Dyes for analysis of protein aggregation

Abstract

Provided are dyes and compositions which are useful in a number of applications, such as the detection and monitoring protein aggregation, kinetic studies of protein aggregation, neurofibrillary plaques analysis, evaluation of protein formulation stability, and analysis of molecular chaperone activity.

1 . A compound comprising
 (a) the structure of compound S13 or   (b) the structure   
       
         
           
           
               
               
           
         
         wherein
 m and n are independently 1, 2 or 3; 
 L is —(CH 2 ) 3 , —(CH 2 ) 4 , —(CH 2 ) 6 , —CH 2 —CH 2 —O—CH 2 —CH 2 , or —CH 2 -(phenylene)-CH 2 —; 
 R 1 , R 2 , R 3 , R 4 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 19 , R 20 , R 21  and R 22  are independently hydrogen, halogen, amino, ammonium, nitro, sulfo, sulfonamide, carboxy, ester, cyano, phenyl, benzyl, an alkyl group wherein the alkyl group is saturated or unsaturated, linear or branched, substituted or unsubstituted, an alkoxy group wherein the alkoxy group is saturated or unsaturated, branched or linear, substituted or unsubstituted, or when taken in combination R 1  and R 2 , or R 3  and R 4 , or R 9  and R 10 , or R 11  and R 12 , or R 13  and R 14 , or R 15  and R 16 , or R 19  and R 20 , or R 21  and R 22  form a five or six membered ring wherein the ring is saturated or unsaturated, substituted or unsubstituted, and wherein R 9  and R 10 , or R 11  and R 12 , or R 13  and R 14 , or R 15  and R 16  can comprise alkyl chains that are joined together, wherein a quinoline moiety can be formed; 
 R 7 , R 8 , R 17  and R 18  are independently hydrogen, Z, an alkyl group wherein the alkyl group is saturated or unsaturated, linear or branched, substituted or unsubstituted, an alkoxy group wherein the alkoxy group is saturated or unsaturated, branched or linear, substituted or unsubstituted, or when taken together, R 7  and R 8  and R 17  and R 18 , may form a 5 or 6 membered ring wherein the ring is saturated or unsaturated, substituted or unsubstituted;
 wherein Z comprises a carboxyl group (CO 2   − ), a carbonate ester (COER 25 ), a sulfonate (SO 3   − ), a sulfonate ester (SO 2 ER 25 ), a sulfoxide (SOR 25 ), a sulfone (SO 2 CR 25 R 26 R 27 ), a sulfonamide (SO2NR 25 R 26 ), a phosphate (PO 4   = ), a phosphate monoester (PO 3   − ER 25 ), a phosphate diester (PO 2 ER 25 ER 26 ), a phosphonate (PO 3   = ) a phosphonate monoester (PO 2   − ER 25 ) a phosphonate diester (POER 25 ER 26 ), a thiophosphate (PSO 3   = ), a thiophosphate monoester (PSO 2   − ER 25 ) a thiophosphate diester (PSOER 25 ER 26 ), a thiophosphonate (PSO 2   = ), a thiophosphonate monoester (PSO − ER 25 ) a thiophosphonate diester (PSER 25 ER 26 ), a phosphonamide (PONR 25 R 26 NR 28 R 29 ), its thioanalogue (PSNR 25 R 26 NR 28 R 29 ), a phosphoramide (PONR 25 R 26 NR 27 NR 28 R 29 ), its thioanalogue (PSNR 25 R 26 NR 27 NR 28 R 29 ), a phosphoramidite (PO 2 R 25 NR 28 R 29 ) or its thioanalogue (POSR 25 NR 28 R 29 ) where E can be independently O or S; 
 wherein R 25 , R 26 , R 27 , R 28 , and R 29  are independently a hydrogen, an unsubstituted straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group, a substituted straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group wherein one or more C, CH or CH 2  groups are substituted with an O atom, N atom, S atom, or NH group, or an unsubstituted or substituted aromatic group; 
 
 Z is attached directly, or indirectly through a second linker arm comprising carbon, sulfur, oxygen, nitrogen, and any combinations thereof and wherein the second linker arm may be saturated or unsaturated, linear or branched, substituted or unsubstituted or any combinations thereof; and 
 R 5 , R 6 , R 23  and R 24  can independently be hydrogen or an alkyl group wherein the alkyl group is saturated or unsaturated, linear or branched, substituted or unsubstituted, or when taken in combination R 5  and R 6  or R 2  and R 5  or R 3  and R 6  or R 23  and R 24  or R 22  and R 23  or R 20  and R 24  form a five or six membered ring wherein the ring is saturated or unsaturated, substituted or unsubstituted. 
 
       
     
     
         2 . The compound of  claim 1 , wherein the compound exhibits increased fluorescence in the presence of an aggregated form of a protein when compared to the fluorescence exhibited when the compound is in the presence of the unaggregated form of the protein. 
     
