Fret protease assays for clostridial toxins

Abstract

The present invention provides clostridial toxin substrates useful in assaying for the protease activity of any clostridial toxin, including botulinum toxins of all serotypes as well as tetanus toxins. A clostridial toxin substrate of the invention contains a donor fluorophore; an acceptor having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore; and a clostridial toxin recognition sequence that includes a cleavage site, where the cleavage site intervenes between the donor fluorophore and the acceptor and where, under the appropriate conditions, resonance energy transfer is exhibited between the donor fluorophore and the acceptor.

1 . A botulinum toxin substrate, comprising: 
 a) a donor fluorophore;    b) an acceptor having an absorbance spectrum overlapping the emission spectrum of said donor fluorophore, wherein the acceptor is an acceptor fluorophore; and    c) a BoNT/B recognition sequence comprising a BoNT/B P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence, said BoNT/B P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence intervening between said donor fluorophore and said acceptor;    wherein, under the appropriate conditions, resonance energy transfer is exhibited between said donor fluorophore and said acceptor fluorophore.    
     
     
         2 . A botulinum toxin substrate, comprising 
 (a) a donor fluorophore;    (b) an acceptor having an absorbance spectrum overlapping the emission spectrum of said donor fluorophore; and    (c) a botulinum toxin recognition sequence comprising a BoNT/B P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence including a BoNT/B P 1 -P 1 ′ cleavage site, said BoNT/B P 1 -P 1 ′ cleavage site intervening between said donor fluorophore and said acceptor;    wherein either said donor fluorophore or said acceptor is not positioned within said BoNT/B P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence; and    wherein, under the appropriate conditions, resonance energy transfer is exhibited between said donor fluorophore and said acceptor.    
     
     
         3 . The substrate of  claim 2 , wherein said donor fluorophore is not positioned within said BoNT/B P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence.  
     
     
         4 . The substrate of  claim 2 , wherein said acceptor is not positioned within said BoNT/B P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence.  
     
     
         5 . The substrate of either  claim 1  or  2 , wherein said BoNT/B P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence comprises at least six consecutive residues of VAMP, or a peptidomimetic thereof, said six consecutive residues comprising Gln-Phe, or a peptidomimetic thereof.  
     
     
         6 . The substrate of  claim 5 , wherein said BoNT/B P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence comprises at least six consecutive residues of human VAMP 2, or a peptidomimetic thereof, said six consecutive residues comprising Gln 76 -Phe 77 , or a peptidomimetic thereof.  
     
     
         7 . The substrate of  claim 5 , wherein said BoNT/B P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence comprises SEQ ID NO: 3, or a peptidomimetic thereof.  
     
     
         8 . The substrate of  claim 5 , wherein said BoNT/B recognition sequence comprises residues 55 to 94 of SEQ ID NO: 4, residues 60 to 94 of SEQ ID NO: 4, residues 60 to 88 of SEQ ID NO: 4, or a peptidomimetic thereof.  
     
     
         9 . The substrate of either  claim 1  or  2 , wherein said substrate comprises at most 20 residues, at most 40 residues, at most 50 residues, at most 100 residues, at most 150 residues, at most 200 residues, at most 300 residues or at most 400 residues.  
     
     
         10 . The substrate of either  claim 1  or  2 , wherein said donor fluorophore and said acceptor are separated by at most 6 residues, at most 8 residues, at most 10 residues, at most 20 residues, at most 30 residues, or at most 40 residues.  
     
     
         11 . The substrate of either  claim 1  or  2 , wherein said substrate can be cleaved with an activity of at least 1 nanomoles/minute/milligram toxin, at least 20 nanomoles/minute/milligram toxin, at least 50 nanomoles/minute/milligram toxin, at least 100 nanomoles/minute/milligram toxin, or at least 150 nanomoles/minute/milligram toxin.  
     
