Methods for identifying inhibitors of botulinum neurotoxins

A method for identifying a botulinum neurotoxin inhibitor 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 synaptosomal associated protein of 25 kD (SNAP-25), a SNAP-25 isoform, syntaxin, a syntaxin isoform, vesicle-associated membrane protein (VAMP), a VAMP isoform, and peptides having at least 80% identity to the foregoing, wherein said peptide substrate comprises a motif of soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptor (a SNARE motif);
• (b) a reporter domain comprising a fluorescent protein covalently attached to one side of said peptide substrate;
• and
• (c) an immobilization domain covalently attached to an opposite side of said peptide substrate,
• wherein said complex is cleaved to produce a cleaved complex in the absence of said test molecule;
• (ii) lysing said cells and collecting cell lysate from said cells;
• (iii)immobilizing the substrate complex on a solid support;
• (iii) comparing the effect of the presence of said test molecule on the amount of said cleaved complex to the amount of said cleaved complex in the absence of said test molecule;
• and
• (iv) 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;
• wherein said botulinum neurotoxin in step (i) is delivered to said cells by means selected from administering a botulinum neurotoxin to said cells and expressing said botulinum neurotoxin in said cells by a recombinant vector;
• and
• wherein said recombinant vector comprises a nucleic acid sequence that is from about 80% to 100% homologous to the nucleic acid sequences represented in the group consisting of:
• SEQ ID NO:
• 1, SEQ ID NO:
• 2, SEQ ID NO:
• 5, SEQ ID NO:
• 7, SEQ ID NO:
• 9, SEQ ID NO:
• 11 and SEQ ID NO:
• 13, wherein said sequence encodes a neurotoxin that is capable of cleaving the specific cleavage site of the endogenous substrate of said neurotoxin. A method for identifying a botulinum neurotoxin inhibitor 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 synaptosomal associated protein of 25 kD (SNAP-25), a SNAP-25 isoform, syntaxin, a syntaxin isoform, vesicle-associated membrane protein (VAMP), a VAMP isoform, and peptides having at least 80% identity to the foregoing, wherein said peptide substrate comprises a motif of soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptor (a SNARE motif);
• (b) a reporter domain comprising a fluorescent protein covalently attached to one side of said peptide substrate;
• and
• (c) an immobilization domain covalently attached to an opposite side of said peptide substrate,
• wherein said complex is cleaved to produce a cleaved complex in the absence of said test molecule;
• (ii) lysing said cells and collecting cell lysate from said cells;
• (iii)immobilizing the substrate complex on a solid support;
• (iii) comparing the effect of the presence of said test molecule on the amount of said cleaved complex to the amount of said cleaved complex in the absence of said test molecule;
• and
• (iv) 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;
• wherein said botulinum neurotoxin in step (i) is delivered to said cells by means selected from administering a botulinum neurotoxin to said cells and expressing said botulinum neurotoxin in said cells by a recombinant vector;
• and
• wherein said botulinum neurotoxin is from about 80% to 100% homologous to an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, and SEQ ID NO:14, wherein said neurotoxin is capable of cleaving a specific cleavage site of said neurotoxin's endogenous substrate.