Thrombin Substrate and Assay for Determining the Level of Bioactive Thrombin in a Sample

Abstract

Substrates for thrombin and assays for determining the level of bioactive thrombin in a sample are disclosed, wherein the substrate has the general formula: A-X—Z-A′ wherein one of either A or A′ comprises a luminescent chelate and the other one of A or A′ comprises a first partner of a binding pair, X forms a tri- or tetra-peptide, and Z comprises a linker.

1 . A substrate for thrombin having the formula:
   A-X—Z-A′
   wherein   one of either A or A′ comprises a luminescent chelate, and   the other one of A or A′ comprises a first partner of a binding pair, optionally including a spacer, and connected via a peptide bond to the remaining part of the substrate;   X forms a tri- or tetra-peptide selected from the group consisting of X′-Phe-Aze-Arg, X′-Phe-Pip-Arg, and X′-Phe-Pro-Arg, wherein   X′ is absent or is selected from the group consisting of Lys, Ahx, Ile, and Val;   Z is NH—R—Z′, wherein   R is selected from the group consisting of C 1 - 6 alkylene-arylene, C 1 - 6 alkylenoxy-arylene, C 1 - 6 alkylene-CONH—C 1 - 6 alkylene-arylene, arylene, arylene-C 1 - 6 alkylene, arylene-C 1 - 6 alkylenoxy, (R 1 ) a -arylene-(NHCO—R 2 ) b , (R 3 ) c arylene-(CONH—R 4 ) d , (R 5 —CONH) e -arylene-(R 6 ) f , and (R 7 —NHCO) g -arylene-(R 8 ) h ,   wherein each R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , and R 8  at each occurrence is independently selected from the group consisting of C 1 - 6 alkylene, C 1 - 6 alkylenoxy, C 1 - 6 thioalkylene, C 1 - 6 thioalkylenoxy, carbonyl-C 1 - 6 alkylene, carbonyl-C 1 - 6 alkylenoxy, C 1 - 6 alkylene-carbonyl, arylene and arylene-C 1 - 6 alkylene, and each of a, b, c, d, e, f, g, and h is independently selected from the group consisting of the integers from 0 to 6,   wherein the arylene is phenylene, biphenylene or naphthylene, wherein the phenylene, biphenylene or naphthylene is optionally mono-, di- or tri-substituted by one or more substituents selected from the group consisting of halogen, OH, SH, CN, NO 2 , C 1 - 6 alkyl, C 1 - 6 alkoxy, and C 1 - 6 alkoxycarbonyl;   Z′ is selected from the group consisting of thiourea (—NH—CS—NH), aminoacetamide (—NH—CO—CH 2 —NH—), amide (—NH—CO—), methylamide (—NCH 3 —CO—) and substituted-triazine-diamine (—NH—(R 9 C 3 N 3 )—NH—),   thioether (—S—), thioacetamide (—S—CH 2 —CO—NH—), disulfide (—S—S—), (—S—CO—CH 2 —NH—) and (—S—(R 9 C 3 N 3 )—NH—) or   amide (—CO—NH—, —CO—NCH 3 —) and an (—CO—O—),   wherein R 9  is selected from the group consisting of hydrogen, halogen, C 1 - 6 alkyl, C 1 - 6 thioalkyl, C 1 - 6 alkoxy, C 1 - 6 thioalkoxy, aryloxy, and amino, which alkyl, thioalkyl, alkoxy, thioalkoxy or aryloxy group is optionally mono-, di- or tri-substituted and which amino group is optionally mono- or di-substituted by one or more substituents selected from the group consisting of halogen, OH, SH, CN, NO 2 , C 1 - 6 alkyl, C 1 - 6 alkoxy, and C 1 - 6 alkoxycarbonyl.   
     
     
         2 . The substrate according to  claim 1 , wherein X is D-Phe-L-2-Aze-L-Arg or D-Phe-L-2-Pip-L-Arg. 
     
