Use of a Recombinant Fluorescent Substrate with Cleavage Sites for All Botulinum Neurotoxins in High-Throughput Screening of Natural Product Extracts for Inhibitors of Serotypes A, B, and E7

The seven serotypes of botulinum neurotoxin (BoNTs) are zinc metalloproteases that cleave and inactivate proteins critical for neurotransmission. Synaptosomal protein of 25 kDA (SNAP-25) is cleaved by BoNTs A, C, and E, while vesicle-associated membrane protein (VAMP) is the substrate for BoNTs B, D. F. and G. BoNTs are not only medically useful drugs, but are also potential bioterrorist and biowarfare threat agents. Because BoNT protease activity is required for toxicity, inhibitors of that activity might be effective for anti-botulinum therapy. To expedite inhibitor discovery, we constructed a hybrid gene encoding (from N-terminus to C-terminus, with respect to the expressed product) green fluorescent protein, then a SNAP-25 fragment encompassing residues met-127 to gly-206, followed by VAMP residues met-1 to lys-94. Cysteine was added as the C-terminus. The expressed product, which contained the protease cleavage sites for all seven botulinum serotypes, was purified and coupled covalently through the C-terminal sulfhydryl group to maleimide-activated 96-well plates. The substrate was readily cleaved by BoNTs A, B, D, E, and F. Using this assay and an automated 96-well pipettor, we screened 528 natural product extractsfor inhibitors of BoNTs A, B, and E protease activities. Serotype-specific inhibition was found in 30 extracts, while five others inhibited two serotype. Pub. in Applied and Environmental Microbiology, v74 n3, p653-659, Feb 2008.