title: High-throughput assays for botulinum neurotoxin proteolytic activity: serotypes A, B, D, and F.
abstract: Botulinum neurotoxins (BoNT) are zinc metalloproteases that cleave and inactivate cellular proteins essential for neurotransmitter release. Because the paralytic effect of BoNT is a consequence of its enzymatic activity, selective inhibitors may be useful as drugs or as tools for further research. To expedite inhibitor discovery, we developed high-throughput, solid-phase protease activity assays for four of the seven BoNT serotypes: A, B, D, and F. Each assay consisted of a cleavable oligopeptide, based on the natural substrate sequence, labeled with fluorescein and covalently attached to maleimide-activated multiwell plates. Solutions of holotoxin or nontoxic catalytic domain of BoNT were incubated in substrate-coated wells, with or without test compounds, followed by transfer and assay of solubilized product in a multiwell fluorometer. Routine toxin concentrations ranged from 10 to 100 ng/ml, but concentrations as low as 2 ng/ml gave reproducible signals. The fluorescence assays were selective, gave very low background readings, and were stable upon prolonged storage. Using the nontoxic catalytic domain of BoNT A, we determined the relative inhibitory potencies of a family of structurally related pseudotripeptide compounds. Unlike previous methods, our assays did not employ antibodies or reverse-phase extraction steps, only well-to-well transfers, and were easily adapted to a high-throughput automated environment.
authors.affiliation: Department of Cell Biology and Biochemistry, Toxinology and Aerobiology Division, U.S. Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702-5011, USA.
authors.forename: J J
authors.initials: JJ
authors.lastname: Schmidt
authors.forename: R G
authors.initials: RG
authors.lastname: Stafford
authors.forename: C B
authors.initials: CB
authors.lastname: Millard
mesh_terms: D001905:Botulinum Toxins / Q000032:analysis* / Q000037:antagonists & inhibitors; D019274:Botulinum Toxins, Type A; D004353:Drug Evaluation, Preclinical; D010447:Peptide Hydrolases / Q000032:analysis*; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
chemical_list: D010455:Peptides; D011480:Protease Inhibitors; C096323:rimabotulinumtoxinB; C096325:botulinum toxin type D; D010447:Peptide Hydrolases; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; C096321:botulinum toxin type F