title: Evaluation of phosphoramidon and three synthetic phosphonates for inhibition of botulinum neurotoxin B catalytic activity.
abstract: Three putative metalloprotease inhibitors were synthesized and tested for their ability to inhibit the catalytic activity of botulinum neurotoxin B light chain (BoNT/B LC). The compounds were designed to emulate the naturally occurring metalloprotease inhibitor phosphoramidon, which has been reported to be a weak antagonist of BoNT/B action. All three analogs contained the dipeptide Phe-Glu in place of Leu-Trp of phosphoramidon and possessed a phenyl, ethyl or methyl group in place of the rhamnose sugar of the parent compound. The inhibitors were evaluated in a cell-free assay based on the detection of a fluorescent product following cleavage of a 50-mer synaptobrevin peptide ([Pya(88)] S 39-88) by BoNT/B LC. This peptide corresponds to the hydrophilic core of synaptobrevin-2 and contains a fluorescent analog L-pyrenylalanine (Pya) in place of Tyr(88). Cleavage of [Pya(88)] S 39-88 by BoNT/B LC gives rise to fragments of 38 and 12 amino acid residues. Quantification of BoNT/B-mediated substrate cleavage was achieved by separating the 12-mer fragment (FETSAAKLKRK-Pya) that contains the C-terminal fluorophore and measuring fluorescence at 377 nm. The results indicate that the phenyl-substituted synthetic compound ICD 2821 was slightly more active than phosphoramidon, but analogs with methyl or ethyl substitutions were relatively inactive. These findings suggest that phosphonate monoesters may be useful for providing insights into the structural requirement of BoNT/B protease inhibitors.
authors.affiliation: Neurotoxicology Branch, Pharmacology Division, US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground MD, 21010, USA.
authors.forename: M
authors.initials: M
authors.lastname: Adler
authors.forename: J D
authors.initials: JD
authors.lastname: Nicholson
authors.forename: D F
authors.initials: DF
authors.lastname: Starks
authors.forename: C T
authors.initials: CT
authors.lastname: Kane
authors.forename: F
authors.initials: F
authors.lastname: Cornille
authors.forename: B E
authors.initials: BE
authors.lastname: Hackley
mesh_terms: D001905:Botulinum Toxins / Q000037:antagonists & inhibitors*; D019274:Botulinum Toxins, Type A; D002384:Catalysis; D006020:Glycopeptides / Q000494:pharmacology*; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D063065:Organophosphonates / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D019287:Zinc Sulfate / Q000494:pharmacology
chemical_list: D006020:Glycopeptides; D063065:Organophosphonates; D011480:Protease Inhibitors; C096323:rimabotulinumtoxinB; D019287:Zinc Sulfate; D008666:Metalloendopeptidases; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; C008890:phosphoramidon