Methods for measuring enzyme activity

Abstract

The invention relates to fluorescence methods for measuring enzyme activity, in particular enzyme cleaving and joining activities. The invention also relates to fluorogenic substrates which are useful for measuring enzyme activity and as in vitro and in vivo imaging probes.

1 . In a method of measuring the activity of an enzyme in cleaving a substrate, said substrate including at least one fluorescent label bound to a polymer which includes one or more tyrosine, tryptophan, phenoxy, indolyl or nitro-phenylalanine moieties, said moieties being separated from the said at least one fluorescent label by a linkage group cleavable by said enzyme, the improvement comprising the steps of:
 i) measuring the fluorescence lifetime of the at least one label of the substrate in a reaction mixture which facilitates enzyme activity;   ii) adding the enzyme to said reaction mixture, and   iii) measuring any increase in fluorescence lifetime of the at least one fluorescent label following step ii);   
       wherein said increase in fluorescence lifetime indicates substrate cleavage and can be used to determine enzyme activity. 
     
     
         2 . The method of  claim 1 , wherein said polymer includes one or more phenoxy or indolyl moeities. 
     
     
         3 . The method of  claim 1 , wherein the fluorescent label is an acridone dye of formula: 
       
         
           
           
               
               
           
         
       
       wherein:
 groups R 2  and R 3  are attached to the Z 1  ring structure and groups R 4  and R 5  are attached to the Z 2  ring structure; 
 Z 1  and Z 2  independently represent the atoms necessary to complete one or two fused ring aromatic or heteroaromatic systems, each ring having five or six atoms selected from carbon atoms and optionally no more than two atoms selected from oxygen, nitrogen and sulphur; 
 R 1 , R 2 , R 3 , R 4  and R 5  are independently selected from hydrogen, halogen, amide, hydroxyl, cyano, amino, mono- or di-C 1 -C 4  alkyl-substituted amino, sulphydryl, carbonyl, C 1 -C 6  alkoxy, aryl, heteroaryl, C 1 -C 20  alkyl, aralkyl; the group -E-F where E is a spacer group having a chain from 1-60 atoms selected from the group consisting of carbon, nitrogen, oxygen, sulphur and phosphorus atoms and F is a target bonding group; and the group —(CH 2 —) n Y where Y is selected from sulphonate, sulphate, phosphonate, phosphate, quaternary ammonium and carboxyl and n is zero or an integer from 1 to 6. 
 
     
     
         4 . The method of  claim 1 , wherein the fluorescent label is a quinacridone dye of formula: 
       
         
           
           
               
               
           
         
       
       wherein:
 groups R 3  and R 4  are attached to the Z 1  ring structure and groups R 5  and R 6  are attached to the Z 2  ring structure; 
 Z 1  and Z 2  independently represent the atoms necessary to complete one or two fused ring aromatic or heteroaromatic systems, each ring having five or six atoms selected from carbon atoms and optionally no more than two atoms selected from oxygen, nitrogen and sulphur; 
 R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7  and R 8  are independently selected from hydrogen, halogen, amide, hydroxyl, cyano, amino, mono- or di-C 1 -C 4  alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C 1 -C 6  alkoxy, aryl, heteroaryl, C 1 -C 20  alkyl, aralkyl; the group -E-F where E is a spacer group having a chain from 1-60 atoms selected from the group consisting of carbon, nitrogen, oxygen, sulphur and phosphorus atoms and F is a target bonding group; and the group —(CH 2 —) n Y where Y is selected from sulphonate, sulphate, phosphonate, phosphate, quaternary ammonium and carboxyl and n is zero or an integer from 1 to 6. 
 
     
     
         5 . The method of  claim 1 , wherein said polymer is selected from the group consisting of peptides, polypeptides, proteins, nucleic acids, oligonucleic acids, protein nucleic acids, polysaccharides and polyglycerides. 
     
     
         6 . The method of  claim 1 , wherein the polymer contains 4 to 40 amino acid residues. 
     
     
         7 . The method of  claim 1 , wherein said linkage group is cleavable by an enzyme of EC Class 3. 
     
     
         8 . The method of  claim 7 , wherein the enzyme is a hydrolase enzyme selected from the group consisting of esterases, peptidases, amidases, nucleases and glycosidases. 
     
     
         9 . The method of  claim 8 , wherein said enzyme is a peptidase selected from the group consisting of angiotensin converting enzyme (ACE), caspase, cathepsin D, chymotrypsin, pepsin, subtilisin, proteinase K, elastase, neprilysin, thermolysin, asp-n, matrix metallo protein 1 to 20, papain, plasmin, trypsin, enterokinase and urokinase. 
     
     
         10 . The method of  claim 8 , wherein said enzyme is an endonuclease or exonuclease selected from the group consisting of exodeoxyribonuclease III (E.C.3.1.112), exodeoxyribonuclease I (E.C.3.1.11.1), exodeoxyribonuclease V (E.C.3.1.11.5), venom exonuclease (E.C.3.1.15.1), deoxyribonuclease I (E.C.3.1.21.1), deoxyribonuclease II (E.C.3.1.22.1), ribonuclease H (E.C. 3.1.26.4), ribonuclease T1 (E.C.3.1.27.3), pancreatic ribonuclease (E.C.3.1.27.5) and micrococcal nuclease (E.C.3.1.31.1). 
     
