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Evaluation of phosphoramidon and three synthetic phosphonates for inhibition of botulinum neurotoxin B catalytic activity
Author(s) -
Adler Michael,
Nicholson James D.,
Starks David F.,
Kane Charles T.,
Cornille Fabrice,
Hackley, Jr Brennie E.
Publication year - 1999
Publication title -
journal of applied toxicology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.784
H-Index - 87
eISSN - 1099-1263
pISSN - 0260-437X
DOI - 10.1002/(sici)1099-1263(199912)19:1+<s5::aid-jat606>3.0.co;2-m
Subject(s) - phosphoramidon , dipeptide , chemistry , peptide , biochemistry , stereochemistry , protease , cleavage (geology) , pngase f , metalloproteinase , enzyme , biology , neprilysin , glycoprotein , paleontology , fracture (geology) , glycan
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.