putative ligands.  
     
     
         38 . The method of  claim 32 , 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.  
     
     
         39 . The method of  claim 32 , wherein the compound library comprises putative ligands selected from the group consisting of natural products.  
     
     
         40 . The method of  claim 32 , wherein the compound library comprises putative ligands selected from the group consisting of synthetic small molecule organic compounds.  
     
     
         41 . The method of  claim 32 , 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.  
     
     
         42 . The method of  claim 32 , wherein each target receptor is bound to a solid phase support.  
     
     
         43 . The method of  claim 32 , wherein the column contains from about 1 fmol to about 10 nmol of target receptor active sites.  
     
     
         44 . The method of  claim 32 , wherein the pre-determined break through time for the indicator compound in the absence of the compound library is less than about 5 minutes.  
     
     
         45 . A method for screening a plurality of compound libraries 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 plurality of compound libraries 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 predetermined affinity for the target receptor and having a pre-determined break through time on each column in the absence of the compound library relative to a void marker compound;    (d) applying each compound library to a separate 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 and the indicator compound or compounds, or (ii) the void marker and the indicator compound or compounds, to each column under frontal chromatography conditions to provide an effluent;    (f) analyzing the effluent from each column to determine a break through time for the indicator compound or compounds.    
     
     
         46 . The method of  claim 47 , wherein said method further comprises the step of: 
 (g) determining whether any putative ligands of a 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.    
     
     
         47 . The method of  claim 45 , wherein the analysis of the effluent is conducted using a mass spectrometer.  
     
     
         48 . The method of  claim 47 , wherein the mass spectrometer is an electrospray mass spectrometer.  
     
     
         49 . The method of  claim 45 , wherein each compound library independently comprises less than about 50,000 putative ligands.  
     
     
         50 . The method of  claim 49 , wherein each compound library independently comprises about 5 to about 100 putative ligands.  
     
     
         51 . The method of  claim 45 , wherein each compound library independently 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, proteoglycans, and synthetic analogs or derivatives thereof.  
     
     
         52 . The method of  claim 45 , wherein each compound library independently comprises putative ligands selected from the group consisting of synthetic small molecule organic compounds.  
     
     
         53 . The method of  claim 45 , 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, tissues, bacteria, fungi, viruses, parasites, preons, and synthetic analogs or derivatives thereof.  
     
     
         54 . The method of  claim 45 , wherein each target receptor is bound to a solid phase support.  
     
     
         55 . The method of  claim 45 , wherein each column contains from about 1 pmol to about 10 nmol of target receptor active sites.  
     
     
         56 . The method of  claim 45 , wherein the pre-determined break through time for the indicator compound in the absence of the compound library is less than about 5 minutes.  
     
     
         57 . A method for screening a compound library to determine the relative affinity of a plurality of putative ligands to a target receptor relative to an indicator compound having a pre-determined affinity for the target receptor, which method comprises: 
 (a) providing a compound library comprising a plurality of putative ligands;    (b) providing at least one void marker compound;    (c) providing a column comprising a target receptor optionally bound to a solid phase support;    (d) providing an indicator compound having a pre-determined affinity for the target receptor and having a pre-determined break through time on each column in the absence of the compound library relative to a void marker compound and having a pre-determined signal intensity in the presence of the compound library;    (e) applying a mixture comprising the compound library and the indicator compound to the column under frontal chromatography conditions to provide an effluent;    (f) analyzing the effluent to determine a break through time and/or signal intensity for the indicator compound.    
     
     
         58 . The method of  claim 57 , 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 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.    
     
     
         59 . The method of  claim 57 , wherein said method further comprises the step of: 
 (h) determining whether the affinity for the target receptor is due to a plurality of ligands having weaker affinity for the target receptor relative to the indicator compound or to one or more ligands having stronger affinity for the target receptor relative to the indicator compound by comparing the signal intensity of the indicator in the effluent with the pre-determined signal intensity for the indicator.    
     
     
         60 . The method of  claim 57 , wherein the analysis of the effluent is conducted using a mass spectrometer.  
     
     
         61 . The method of  claim 60 , wherein the mass spectrometer is an electrospray mass spectrometer.  
     
     
         62 . The method of  claim 57 , wherein the compound library comprises less than about 10,000 putative ligands.  
     
     
         63 . The method of  claim 62 , wherein the compound library comprises about 5 to about 100 putative ligands.  
     
     
         64 . The method of  claim 57 , 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.  
     
     
         65 . The method of  claim 57 , wherein the compound library comprises putative ligands selected from the group consisting of natural products.  
     
     
         66 . The method of  claim 57 , wherein the compound library comprises putative ligands selected from the group consisting of synthetic small molecule organic compounds.  
     
     
         67 . The method of  claim 57 , wherein the target receptor is selected from the group consisting of proteins, glycoproteins, glycosaminoglycans, proteoglycans, integrins, enzymes, lectins, selecting, 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.  
     
     
         68 . The method of  claim 57 , wherein the target receptor is bound to a solid phase support.  
     
     
         69 . The method of  claim 57 , wherein the column contains from about 1 fmol to about 10 nmol of target receptor active sites.  
     
     
         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 . The method of  claim 71 , wherein the mass spectrometer is an electrospray mass spectrometer.  
     
     
         73 . The method of  claim 70 , wherein the compound library comprises less than about 50,000 putative ligands.  
     
     
         74 . The method of  claim 73 , wherein each compound library comprises about 5 to about 100 putative ligands.  
     
     
         75 . The method of  claim 70  wherein the target receptor is an enzyme.  
     
     
         76 . The method of  claim 75  wherein the first indicator compound a substrate for the enzyme.  
     
     
         77 . A method for screening a target receptor or a plurality of target receptors to determine the relative affinity of the receptor or receptors to an immobilized ligand or ligands relative to an indicator compound or a plurality of indicator compounds, which method comprises: 
 (a) providing a target receptor or a plurality of target receptors;    (b) providing a column comprising a ligand or a plurality of ligands, each ligand bound to a solid phase support;    (c) providing at least one void marker compound;    (d) providing an indicator compound or a plurality of indicator compounds for each ligand each indicator compound having a pre-determined affinity for the ligand and having a pre-determined break through time on the column relative to a void marker compound;    (e) applying the target receptor or receptors to the column under frontal chromatography conditions to equilibrate or partially equilibrate the column with the target receptor or receptors;    (f) applying (i) a mixture comprising the target receptor or receptors, 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 conditions to provide an effluent;    (g) analyzing the effluent to determine a break through time for the indicator compound or compounds.    
     
     
         78 . The method of  claim 77 , wherein said method further comprises the step of: 
 (h) determining whether any of the target receptors has an affinity for the ligand or ligands as measured by the indicator compound or compounds by comparing the break through time for the indicator compound or compounds from step (g) with the pre-determined break through time for the indicator compound or compounds.