     
         3 . The substrate according to  claim 1 , wherein Z′ is N and R is arylene, C 1 - 6 alkylene-arylene, or (R 1 ) a -arylene-(NHCO—R 2 ) b , wherein a and b are independently 0, 1 or 2. 
     
     
         4 . The substrate according to  claim 1 , wherein said luminescent chelate is a fluorescent lanthanide chelate. 
     
     
         5 . The substrate according to  claim 4 , wherein said lanthanide chelate is selected from the group consisting of 
       
         
           
           
               
               
           
         
       
     
     
         6 . The substrate according to  claim 1 , wherein said first partner of a binding pair is selected from the group consisting of biotin/streptavidin, biotin/avidin, biotin/biotin acceptor peptide, and streptavidin derivative/acceptor peptide. 
     
     
         7 . The substrate according to  claim 1 , wherein said first partner of a binding pair is connected to the remaining part of the substrate via a spacer of the formula NH—R 10 —CONH—R 11- CO—(NH—R 12 —NH) i ,
 wherein each of R 10 , R 11  and R 12  is independently selected from the group consisting of C 1 - 6 alkylene, C 1 - 6 alkylenoxy, C 1 - 6 thioalkylene, C 1 - 6 thioalkylenoxy, carbonyl-C 1 - 6 alkylene, carbonyl-C 1 - 6 alkylenoxy, C 1 - 6 alkylene-carbonyl, arylene and arylene-C 1 - 6 alkylene, 
 wherein the arylene is phenylene, biphenylene or naphthylene, wherein the phenylene, biphenylene or naphthylene is optionally mono-, di- or tri-substituted by one or more substituents selected from the group consisting of halogen, OH, SH, CN, NO 2 , C 1 - 6 alkyl, C 1 - 6 alkoxy, and C 1 - 6 alkoxycarbonyl, and wherein i is an integer from 0 to 6, 
 or the spacer is a W-mer peptide, wherein W is an integer from 2 to 40. 
 
     
     
         8 . The substrate according to  claim 7 , wherein R 10 , R 11  and R 12  are identical or different straight-chained C 1 - 6 alkylene, arylene or arylene-C 1 - 6 alkylene and i is 0 or 1. 
     
     
         9 . The substrate according to  claim 1  selected from the group consisting of S1V6a, S1V6b, S1V6c, S1V9a, S1V9b, S1V12a, S1V12b and S1V12c 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         10 . A one-step assay method for determining the level of bioactive thrombin in a test sample comprising the steps:
 a) combining, sequentially or simultaneously, a substrate according to  claim 1 , an activator and said test sample;   b) incubating the resulting reaction mixture to release thrombin-cleaved, chelate-containing substrate fragments and immobilising the binding partner-containing substrate fragment and non-thrombin-cleaved intact substrate on an immobilisation matrix;   c) washing off non-immobilised, thrombin-cleaved chelate-containing substrate fragment and non-immobilised, non-thrombin-cleaved substrate, if present;   d) measuring the level of luminescent emission from immobilized intact substrate; and   e) calculating thrombin activity from the reduction of intensity of luminescent emission compared to a thrombin-free standard sample.   
     
     
         11 . The assay method according to  claim 10 , wherein the addition of said sample and activator is performed simultaneously with the addition of the substrate to an immobilisation matrix. 
     
     
         12 . The assay method according to  claim 10 , wherein the addition of said sample and activator is performed after immobilisation of the substrate on an immobilisation matrix. 
     
     
         13 . A two-step assay method for determining the level of bioactive thrombin in a test sample comprising the steps:
 a) adding said sample and an activator to a substrate according to  claim 1  in liquid phase,   b) incubating the resulting reaction mixture to release thrombin-cleaved, chelate-containing substrate fragment,   c) adding the reaction mixture to an immobilisation matrix,   d) washing off non-immobilised, thrombin-cleaved chelate-containing substrate fragment and non-immobilised, non-thrombin-cleaved substrate, if present,   e) measuring the level of luminescent emission from immobilised intact substrate, and   f) calculating thrombin activity from the reduction of intensity of luminescent emission compared to a thrombin-free standard sample.   
     
