Fluorescent molecular probes for use in assays that measure test compound competitive binding with sam-utilizing proteins

Abstract

Assay methods may generally comprise forming homogeneous assay mixtures comprising target SAM-utilizing protein, fluorescent detection analyte, and test compound, incubating, and measuring FP or TR-FRET signal emitted in order to determine a measure of test compound-SAM-utilizing protein binding. Assay mixtures comprise a SAM-utilizing protein, and a fluorescent detection analyte that binds with the SAM-utilizing protein in the absence of test compound. Assay mixtures may further comprise a test compound. Assay mixture embodiments may generate FP or TR-FRET signal properties that are a function of the inherent binding interactions of both the test compound and the detection analyte with the SAM-utilizing protein. Fluorescent detection analytes comprise a fluorophore moiety, a covalent linker moiety, and a SAM-utilizing protein ligand moiety and could be utilized in FP or TR-FRET assays to measure test compound binding.

1 - 23 . (canceled) 
     
     
         24 . An assay mixture for identifying compounds that bind to a SAM-utilizing protein comprising:
 (a) a detection analyte comprising:
 (1) a fluorophore moiety; 
 (2) a SAM-utilizing protein ligand moiety; and 
 (3) a linker moiety that covalently links the fluorophore moiety with the SAM-utilizing protein ligand moiety; 
   (b) a SAM-utilizing protein or a SAM-utilizing protein labeled with a donor fluorophore or an acceptor fluorophore; and   (c) a test compound.   
     
     
         25 . The assay mixture of  claim 24 , wherein the SAM-utilizing protein ligand moiety comprises a nucleoside-type moiety. 
     
     
         26 . The assay mixture of  claim 25 , wherein the nucleoside-type moiety is selected from the group consisting of a sinefungin moiety, a sulfur-based moiety, and a nitrogen-based moiety. 
     
     
         27 . The assay mixture of  claim 24 , wherein the SAM-utilizing protein ligand moiety (2) comprises a sinefungin moiety and wherein the linker moiety that covalently links the fluorophore moiety with the sinefungin moiety (3) comprises a linker moiety that covalently links the fluorophore moiety with the sinefungin moiety through an atom on the sinefungin moiety selected from the group consisting of a ribose 2′-hydroxy oxygen atom and a ribose 3′-hydroxy oxygen atom. 
     
     
         28 . The assay mixture of  claim 24 , wherein the SAM-utilizing protein ligand moiety (2) comprises a sinefungin moiety and wherein the linker moiety that covalently links the fluorophore moiety with the sinefungin or sinefungin analog moiety (3) comprises a linker moiety that covalently links the fluorophore moiety with the sinefungin moiety through an atom on a base ring or a base ring replacement portion of the sinefungin moiety. 
     
     
         29 . The assay mixture of  claim 24 , wherein the SAM-utilizing protein comprises a SAM-utilizing methyltransferase enzyme and wherein the SAM-utilizing protein labeled with a donor fluorophore or an acceptor fluorophore comprises a SAM-utilizing methyltransferase enzyme labeled with a donor fluorophore or an acceptor fluorophore (c). 
     
     
         30 . (canceled) 
     
     
         31 . The assay mixture of  claim 24 , wherein the SAM-utilizing protein comprises a SET domain-containing lysine methyltransferase enzyme and wherein the SAM-utilizing protein labeled with a donor fluorophore or an acceptor fluorophore comprises a SET domain-containing lysine methyltransferase enzyme labeled with a donor fluorophore or an acceptor fluorophore (c). 
     
     
         32 . The assay mixture of  claim 31 , wherein the SET domain-containing lysine methyltransferase enzyme is selected from the group consisting of SET7/9, MLL, GLP and G9a. 
     
     
         33 - 34 . (canceled) 
     
     
         35 . The assay mixture of  claim 24 , wherein the SAM-utilizing protein comprises lysine methyltransferase enzyme SET7/9 and wherein the SAM-utilizing protein labeled with a donor fluorophore or an acceptor fluorophore comprises lysine methyltransferase enzyme SET7/9 labeled with a donor fluorophore or an acceptor fluorophore. 
     
     
         36 . The assay mixture of  claim 24 , wherein the SAM-utilizing protein comprises lysine methyltransferase enzyme G9a and wherein the SAM-utilizing protein labeled with a donor fluorophore or an acceptor fluorophore comprises lysine methyltransferase enzyme G9a labeled with a donor fluorophore or an acceptor fluorophore. 
     
     
         37 . The assay mixture of  claim 24 , wherein the SAM-utilizing protein comprises lysine methyltransferase enzyme GLP and wherein the SAM-utilizing protein labeled with a donor fluorophore or an acceptor fluorophore comprises lysine methyltransferase enzyme GLP labeled with a donor fluorophore or an acceptor fluorophore; and
 (c) a test compound.   
     