     
         3 . The compound of  claim 2 , wherein the protein is immunoglobulin, a DNA polymerase or a fragment thereof, α-synuclein, synphilin-1, TCRα, P23H mutant of rhodopsin, ΔF508 mutant of CFTR, amyloid-Jβ, prion protein, Tau, SOD1, Ig light chains, ataxin-1, ataxin-3, ataxin-7, calcium channel, atrophin-1, androgen receptor, p62/sequestosome1 (SQSTM1), Pael receptor, serum amyloid A, transthyretin, β2-microglobulin, apolipoprotein A-1, gelsolin, atrial natriuretic factor, lysozyme, insulin, fibrinogen, crystallin, surfactant protein C, lactoferrin, βig-h3, PAPB2, corneodesmosin, neuroserpin, cochlin, RET, myelin, protein 22/0, SCAD, prolactin, lactadherin, p53, procalcitonin, cytokeratin, GFAP, ATP7B, prolyl hydroxylase PHD3, presenilin, or huntingtin. 
     
     
         4 . The compound of  claim 1 , comprising the structure 
       
         
           
           
               
               
           
         
       
     
     
         5 . The compound of  claim 4 , wherein each of R 5 , R 6 , R 23  and R 24  are a methyl or an ethyl moiety. 
     
     
         6 . The compound of  claim 1 , wherein the compound comprises S25, S43, TOL3, YAT2134, YAT2148, YAT2149, S13, YAT2135, YAT2324 or YAT2150. 
     
     
         7 . The compound of  claim 1 , wherein the compound is S25. 
     
     
         8 . The compound of  claim 1 , wherein the compound is YAT2150. 
     
     
         9 . The compound of  claim 1 , wherein the compound is TOL3. 
     
     
         10 - 16 . (canceled) 
     
     
         17 . A compound exhibiting at least three times increased fluorescence in the presence of an aggregated form of a protein when compared to the fluorescence exhibited when the compound is in the presence of the unaggregated form of the protein, wherein the compound is D95, D97, L-30, L-33, Lu-1, Lu-2, S-8, S13. S22, S25, S33, S39, S42, S43, S48, S49, SL2131, SL2592, Tio-1, TOL-2, TOL-3, TOL-5, TOL-6, TOL-7, TOL-11, YA-1, YA-3, YAT2134, YAT2135, YAT2148, YAT2149, YAT2150, YAT2213, YAT2214 or YAT2324. 
     
     
         18 . A multi-dye composition comprising at least three dyes, wherein each of the at least three dyes exhibits increased fluorescence in the presence of an aggregated form of a protein when compared to the fluorescence exhibited when the compound is in the presence of the unaggregated form of the protein. 
     
     
         19 - 29 . (canceled) 
     
     
         30 . The compound of  claim 17 , further comprising a reactive group. 
     
     
         31 - 34 . (canceled) 
     
     
         35 . A labeled target molecule comprising a target molecule attached to the compound of  claim 30  through the reactive group. 
     
     
         36 - 38 . (canceled) 
     
     
         39 . A solid support attached to the compound of  claim 30  through the reactive group. 
     
     
         40 - 42 . (canceled) 
     
     
         43 . A kit for assaying aggregation of a protein, comprising in packaged combination:
 at least two of the compounds of  claim 17 .   
     
     
         44 - 56 . (canceled) 
     
     
         57 . A method for detecting an aggregate of a protein in a sample, the method comprising
 (a) combining the sample with the compound of  claim 2 ;   (b) measuring the amount of fluorescence in the dye-sample mixture;   (c) comparing the amount of fluorescence determined in (b) with the amount of fluorescence in
 (i) a mixture of the compound with a control sample without aggregated protein, or 
 (ii) a mixture of the compound with a known standard quantity of aggregated protein; and 
   (d) determining the aggregation of the protein in the sample based on the comparison in (c).   
     
     
         58 - 114 . (canceled) 
     
     
         115 . A method for separating aggregates of a protein from monomeric forms of the protein in a sample, the method comprising
 (a) combining the sample to the solid support of  claim 39  under conditions where aggregates of the protein preferentially bind to the compound; and   (b) separating sample protein bound to the solid support from unbound protein,   wherein protein bound to the solid support are substantially aggregates and unbound protein is substantially monomers.   
     
     
         116 . The compound of  claim 1 , wherein L is —CH 2 -(o-phenylene)-CH 2 — or —CH 2 -(p-phenylene)-CH 2 —. 
     
     
         117 . A combination of two compounds of  claim 1 , wherein each compound exhibits at least three times increased fluorescence in the presence of an aggregated form of a protein when compared to the fluorescence exhibited when the compound is in the presence of the unaggregated form of the protein. 
     
     
         118 . The combination of two compounds of  claim 117 , wherein the two compounds are S25 and TOL3.