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 - 44 . (canceled) 
     
     
         45 . A fluorescent detection analyte having the structure of Formula (I): 
       
         
           
           
               
               
           
         
         wherein the ----- between two atoms in either the A ring or the B ring represents the bond involving the two atoms is either a single or a double bond, and it may only represent a double bond between the 4′-carbon and X 9  when X 9  is CH; 
         wherein X 9  is O, NR 7 , S, CH (allowed when its bond to the 4′-carbon is a carbon-carbon double bond), or CH 2  (allowed when its bond to the 4′-carbon is a carbon-carbon single bond);
 wherein R 7  is hydrogen or methyl; 
 
         wherein each of X 5 , X 6 , X 7 , and X 8  is independently a carbon or a nitrogen;
 wherein no more than three of X 5 , X 6 , X 7 , and X 8  is nitrogen; 
 wherein when any X 5 , X 6 , X 7 , or X 8  is nitrogen, the associated W 5 , W 6 , W 7 , and W 8 , respectively, is not present; 
 
         wherein when X 5  is carbon, W 5  may be hydrogen, methyl, amino, or chloro; 
         wherein when X 6  is carbon, W 6  may be hydrogen, methyl, amino, acetyl, carboxy, carboxamide, or hydroxy; 
         wherein when X 7  or X 8  is carbon, W 7  or W 8 , respectively, may independently be hydrogen, C 1-6  alkyl, C 2-6  alkenyl, C 2-6  alkynyl, C 3-6  cycloalkyl, (C 3-6  cycloalkyl)methyl, phenyl, benzyl, five- or six-membered heterocyclyl, five- or six-membered heteroaryl, cyano, amino, acetyl, carboxy, hydroxy or CONR 8 R 9 ;
 wherein each of R 8  and R 9  is independently hydrogen, C 1-6  alkyl, C 2-6  alkenyl, C 2-6  alkynyl, C 3-6  cycloalkyl, (C 3-6  cycloalkyl)methyl, phenyl, benzyl, five- or six-membered heterocyclyl, five- or six-membered heteroaryl, or OR 10 , or together with the nitrogen atom form a pyrrolidine, piperidine, morpholine, or pyrazine ring;
 wherein R 10  is hydrogen, C 1-6  alkyl, C 2-6  alkenyl, C 2-6  alkynyl, C 3-6  cycloalkyl, (C 3-6  cycloalkyl)methyl, phenyl, benzyl, five- or six-membered heterocyclyl, or five- or six-membered heteroaryl; 
 
 or wherein W 7  or W 8  together form a five- or six-membered aryl, carbocyclic, heterocyclic, or heteroaryl ring fused with the B ring; 
 
         wherein X 2′  is hydrogen, hydroxy, or OR 2′ ; 
         wherein X 3′  is hydrogen, hydroxy, or OR 3′ ; 
         wherein X 5′  is C(═X 4 )X 10 R;
 wherein X 4  is O or H 2 ; 
 wherein X 10  is C(H)NR 11 R 12 , NR 1 , or S;
 wherein R 11  and R 12  each is independently hydrogen, C 1-4  alkyl, C 2-3  alkenyl, C 2-3  alkynyl, C 3-6  cycloalkyl, phenyl, benzyl, or acetyl, or together with the nitrogen atom form an aziridine, azetidine, pyrrolidine, piperidine, morpholine, or pyrazine ring; 
 wherein R and R 1  each is independently 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, 
 
 
         wherein any alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl ring is optionally substituted with one or more of fluoro, chloro, bromo, iodo, C 1-6  alkyl, C 2-6  alkenyl, C 2-6  alkynyl, methoxy, ethoxy, trifluoromethoxy, trifluoromethyl, hydroxy, thiomethyl, cyano, NR 8 R 9 , —N(H)C(═O)X 11 , acetyl, carboxy, carboxy(C 1-4  alkyl), or CONR 8 R 9 ;
 wherein X 11  is C 1-6  alkyl, C 2-6  alkenyl, C 2-6  alkynyl, C 3-6  cycloalkyl, (C 3-6  cycloalkyl)methyl, phenyl, benzyl, OR 10 , or NR 8 R 9 ; 
 
         wherein one of R 2 , R 2′  (if it exists), and R 3′  (if it exists), comprises a linker component, wherein the linker component comprises a linker moiety bonded to a fluorophore moiety, and the other existing of R 2 , R 2′ , and R 3′  are hydrogen, and wherein R 2 , when not hydrogen, substitutes a hydrogen atom of the B ring or a ring fused to the B ring or a functional group covalently bound to the B ring or a ring fused to the B ring; 
         wherein the linker moiety comprises a structure as illustrated in Formula (VI) or Formula (VII): 
       
