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 E.
The seven serotypes of botulinum neurotoxin (BoNTs) are zinc metalloproteases that cleave and inactivate proteins critical for neurotransmission. The 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 not only are medically useful drugs but also are potential bioterrorist and biowarfare threat agents. Because BoNT protease activity is required for toxicity, inhibitors of that activity might be effective for antibotulinum therapy. To expedite inhibitor discovery, we constructed a hybrid gene encoding (from the N terminus to the C terminus, with respect to the expressed product) green fluorescent protein, then a SNAP-25 fragment encompassing residues Met-127 to Gly-206, and then 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 extracts for inhibitors of BoNT A, B, and E protease activities. Serotype-specific inhibition was found in 30 extracts, while 5 others inhibited two serotypes.
Hines
Harry B
HB
Department of Cell Biology and Biochemistry, Integrated Toxicology Division, USAMRIID, Frederick, MD 21702-5011, USA.
Kim
Alexander D
AD
Stafford
Robert G
RG
Badie
Shirin S
SS
Brueggeman
Ernst E
EE
Newman
David J
DJ
Schmidt
James J
JJ
D000992:Antitoxins / Q000737:chemistry / Q000378:metabolism; D001905:Botulinum Toxins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019274:Botulinum Toxins, Type A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D050825:Synaptosomal-Associated Protein 25 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050988:Vesicle-Associated Membrane Protein 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000992:Antitoxins; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D050825:Synaptosomal-Associated Protein 25; D050988:Vesicle-Associated Membrane Protein 2; C096323:rimabotulinumtoxinB; D049452:Green Fluorescent Proteins; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; C096326:botulinum toxin type E