     
         12 . A method of determining BoNT/B protease activity, the method comprising the steps of: 
 a) treating a sample, under conditions suitable for BoNT/B protease activity, with a BoNT/B botulinum toxin substrate, said BoNT/B botulinum toxin substrate according to any one of claims  1 - 11 ;    b) exciting said donor fluorophore; and    c) determining resonance energy transfer of said treated substrate relative to a control substrate, 
 wherein a difference in resonance energy transfer of said treated substrate as compared to said control substrate is indicative of BoNT/B toxin protease activity.  
   
     
     
         13 . The method of  claim 12 , wherein said sample is a crude cell lysate.  
     
     
         14 . The method of  claim 12 , wherein said sample is isolated BoNT/B or isolated BoNT/B light chain.  
     
     
         15 . The method of  claim 12 , wherein said sample is a formulated BoNT/B product.  
     
     
         16 . The method of  claim 12 , wherein said acceptor is a fluorophore and step (c) comprises detecting donor fluorescence intensity of said treated substrate, wherein an increase in substrate cleavage results in an increase in donor fluorescence intensity of said treated substrate as compared to said control substrate, said increased donor fluorescence intensity being indicative of BoNT/B protease activity.  
     
     
         17 . The method of  claim 12 , wherein said acceptor is a fluorophore and step (c) comprises detecting acceptor fluorescence intensity of said treated substrate, wherein an increase in substrate cleavage results in a decrease in acceptor fluorescence intensity of said treated substrate as compared to said control substrate, said decreased acceptor fluorescence intensity being indicative of BoNT/B protease activity.  
     
     
         18 . The method of  claim 12 , wherein said acceptor is a fluorophore and step (c) comprises detecting an acceptor emission maximum and a donor fluorophore emission maximum, wherein an increase in substrate cleavage results in a shift in emission maxima from near said acceptor emission maximum to near said donor fluorophore emission maximum, said shift in emission maxima being indicative of BoNT/B protease activity.  
     
     
         19 . The method of  claim 12 , wherein said acceptor is a fluorophore and step (c) comprises detecting the ratio of fluorescence amplitudes near an acceptor emission maximum over the fluorescence amplitudes near a donor fluorophore emission maximum, wherein an increase in substrate cleavage results in a decreased ratio of said treated substrate as compared to said control substrate, said decreased ratio being indicative of BoNT/B protease activity.  
     
     
         20 . The method of  claim 12 , wherein said acceptor is a fluorophore and step (c) comprises detecting the ratio of fluorescence amplitudes near an donor emission maximum over the fluorescence amplitudes near a acceptor fluorophore emission maximum, wherein an increase in substrate cleavage results in an increased ratio in said treated substrate as compared to the control substrate, said increased ratio being indicative of BoNT/B protease activity.  
     
     
         21 . The method of  claim 12 , wherein said acceptor is a fluorophore and step (c) comprises detecting the excited state lifetime of the donor fluorophore of said treated substrate, wherein an increase in substrate cleavage results in an increase in donor fluorophore excited state lifetime of said treated substrate as compared to said control substrate, said increased excited state lifetime being indicative of BoNT/B protease activity.  
     
     
         22 . The method of  claim 12 , wherein said acceptor is a quencher and step (c) comprises detecting donor fluorescence intensity of said contacted cell, wherein an increase in substrate cleavage results in an increase in donor fluorescence intensity of said treated substrate as compared to said control substrate, said increased donor fluorescence intensity being indicative of BoNT/B protease activity.  
     
     
         23 . The method of  claim 12 , further comprising repeating step (c) at one or more later time intervals.  
     
     
         24 . The method of  claim 12 , wherein at least 90% of said BoNT/B substrate is cleaved.  
     
     
         25 . The method of  claim 12 , wherein at most 25% of said BoNT/B substrate is cleaved, at most 15% of said BoNT/B substrate is cleaved, or at most 5% of said BoNT/B substrate is cleaved.  
     
     
         26 . The method of  claim 12 , wherein the conditions suitable for Clostridial toxin protease activity are selected such that the assay is linear.