     
         14 . The assay method according to  claim 10 , wherein said activator is selected from the group consisting of thromboplastin, partial thromboplastin reagents and contact activators. 
     
     
         15 . The assay method according to  claim 14 , wherein said partial thromboplastin reagents comprise phospholipids, and said contact activators comprise silica, kaolin, celite or ellagic acid. 
     
     
         16 . A test kit for determining the level of bioactive thrombin in a sample comprising
 a) a luminescent substrate according to  claim 1 , and   b) a solid immobilisation matrix.   
     
     
         17 . A substrate for an enzyme having the formula:
   A-X—Z-A′
   wherein   one of either A or A′ comprises a luminescent chelate, and   the other one of A or A′ comprises a first partner of a binding pair, optionally including a spacer, and connected via a peptide bond to the remaining part of the substrate;   X forms a tri- or tetra-peptide;   Z is NH—R—Z′, wherein   R is selected from the group consisting of C 1 - 6 alkylene-arylene, C 1 - 6 alkylenoxy-arylene, C 1 - 6 alkylene-CONH—C 1 - 6 alkylene-arylene, arylene, arylene-C 1 - 6 alkylene, arylene-C 1 - 6 alkylenoxy, (R 1 ) a -arylene-(NHCO—R 2 ) b , (R 3 ) c -arylene-(CONH—R 4 ) d , (R 5 —CONH) e -arylene-(R 6 ) f , and (R 7 —NHCO) g -arylene-(R 8 ) h ,   wherein each R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , and R 8  at each occurrence is independently selected from the group consisting of C 1 - 6 alkylene, C 1 - 6 alkylenoxy, C 1 - 6 thioalkylene, C 1 - 6 thioalkylenoxy, carbonyl-C 1 - 6 alkylene, carbonyl-C 1 - 6 alkylenoxy, C 1 - 6 alkylene-carbonyl, arylene and arylene-C 1 - 6 alkylene, and each of a, b, c, d, e, f, g, and h is independently selected from the integers from 0 to 6,   wherein the arylene is phenylene, biphenylene or naphthylene, wherein the phenylene, biphenylene or naphthylene is optionally mono-, di- or tri-substituted by one or more substituents selected from the group consisting of halogen, OH, SH, CN, NO 2 , C 1 - 6 alkyl, C 1 - 6 alkoxy, and C 1 - 6 alkoxycarbonyl,   Z′ is selected from the group consisting of thiourea (—NH—CS—NH), aminoacetamide (—NH—CO—CH 2 —NH—), amide (—NH—CO—), methylamide (—NCH 3 —CO—) and substituted-triazine-diamine (—NH—(R 9 C 3 N 3 )—NH—),   thioether (—S—), thioacetamide (—S—CH 2 —CO—NH—), disulfide (—S—S—), (—S—CO—CH 2 —NH—) and (—S—(R 9 C 3 N 3 )—NH) or   amide (—CO—NH—, —CO—NCH 3 —) and an ester (—CO—O—),   wherein R 9  is selected from the group consisting of hydrogen, halogen, C 1 - 6 alkyl, C 1 - 6 thioalkyl, C 1 - 6 alkoxy, C 1 - 6 thioalkoxy, aryloxy, and amino, which alkyl, thioalkyl, alkoxy, thioalkoxy or aryloxy group is optionally mono-, di- or tri-substituted and which amino group is optionally mono- or di-substituted, by one or more substituents selected from the group consisting of halogen, OH, SH, CN, NO 2 , C 1 - 6 alkoxy and C 1 - 6 alkoxycarbonyl.