List Labs POSTER - Presented at the 47th Annual Interagency Botulinum Research Coordinating Committee Meeting, November 2010 in Atlanta, GA
Botulinum neurotoxins are among the most toxic substances known to man. Four of the seven immunologically distinct serotypes, A, B, E, and F, cause botulism in humans. A zinc endoprotease on the 50 kDa light chain of the 150 kDa holotoxin, cleaves a single target protein essential for synaptic vesicle membrane fusion. This inhibits neurotransmitter release and leads to muscular paralysis. Measurement of this proteolytic activity provides a potentially sensitive and direct means for detecting these potent toxins. Also, the neurotoxin light chains present an ideal target for the development of potential therapeutic inhibitors. The classic approach for monitoring enzymatic activity utilizes short peptides containing a chromophore or fluorophore moiety at the site of cleavage. However, for all the botulinum serotypes, efficient cleavage requires larger peptide substrates; hydrolysis occurs only for peptides that span both sides of the cleavage site. An alternative approach is to use fluorescence resonance energy transfer (FRET) peptides. These fluorogenic peptides contain a fluorescent group at one end and a suitable chromogenic acceptor group at the other, allowing for the inclusion of amino acids on either side of the enzymatically cleaved bond. Fluorescence is quenched initially by intramolecular energy transfer between the donor/acceptor pair. Upon cleavage, the fluorescence is recovered. A FRET peptide substrate for botulinum toxin type A (BoNT/A), SNAPtide, has been designed based on the native synaptosomal substrate SNAP-25. Crystal structures generated with the light chain of BoNT/A and SNAPtide show that part of the cleaved FRET substrate remains bound in the active site after hydrolysis. Based on these results, two potential peptide inhibitors have been synthesized. Data comparing the IC50 and Ki values for both inhibitors are presented. The peptide inhibitors can be used as positive controls in inhibitor screening assays and as lead compounds for potential BoNT/A inhibitors. The SNAPtide sequence was designed as an efficiently cleaved substrate for BoNT/A which contains a minimum of sites for non-specific hydrolysis. A control peptide which contains all potential non-specific cleavage sites but is not cleaved by BoNT/A has also been evaluated. Data comparing the cleavage of both the SNAPtide substrate and the control peptide demonstrate that only minimal cleavage of the control occurs after extended exposure to relatively high amounts of BoNT/A light chain. This control peptide can be used in complex matrices to indicate the presence of contamination by non-specific enzymes.