Microplate thermal shift assay for ligand development using 5- (4"-dimethylaminophenyl) -2- (4' -phenyl) oxazole derivative fluorescent dyes

Abstract

The present invention provides a method for ranking the affinity of each of a multiplicity of different molecules for a target molecule which is capable of unfolding due to a thermal change. The method comprises (a) contacting the target molecule with one molecule of a multiplicity of different molecules, in the presence of a 5-(4″-dimethylaminophenyl)-2-(4′-phenyl)oxazole derivative dye, in each of a multiplicity of containers; (b) simultaneously heating the multiplicity of containers; (c) measuring the fluorescence in each of the containers; (d) generating thermal unfolding information for the target molecule as a function of temperature for each of the containers; (e) comparing the thermal unfolding information obtained for each of the containers to the thermal unfolding information for the target molecule in the absence of any of the molecules in the multiplicity of different molecules; and (f) ranking the affinities of each of the molecules according to the difference in the thermal unfolding information between the target molecule in each of the containers and the target molecule in the absence of any of the molecules in the multiplicity of different molecules.

What is claimed is:  
     
         1 . A method for ranking the affinity of each of a multiplicity of different molecules for a target molecule which is capable of unfolding due to a thermal change, said method comprising 
 (a) contacting the target molecule with one molecule of a multiplicity of different molecules, in the presence of a 5-(4″-dimethylaminophenyl)-2-(4′-phenyl)oxazole derivative dye, in each of a multiplicity of containers;    (b) simultaneously heating said multiplicity of containers;    (c) measuring the fluorescence in each of said containers;    (d) generating thermal unfolding information for the target molecule as a function of temperature for each of said containers;    (e) comparing the thermal unfolding information obtained for each of said containers to (i) the thermal unfolding information obtained for each of the other containers, and (ii) the thermal unfolding information obtained for the target molecule in the absence of any of said molecules in said multiplicity of different molecules; and    (f) ranking the affinities of each of said molecules according to the difference in said thermal unfolding information between the target molecule in each of said containers and the target molecule in the absence of any of said molecules in the multiplicity of different molecules.    
     
     
         2 . The method of  claim 1 , wherein said thermal unfolding information is the thermal unfolding T m .  
     
     
         3 . The method of  claim 1 , wherein said target molecule is a protein.  
     
     
         4 . The method of  claim 1 , wherein said multiplicity of different molecules comprises a combinatorial library.  
     
     
         5 . The method of  claim 1 , wherein said fluorescence in each of said containers is measured simultaneously.  
     
     
         6 . The method of  claim 1 , wherein said heating, said measuring, said generating, and said comparing are performed automatically.  
     
     
         7 . The method of  claim 6 , wherein said contacting is performed automatically.  
     
     
         8 . The method of  claim 6 , wherein said ranking is performed automatically.  
     
     
         9 . The method of  claim 6 , wherein said method is high-throughput.  
     
     
         10 . The method of  claim 1 , wherein said 5-(4″-dimethylaminophenyl)-2-(4′-phenyl)oxazole derivative dye is selected from the group consisting of 5-(4″-dimethylaminophenyl)-2-(4′-phenyl)oxazole butylsulfonamide, or a salt or ester thereof; 5-(4″-dimethylaminophenyl)-2-(4′-phenyl)oxazole-(2-aminoethyl)sulfonamide, or a salt or ester thereof, 5-(4″-dimethylaminophenyl)-2-(4′-phenyl)oxazole-3-sulfonamidophyenylboronic acid, or a salt or ester thereof; 5-(4″-dimethylaminophenyl)-2-(4′-phenyl)oxazole sulfonic acid, or a salt or ester thereof; 5-(4″-dimethylaminophenyl)-2-(4′-phenyl)oxazole sulfonyl hydrazine, 5-(4″-dimethylaminophenyl)-2-(4′-phenyl)oxazole-(2-bromoacetamidoethyl)sulfonamide, or a salt or ester thereof; 5-(4″-dimethylaminophenyl)-2-(4′-phenyl)oxazole-2-(3-(2-pyridyldithio) propionamidoethyl)sulfonamide, or a salt or ester thereof; 5-(4″-dimethylaminophenyl)-2-(4′-phenyl)oxazole sulfonyl chloride, or a salt or ester thereof; 5-(4″-dimethylaminophenyl)-2-(4′-phenyl)oxazole-3-sulfonamidopropionic acid, or a salt or ester thereof; 5-(4″-dimethylaminophenyl)-2-(4′-phenyl)oxazole carboxylic acid, or a salt or ester thereof; and 5-(4″-dimethylaminophenyl)-2-(4′-phenyl)oxazole sulfonate, or a salt or ester thereof.  
     
