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.

What is claimed is: 
     
         1 . A composition of matter for the fluorescent detection of protein aggregation in a test sample, comprising:
 (a) a test sample comprising protein; and   (b) at least one protein aggregation indicator compound having the structural formula   
       
         
           
           
               
               
           
         
       
       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 (SO 2 NR 25 R 26 ), a phosphate, a phosphate monoester (PO 3   − ER 25 ), a phosphate diester (PO 2 ER 25 ER 26 ), a phosphonate, a phosphonate monoester (PO 2   − ER 25 ), a phosphonate diester (POER 25 ER 26 ), a thiophosphate, a thiophosphate monoester (PSO 2   − ER 25 ), a thiophosphate diester (PSOER 25 ER 26 ), a thiophosphonate, a thiophosphonate monoester (PSO − ER 25 ), a thiophosphonate diester (PSER 25 ER 26 ), a phosphonamide (PONR 25 R 26 NR 28 R 29 ) or its thioanalogue (PSNR 25 R 26 NR 28 R 29 ), a phosphoramide (PONR 25 R 26 NR 27 NR 28 R 29 ) or its thioanalogue (PSNR 25 R 26 NR 27 NR 28 R 29 ), or 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, 
 wherein the at least one protein aggregation indicator compound exhibits increased fluorescence when contacted with aggregated protein compared to the fluorescence exhibited by the compound when contacted with the unaggregated protein, and 
 wherein the at least one protein aggregation indicator compound is in contact with the protein of the test sample. 
 
     
     
         2 . The composition of matter of  claim 1 , wherein the protein comprises immunoglobulin, a DNA polymerase or a fragment thereof, α-synuclein, synphilin-1, TCRα, P23H mutant of rhodopsin, ΔF508 mutant of CFTR, amyloid-β, 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. 
     
     
         3 . The composition of matter of  claim 1 , wherein the test sample is a bodily fluid of a mammal. 
     
     
         4 . The composition of matter of  claim 3 , wherein the bodily fluid of the mammal is selected from the group consisting of blood, serum and plasma. 
     
     
         5 . The composition of matter of  claim 1 , wherein the test sample comprises mammalian cells. 
     
     
         6 . The composition of matter of  claim 5 , wherein the mammalian cells are intact. 
     
     
         7 . The composition of matter of  claim 6 , wherein the intact mammalian cells are living. 
     
     
         8 . The composition of matter of  claim 1 , wherein the test sample comprises homogenized mammalian cells of a mammal. 
     
     
         9 . The composition of matter of  claim 1 , wherein the test sample comprises cells. 
     
     
         10 . The composition of matter of  claim 1 , wherein the test sample is a mammalian tissue sample. 
     
     
         11 . The composition of matter of  claim 1 , wherein the protein of the test sample is a recombinant protein. 
     
     
         12 . The composition of matter of  claim 1 , wherein the at least one protein aggregation indicator compound comprises two different protein aggregation indicator compounds of the structural formula, wherein each of said two compounds exhibits increased fluorescence when contacted with aggregated protein compared to the fluorescence exhibited when contacted with unaggregated protein. 
     
     
         13 . The composition of matter of  claim 1 , wherein the at least one protein aggregation indicator compound comprises three different protein aggregation indicator compounds of the structural formula, wherein each of said three compounds exhibits increased fluorescence when contacted aggregated protein compared to the fluorescence exhibited when contacted with unaggregated protein. 
     
     
         14 . The composition of matter of  claim 1 , wherein the at least one protein aggregation indicator compound comprises a compound having the structural formula 
       
         
           
           
               
               
           
         
         wherein X −  is a anion. 
       
     
     
         15 . The composition of matter of  claim 14 , wherein X −  is Br − . 
     
     
         16 . The composition of matter of  claim 1 , wherein the at least one protein aggregation indicator compound comprises a compound having the structural formula 
       
         
           
           
               
               
           
         
         wherein Y −  is a anion. 
       
     
     
         17 . The composition of matter of  claim 16 , wherein Y −  is I − . 
     
     
         18 . The composition of matter of  claim 1 , wherein the at least one protein aggregation indicator compound comprises a compound having the structural formula: 
       
         
           
           
               
               
           
         
         wherein X −  is an anion. 
       
     
     
         19 . The composition of matter of  claim 1 , wherein the at least one protein aggregation indicator compound comprises one or both of: 
       
         
           
           
               
               
           
         
       
     
     
         20 . The composition of matter of  claim 19 , wherein each of R 5 , R 6 , R 23  and R 24  is independently a methyl or an ethyl moiety. 
     