     
         11 . The method of  claim 8 , wherein said enzyme is a glycosidase selected from the group consisting of α-amalyse (E.C.3.2.1.1), β-amalyse (E.C.3.2.1.2), glucan 1,4-α-glucosidase (E.C.3.2.1.3), cellulase (E.C.3.2.1.4), endo-1,3-β-glucanase (E.C.3.2.1.6), oligo-1,6-glucosidase (E.C.3.2.1.10), and lysozyme (E.C.3.2.1.17). 
     
     
         12 . The method of  claim 1 , wherein said substrate is selected from the group consisting of 6-(9-oxo-9H-acridin-10-yl) hexanoyl-AAFFAAY, 6-(9-oxo-9H-acridin-10-yl) hexanoyl-AAFFAAF(Nitro), 6-(9-oxo-9H-acridin-10-yl) hexanoyl-CHLDIIW and 6-(9-oxo-9H-acridin-10-yl) hexanoyl-RPKPVE(Nva)WRK. 
     
     
         13 . The method of  claim 12 , wherein the substrate further contains a cell entry peptide. 
     
     
         14 . The method of  claim 13 , wherein said cell entry peptide is selected from the group consisting of TAT and Chariot. 
     
     
         15 . A method for measuring cellular location and distribution of a substrate, wherein the substrate is capable of being taken up by a living cell, the method comprising the steps of:
 i) measuring the fluorescence intensity and/or the fluorescence lifetime of the label in a cell-free environment;   ii) adding the substrate to one or more cells, and   iii) measuring the fluorescence intensity and/or the lifetime of the fluorescent label following step ii);   
       wherein an increase in fluorescence intensity and/or fluorescence lifetime indicates substrate cleavage and can be used to determine both enzyme activity and localisation. 
     
     
         16 . The method of  claim 15 , wherein said cell is selected from the group consisting of mammalian, plant, insect, fish, avian, bacterial and fungal cells. 
     
     
         17 - 18 . (canceled) 
     
     
         19 . A method of measuring the activity of an enzyme in joining a substrate to a reactant, said substrate including at least one fluorescent label and said reactant including one or more tyrosine, tryptophan, phenoxy, indolyl or nitro-phenylalanine, moieties, the method comprising the steps of:
 i) measuring the fluorescence intensity and/or the fluorescence lifetime of the label in a reaction mixture which facilitates enzyme activity;   ii) adding the enzyme to said reaction mixture, and   iii) measuring any decrease in fluorescence intensity and/or lifetime of the fluorescent label following step ii);   
       wherein said decrease in fluorescence intensity and/or lifetime indicates joining of the substrate to the reactant and can be used to determine enzyme activity. 
     
     
         20 . The method of  claim 19 , wherein the fluorescent label is an acridone dye or a quinacridone dye. 
     
     
         21 . The method of  claim 19 , wherein the substrate and/or the reactant is selected from the group consisting of peptides, polypeptides, proteins, nucleic acids, oligonucleic acids, protein nucleic acids, polysaccharides and polyglycerides. 
     
     
         22 . The method of  claim 19 , wherein the enzyme is a ligase of EC Class 6 or a transferase of EC Class 2. 
     
     
         23 . The method of  claim 19 , wherein said substrate and/or reactant additionally comprise a cell entry peptide. 
     
     
         24 . The method of  claim 23 , wherein said cell entry peptide is selected from the group consisting of TAT and Chariot. 
     
     
         25 . A method for measuring cellular location and/or distribution of the substrate and/or reactant, wherein the substrate and the reactant are capable of being taken up by a living cell, the method comprising the steps of:
 i) measuring the fluorescence intensity and/or the fluorescence lifetime of the label in a cell-free environment;   ii) adding the substrate and the reactant to one or more cells, and   iii) measuring the fluorescence intensity and/or the lifetime of the fluorescent label following step ii);   
       wherein a decrease in fluorescence intensity and/or fluorescence lifetime indicates substrate joining to reactant and can be used to determine both enzyme activity and localisation. 
     
     
         26 . The method of  claim 25 , wherein said cell is selected from the group consisting of mammalian, plant, insect, fish, avian, bacterial and fungal cells. 
     
     
         27 . The method of  claim 19 , further comprising the use of a plurality of different substrates and/or reactants each bound to a plurality of different labels, wherein each said label is individually distinguishable from the others by its unique fluorescence emission and/or its fluorescence lifetime thereby enabling simultaneous measurement of a plurality of enzyme joining activities. 
     
     
         28 . The method of  claim 27 , wherein each of said labels is selected from the group consisting of 6-(9-oxo-9H-acridin-4-carboxamido) hexanoic acid, 6-(2-(acetamido)-9-oxo-9H-acridin-10-yl) hexanoic acid, 6-(9-oxo-9H-acridin-10-yl) hexanoic acid, 6-(2-bromo-9-oxo-9H-acridin-10-yl) hexanoic acid and 6-(12-ethyl-7,14-Dioxo-2,9-disulpho-7,14-dihydroquino[2,3-b]acridin-5(12H)-yl) hexanoic acid.