       
         
           
           
               
               
           
         
         wherein when R 2  comprises the linker component, the linker moiety may alternatively comprise a structure as illustrated in Formula (VIII): 
       
       
         
           
           
               
               
           
         
       
       wherein the (CH 2 ) n  group of Formula (IV) or the (CH 2 ) s  group of Formula (V) comprises the site of covalent attachment at one of R 2 , R 2′ , and R 3′  of Formulas (I), (II), and (III);
 wherein X is CH 2  or O; 
 wherein X 1  is N—H, N—CH 3 , O, or S; 
 wherein X 2  is N—H, N—CH 3 , or, when r is 0 and X 1  is N—H or N-Me, X 2  may alternatively be O; 
 wherein X 3  is NH or O; 
 wherein Z 1  is a carbonyl, thiocarbonyl, or sulfonyl group; 
 wherein Y is a covalent bond that binds the linker moiety to the fluorophore moiety, or (CH 2 ) n  wherein the last CH 2  group in the chain (when n is not 0) is farthest from Z 1  is covalently bound to the fluorophore moiety, or (CH 2 ) n —N(H)—Z 2 , or is of the chemical structure: 
 
       
         
           
           
               
               
           
         
         
           wherein the oxygen atom end is covalently bound to Z 1 ; 
           wherein each R 5  and R 6  is independently H, methyl, or together are (CH 2 ) q ; 
           wherein q is 1, 2, or 3; 
           wherein p is 1 or 2; and 
           wherein Z 2  is a carbonyl or thiocarbonyl group covalently bound by its carbon atom to the fluorophore moiety, or a sulfonyl group covalently bound by sulfur atom to the fluorophore moiety; 
         
         wherein each n is independently 0, 1, 2, 3, 4, or 5; 
         wherein s is 1, 2, or 3; 
         wherein m is 1, 2, or 3; 
         wherein each r is independently 0 or 1; 
         wherein the fluorophore moiety is a structure selected from the group of chemical structures consisting of: 
       
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         wherein * represents the position at which the linker moiety is covalently bound to the fluorophore moiety; 
         wherein A −  is a PF 6   − , trifluoroacetate, acetate, or halide anion; 
         wherein B +  is a sodium, potassium, cesium, ammonium, or  + N(R 4 ) 4  cation; and 
         wherein each R 4  is independently H or C 1-4  alkyl. 
       
     
     
         46 . A fluorescent detection analyte according to  claim 45 , wherein the fluorophore moiety comprises a mixture of chemical structure (b) and (c), a mixture of chemical structure (b) and (n), a mixture of chemical structure (d) and (e), a mixture of chemical structure (f) and (g), a mixture of chemical structure (h) and (i), or a mixture of chemical structure (j) and (k). 
     
     
         47 - 58 . (canceled) 
     
     
         59 . The fluorescent detection analyte of  claim 45 , wherein X 9  is O. 
     
     
         60 . The fluorescent detection analyte of  claim 59 , wherein X 6  is N and wherein each of X 5 , X 7  and X 8  is C. 
     
     
         61 . The fluorescent detection analyte of  claim 45 , wherein X 9  is S. 
     
     
         62 . The fluorescent detection analyte of  claim 61 , wherein X 6  is N and wherein each of X 5 , X 7  and X 8  is C. 
     
     
         63 . The fluorescent detection analyte of  claim 45 , wherein X 9  is NR 7 . 
     
     
         64 . The fluorescent detection analyte of  claim 63 , wherein X 6  is N and wherein each of X 5 , X 7  and X 8  is C. 
     
     
         65 . The fluorescent detection analyte of  claim 45 , wherein X 6  is N and wherein each of X 5 , X 7  and X 8  is C.