     
         38 . The assay mixture of  claim 24 , wherein the SAM-utilizing protein comprises lysine methyltransferase enzyme MLL and wherein the SAM-utilizing protein labeled with a donor fluorophore or an acceptor fluorophore comprises lysine methyltransferase enzyme MLL labeled with a donor fluorophore or an acceptor fluorophore. 
     
     
         39 . (canceled) 
     
     
         40 . The assay mixture of  claim 24 , wherein the SAM-utilizing protein comprises a SET domain-containing lysine methyltransferase enzyme PRDM9 and wherein the SAM-utilizing protein labeled with a donor fluorophore or an acceptor fluorophore comprises a SET domain-containing lysine methyltransferase enzyme PRDM9 labeled with a donor fluorophore or an acceptor fluorophore (c). 
     
     
         41 . (canceled) 
     
     
         42 . The assay mixture of  claim 24 , wherein the SAM-utilizing protein comprises an arginine methyltransferase enzyme and wherein the SAM-utilizing protein labeled with a donor fluorophore or an acceptor fluorophore comprises an arginine methyltransferase enzyme labeled with a donor fluorophore or an acceptor fluorophore (c). 
     
     
         43 . The assay mixture of  claim 42 , wherein the arginine methyltransferase enzyme is selected from the group consisting of PRMT1 and PRMT4. 
     
     
         44 - 46 . (canceled) 
     
     
         47 . The assay mixture of  claim 24 , wherein the detection analyte comprises a fluorescent detection analyte having the structure of Formula (II): 
       
         
           
           
               
               
           
         
         wherein any two of R 2 , R 2′ , and R 3′  are hydrogen and the third is a linker component having a linker moiety bonded to a fluorophore moiety. 
       
     
     
         48 . (canceled) 
     
     
         49 . The assay mixture of  claim 24 , wherein the detection analyte comprises a fluorescent detection analyte having the structure of Formula (III): 
       
         
           
           
               
               
           
         
         wherein any two of R 2 , R 2′ , and R 3′  are hydrogen and the third is a linker component having a linker moiety bonded to a fluorophore moiety. 
       
     
     
         50 . (canceled) 
     
     
         51 . The assay mixture of  claim 24 , wherein the detection analyte comprises a fluorescent detection analyte having the structure of Formula (IV): 
       
         
           
           
               
               
           
         
         wherein any two of R 2 , R 2′ , and R 3′  are hydrogen and the third is a linker component having a linker moiety bonded to a fluorophore moiety and wherein R is hydrogen, C 1-8  alkyl, C 2-6  alkenyl, C 2-6  alkynyl, C 3-6  cycloalkyl, C 6-10  aryl, five-to-ten-membered heteroaryl, five- to ten-membered heterocyclyl, C 1-8  acyl, or [(S)-2-aminobutanoic acid]-4-yl. 
       
     
     
         52 - 54 . (canceled) 
     
     
         55 . The assay mixture of  claim 24 , wherein the detection analyte comprises a fluorescent detection analyte selected from the group consisting of Sinefungin Probe 1A, Sinefungin Probe 1B, Sinefungin Probe 2A, Sinefungin Probe 2B, Sinefungin Probe 3, Sinefungin Probe 4, Sinefungin Probe 5, Sinefungin Probe 6, Sinefungin Probe 7, Sinefungin Probe 8, Sinefungin Probe 9, Sinefungin Probe 10, Sinefungin Probe 11, Sinefungin Probe 12, and Sinefungin Probe 13; wherein any A −  is PF 6   −  or trifluoroacetate, or a mixture thereof, each respectively illustrated below: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         56 . The assay mixture of  claim 24 , wherein the detection analyte comprises a fluorescent detection analyte selected from the group consisting of Thioadenosine Probe 1 and Thioadenosine Probe 2 each respectively illustrated below: 
       
         
           
           
               
               
           
         
       
     
     
         57 . The assay mixture of  claim 24 , wherein the detection analyte comprises a fluorescent detection analyte selected from the group consisting of Aza-adenosine Probe 1, Aza-adenosine Probe 3, Aza-adenosine Probe 5, Aza-adenosine Probe 6, Aza-adenosine Probe 7, Aza-adenosine Probe 8, Aza-adenosine Probe 9, Aza-adenosine Probe 10, and Aza-adenosine Probe 11 each respectively illustrated below: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         58 . The assay mixture of  claim 24 , wherein the detection analyte comprises a fluorescent detection analyte selected from the group consisting of Aza-adenosine Probe 2, Aza-adenosine Probe 4, Aza-adenosine Probe 12, Aza-adenosine Probe 13, Aza-adenosine Probe 14, Aza-adenosine Probe 15, Aza-adenosine Probe 16, Aza-adenosine Probe 17, Aza-adenosine Probe 18, Aza-adenosine Probe 19, Aza-adenosine Probe 20, and Aza-adenosine Probe 21 each respectively illustrated below: