EFFICACY OF A NOVEL METALLOPROTEASE INHIBITOR ON BOTULINUM NEUROTOXIN-B ACTIVITY

The novel inhibitor enylcarbamoylamino-4-chloro-3-propyloxyisocoumarin (ICD 1578) was tested for its ability to antagonize the zinc metallop rotease activity of botulinum toxin B (BoNT/B). The efficacy of this c ompound was tested in a cell-free system using a 50-mer synaptobrevin peptide as substrate. The peptide, designated as [Pya(88)] S 39-88, ha d a fluorescent amino acid analog, L-pyrenylalanine (Pya), substituted for the normal Phe(88) of synaptobrevin-2. Cleavage by BoNT light cha in yielded fragments of 38 and If amino acids, respectively. The small er 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% inhib ition. For comparison, captopril, a well-known zinc metalloprotease in hibitor, generated less than 10% inhibition at a concentration of 5 mM . ICD 1578 is the most potent antagonist of BoNT/B light chain thus fa r described. (C) 1998 Federation of European Biochemical Societies.