     
         21 . The composition of matter of  claim 1 , wherein the at least one protein aggregation indicator compound comprises a compound having the structural formula 
       
         
           
           
               
               
           
         
       
     
     
         22 . The composition of matter of  claim 1 , wherein the at least one protein aggregation indicator compound comprises a compound having the structural formula 
       
         
           
           
               
               
           
         
       
     
     
         23 . The composition of matter of  claim 1 , wherein the at least one protein aggregation indicator compound comprises a compound having the structural formula 
       
         
           
           
               
               
           
         
       
     
     
         24 . The composition of matter of  claim 1 , wherein L is —CH 2 -(ortho-phenylene)-CH 2 — or —CH 2 -(para-phenylene)-CH 2 —. 
     
     
         25 . The composition of matter of  claim 1 , wherein the at least one protein aggregation indicator compound comprises both 
       
         
           
           
               
               
           
         
       
     
     
         26 . A composition of matter comprising:
 (a) a reference sample comprising a known standard quantity of aggregated protein; and   (b) at least one protein aggregation indicator compound having the structural formula   
       
         
           
           
               
               
           
         
       
       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 (SO 2 NR 25 R 26 ), a phosphate, a phosphate monoester (PO 3   − ER 25 ), a phosphate diester (PO 2 ER 25 ER 26 ), a phosphonate, a phosphonate monoester (PO 2   − ER 25 ), a phosphonate diester (POER 25 ER 26 ), a thiophosphate, a thiophosphate monoester (PSO 2   − ER 25 ), a thiophosphate diester (PSOER 25 ER 26 ), a thiophosphonate, a thiophosphonate monoester (PSOER 25 ), a thiophosphonate diester (PSER 25 ER 26 ), a phosphonamide (PONR 25 R 26 NR 28 R 29 ) or its thioanalogue (PSNR 25 R 26 NR 28 R 29 ), a phosphoramide (PONR 25 R 26 NR 27 NR 28 R 29 ) or its thioanalogue (PSNR 25 R 26 NR 27 NR 28 R 29 ), or 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, 
 wherein the at least one protein aggregation indicator compound exhibits increased fluorescence when contacted with aggregated protein compared to the fluorescence exhibited by the compound when contacted with unaggregated protein, and 
 wherein the at least one protein aggregation indicator compound is in contact with the protein of the reference sample. 
 
     
     
         27 . The composition of matter of  claim 25 , wherein the protein of the reference sample is a recombinant protein. 
     
     
         28 . The composition of matter of  claim 26 , wherein the at least one protein aggregation indicator compound comprises a compound having the structural formula 
       
         
           
           
               
               
           
         
         wherein X −  is a anion. 
       
     
     
         29 . The composition of matter of  claim 28 , wherein X −  is Br − . 
     
     
         30 . The composition of matter of  claim 26 , wherein the at least one protein aggregation indicator compound comprises a compound having the structural formula 
       
         
           
           
               
               
           
         
         wherein Y −  is a anion. 
       
     
     
         31 . The composition of matter of  claim 30 , wherein Y −  is I − . 
     
     
         32 . The composition of matter of  claim 26 , wherein the at least one protein aggregation indicator compound comprises a compound having the structural formula: 
       
         
           
           
               
               
           
         
         wherein X −  is an anion. 
       
     
     
         33 . A method for detecting or quantifying protein aggregation in a test sample comprising protein, comprising the steps of:
 providing a first composition of matter according to  claim 1 ;   illuminating the first composition of matter;   measuring the amount of fluorescence emitted from the first composition of matter as a result of said illuminating; and   determining the presence or amount of protein aggregation in the test sample based on the measured amount of fluorescence emitted by the first composition of matter.   
     
     
         34 . The method of  claim 33 , wherein the protein of the test sample comprises a recombinant protein. 
     
     
         35 . The method of  claim 33 , further comprising the steps of:
 providing a second composition of matter comprising
 i. a reference sample comprising a known standard quantity of aggregated protein, and 
 ii. the at least one protein aggregation indicator compound, 
 wherein the protein of the reference sample is in contact with the at least one protein aggregation indicator compound; 
   illuminating the second composition of matter; and   measuring the amount of fluorescence emitted from the second composition of matter as a result of illuminating the second composition of matter,   wherein the determining step comprises determining the presence or amount of protein aggregation in the test sample by comparing the measured amount of fluorescence emitted by the first composition of matter with the measured amount of fluorescence emitted by the second composition.   
     
     
         36 . The method of  claim 34 , wherein the known standard quantity of aggregated protein in the reference sample is no aggregated protein. 
     
     
         37 . The method of  claim 35 , wherein the protein of the reference sample comprises a recombinant protein.