     
         11 . The method of  claim 10 , wherein said 5-(4″-dimethylaminophenyl)-2-(4′-phenyl)oxazole derivative dye is 5-(4″-dimethylaminophenyl)-2-(4′-phenyl)oxazole sulfonate, or a salt or ester thereof.  
     
     
         12 . The method of  claim 11 , wherein said 5-(4″-dimethylaminophenyl)-2-(4′-phenyl)oxazole derivative dye is 5-(4″-dimethylaminophenyl)-2-(4′-phenyl)oxazole sulfonate, sodium salt.  
     
     
         13 . A multi-variable method for ranking the affinity of a combination of two or more of a multiplicity of different molecules for a target molecule which is capable of unfolding due to a thermal change, said method comprising: 
 (a) contacting said target molecule with a combination of two or more different molecules of said multiplicity of different molecules, in the presence of 5-(4″-dimethylaminophenyl)-2-(4′-phenyl)oxazole derivative dye, in each of a multiplicity of containers;    (b) simultaneously heating said multiplicity of containers;    (c) measuring the fluorescence in each of said containers;    (d) generating thermal unfolding information for the target molecule as a function of temperature in each of said containers;    (e) comparing said thermal unfolding information obtained for each of said containers to (i) the thermal unfolding information obtained for each of said other containers, and (ii) the thermal unfolding information obtained for the target molecule in the absence of any of the two or more different molecules; and    (f) ranking the affinities of the combinations of said two or more of the multiplicity of different molecules according to the difference in said thermal unfolding information between the target molecule in each of said containers and the thermal unfolding information obtained for the target molecule in the absence of any of said molecules in said multiplicity of different molecules.    
     
     
         14 . The method of  claim 13 , wherein said thermal unfolding information is the thermal unfolding T m .  
     
     
         15 . A method for assaying a collection of a multiplicity of different molecules for a molecule which binds to a target molecule which is capable of unfolding due to a thermal change, said method comprising: 
 (a) contacting said target molecule with a collection of at least two molecules of said multiplicity of different molecules, in the presence of a 5-(4″-dimethylaminophenyl)-2-(4′-phenyl)oxazole derivative dye, in each of a multiplicity of containers;    (b) simultaneously heating said multiplicity of containers;    (c) measuring the fluorescence in each of said containers;    (d) generating thermal unfolding information for the target molecule as a function of temperature for each of said containers;    (e) comparing said thermal unfolding information obtained for each of said containers to (i) the thermal unfolding information obtained for each of said containers, and (ii) the thermal unfolding information obtained for the target molecule in the absence of any of said multiplicity of different molecules; and    (f) ranking the affinities of said collections of different molecules according to the difference in said thermal unfolding information between the target molecule in each of said containers and the thermal unfolding information obtained for the target molecule in the absence of any of said molecules in said multiplicity of different molecules;    (g) selecting the collection of different molecules which contains a molecule with affinity for the target molecule;    (h) dividing the selected collection into smaller collections of molecules in each of a multiplicity of containers; and    (i) repeating the above steps until a single molecule, from said multiplicity of different molecules, is identified.    
     
     
         16 . The method of  claim 15 , wherein said thermal unfolding information is the thermal unfolding T m .  
     
     
         17 . A multi-variable method for ranking the efficacy of one or more of a multiplicity of different biochemical conditions for stabilizing a target molecule which is capable of unfolding due to a thermal change, said method comprising: 
 (a) contacting said target molecule with one or more of said multiplicity of biochemical conditions, in the presence of a 5-(4″-dimethylaminophenyl)-2-(4′-phenyl)oxazole derivative dye, in each of a multiplicity of containers;    (b) simultaneously heating said multiplicity of containers;    (c) measuring the fluorescence in each of said containers;    (d) generating thermal unfolding information for the target molecule as a function of temperature for each of said containers;    (e) comparing said thermal unfolding information obtained for each of said containers to (i) the thermal unfolding information obtained for each of the other containers, and (ii) the thermal unfolding information obtained for said target molecule under a reference set of biochemical conditions; and    (f) ranking the efficacies of each of said biochemical conditions for each of said containers according to the difference in said thermal unfolding information between the target molecule for each of said containers and the target molecule under said reference set of biochemical conditions.    
     
     
         18 . The method of  claim 17 , wherein said thermal unfolding information is the thermal unfolding T m .  
     
     
         19 . A multi-variable method for optimizing the shelf life of a target molecule which is capable of unfolding due to a thermal change, said method comprising: 
 (a) contacting said target molecule with one or more of a multiplicity of different molecules or different biochemical conditions, in the presence of a 5-(4″-dimethylaminophenyl)-2-(4′-phenyl)oxazole derivative dye, in each of a multiplicity of containers;    (b) simultaneously heating said multiplicity of containers;    (c) measuring the fluorescence in each of said containers;    (d) generating thermal unfolding information for the target molecule as a function of temperature for each of said containers;    (e) comparing said thermal unfolding information obtained for each of said containers to (i) the thermal unfolding information obtained for each of the other containers, and (ii) the thermal unfolding information obtained for said target molecule under a reference set of biochemical conditions; and    (f) ranking the efficacies of each of said biochemical conditions for each of said containers according to the difference in said thermal unfolding information between the target molecule for each of said containers and the target molecule under said reference set of biochemical conditions.    
     
     
         20 . The method of  claim 19 , wherein said thermal unfolding information is the thermal unfolding T m .  
     
     
         21 . A multi-variable method for ranking the efficacies of one or more of a multiplicity of different biochemical conditions to facilitate the refolding or renaturation of a sample of a denatured or unfolded protein, said method comprising: 
 (a) placing one of the refolded protein samples, in the presence of a 5-(4″-dimethylaminophenyl)-2-(4′-phenyl)oxazole derivative dye, in each of a multiplicity of containers, wherein each of the refolded protein samples has been previously refolded or renatured according to one or more of the multiplicity of conditions;    (b) simultaneously heating said multiplicity of containers;    (c) measuring the fluorescence in each of said containers;    (d) generating thermal unfolding information for the target molecule as a function of temperature for each of said containers;    (e) comparing said thermal unfolding information obtained for each of said containers to (i) the thermal unfolding information obtained for each of the other containers, and (ii) the thermal unfolding information obtained for said target molecule under a reference set of biochemical conditions; and    (f) ranking the efficacies of each of said biochemical conditions for each of said containers according to the difference in said thermal unfolding information between the target molecule for each of said containers and the target molecule under said reference set of biochemical conditions.    
     
     
         22 . The method of  claim 21 , wherein said thermal unfolding information is the thermal unfolding T m .  
     
     
         23 . A multi-variable method for ranking the efficacies of one or more of a multiplicity of different biochemical conditions to facilitate the refolding or renaturation of a sample of a denatured unfolded protein, said method comprising 
 (a) Determining one or more combinations of a multiplicity of different conditions which promote protein stabililty, incubate the denatured protein under said one or more combinations of biochemical conditions that were identified as promoting protein stabilization;    (b) assessing folded protein yield according to the method of  claim 18;     (c) ranking the efficacies of said multiplicity of different refolding conditions according to folded protein yield; and    (d) repeating these steps until a combination of biochemical conditions that promote optimal protein folding are identified.    
     
     
         24 . The method of  claim 23 , wherein said thermal unfolding information is the thermal unfolding T m .  
     
     
         25 . A multi-variable method for ranking the efficacy of one or more of a multiplicity of different biochemical conditions for facilitating the crystallization of a protein which is capable of unfolding due to a thermal change, said method comprising: 
 (a) contacting the protein with one or more of the multiplicity of different biochemical conditions, in the presence of a 5-(4″-dimethylaminophenyl)-2-(4′-phenyl)oxazole derivative dye, in each of a multiplicity of containers;    (b) simultaneously heating said multiplicity of containers;    (c) measuring the fluorescence in each of said containers;    (d) generating thermal unfolding information for the target molecule as a function of temperature for each of said containers;    (e) comparing said thermal unfolding information obtained for each of said containers to (i) the thermal unfolding information obtained for each of the other containers, and (ii) the thermal unfolding information obtained for said target molecule under a reference set of biochemical conditions; and    (f) ranking the efficacies of each of said biochemical conditions for each of said containers according to the difference in said thermal unfolding information between the target molecule for each of said containers and the target molecule under said reference set of biochemical conditions.    
     
     
         26 . The method of  claim 23 , wherein said thermal unfolding information is the thermal unfolding T m .