Efficacy of a novel metalloprotease inhibitor on botulinum neurotoxin B activity

The novel inhibitor 7-N-phenylcarbamoylamino-4-chloro-3-propyloxyisocoumarin (ICD 1578) was tested for its ability to antagonize the zinc metalloprotease activity of botulinum toxin B (BoNT/B). The efficacy of this compound was tested in a cell-free system using a 50-mer synaptobrevin peptide as substrate. The peptide, designated as [Pya(88)] S 39-88, had a fluorescent amino acid analog, L-pyrenylalanine (Pya), substituted for the normal Phe(88) of synaptobrevin-2. Cleavage by BoNT light chain yielded fragments of 38 and If amino acids, respectively. The smaller fragment, containing the Pya fluorophore, was readily separated and quantified by fluorescence spectroscopy at 377 nm, In the presence of 7-200 mu M ICD 1578, cleavage of [Pya(88)] S 39-88 was progressively reduced (IC50 = 27.6 mu M), and 100 mu M ICD 1578 produced > 95% inhibition. For comparison, captopril, a well-known zinc metalloprotease inhibitor, generated less than 10% inhibition at a concentration of 5 mM. ICD 1578 is the most potent antagonist of BoNT/B light chain thus far described. (C) 1998 Federation of European Biochemical Societies.