Methods for identifying inhibitors of botulinum neurotoxins

A method for identifying a botulinum neurotoxin inhibitor wherein said inhibitor is a molecule that inhibits a botulinum neurotoxin from proteolytically cleaving its endogenous substrate and said endogenous substrate is selected from the group consisting of SNAP-25, syntaxin, VAMP-1 and VAMP-2,

• wherein said method uses a botulinum neurotoxin substrate complex, said method comprising the steps of:
• (i) contacting said complex with a botulinum neurotoxin, in the presence and absence of a test molecule, wherein said complex comprises:
• (a) a peptide substrate selected from the group consisting of SNAP-25, a SNAP-25 isoform, syntaxin, a syntaxin isoform, VAMP, a VAMP isoform, and peptides having at least 80% identity to the foregoing, wherein said peptide substrate is capable of being cleaved at a specific cleavage site by a botulinum neurotoxin;
• (b) a reporter domain on one side of said peptide substrate;
• and (c) an immobilization domain on the opposite side of said peptide substrate,
• wherein said complex is cleaved to produce a cleaved complex in the absence of said test molecule;
• (ii) comparing the effect of the presence of said test molecule on the production of said cleaved complex to the production of said cleaved complex in the absence of said test molecule, and
• (iii) identifying the test molecule as a botulinum neurotoxin inhibitor if the presence of said test molecule decreases the relative amount of cleaved complex as compared to the amount of cleaved complex in the absence of said molecule.

A method for identifying a botulinum neurotoxin inhibitor wherein said inhibitor is a molecule that inhibits a botulinum neurotoxin from proteolytically cleaving its endogenous substrate, wherein said endogenous substrate is selected from the group consisting of SNAP-25, syntaxin, VAMP-1 and VAMP-2,

• wherein said method uses cells that express a botulinum neurotoxin substrate complex, said method comprising the steps of:
• (i) exposing botulinum neurotoxin substrate complex expressing cells to a botulinum neurotoxin, in the presence and absence of a test molecule,
• wherein said botulinum neurotoxin substrate complex comprises:
• (a) a peptide substrate selected from the group consisting of SNAP-25, a SNAP-25 isoform, syntaxin, a syntaxin isoform, VAMP, a VAMP isoform, and peptides having at least 80% identity to the foregoing, wherein said peptide substrate is capable of being cleaved at a specific cleavage site by a botulinum neurotoxin;
• (b) a reporter domain on one side of said peptide substrate;
• and (c) an immobilization domain on the opposite side of said peptide substrate,
• wherein said complex is cleaved to produce a cleaved complex in the absence of said test molecule;
• (ii) comparing the effect of the presence of said test molecule on the production of said cleaved complex to the production of said cleaved complex in the absence of said test molecule;
• and
• (iii) identifying the test molecule as a botulinum neurotoxin inhibitor if the presence of said test molecule decreases the relative amount of cleaved complex produced in said cells as compared to the amount of cleaved complex produced in said cells in the absence of said test molecule.

A method for detecting the presence of botulinum neurotoxin in a sample, wherein said method uses a botulinum neurotoxin substrate complex, said method comprising the steps of:

• (i) immobilizing a botulinum neurotoxin substrate complex a solid support
• wherein said complex comprises:
• (a) a peptide substrate selected from the group consisting of SNAP-25, a SNAP-25 isoform, syntaxin, a syntaxin isoform, VAMP, a VAMP isoform, and peptides having at least 80% identity to the foregoing, wherein said peptide substrate is capable of being cleaved at a specific cleavage site by a botulinum neurotoxin;
• (b) a reporter domain on one side of said peptide substrate;
• and (c) an immobilization domain on the opposite side of said peptide substrate
• and further wherein said complex is capable of being cleaved by a botulinum neurotoxin to produce a cleaved complex;
• (ii) contacting a sample with said immobilized complex;
• and
• (iii) comparing the amount of cleaved complex present in said sample to controls containing known amounts of a botulinum neurotoxin, wherein a decrease in the amount of complex to said solid support or an increase in reporter domain released from said complex detects the presence of botulinum neurotoxin in a sample.

A method for measuring concentration of neurotoxin in a sample, wherein said method uses a botulinum neurotoxin substrate complex, said method comprising the steps of:

• (i) contacting said complex with a sample, wherein said complex comprises:
• (a) a peptide substrate selected from the group consisting of SNAP-25, a SNAP-25 isoform, syntaxin, a syntaxin isoform, VAMP, a VAMP isoform, and peptides having at least 80% identity to the foregoing, wherein said peptide substrate is capable of being cleaved at a specific cleavage site by a botulinum neurotoxin;
• (b) a reporter domain on one side of said peptide substrate;
• and (c) an immobilization domain on the opposite side of said peptide substrate,
• wherein said complex is cleaved to produce a cleaved complex in the presence of a botulinum neurotoxin;
• (ii) comparing the effect of the presence of said sample on the production of said cleaved complex to the production of said cleaved complex in the presence of standard, quantities of a botulinum neurotoxin, and
• (iii) measuring the quantity of neurotoxin in said sample by correlating the amount of cleaved complex produced by said sample with the amount of cleaved complex produced by a standard quantity of a botulinum neurotoxin.