Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.69 (botulinum neurotoxin)
 1,901 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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 [Pya88] S 39-88, had a fluorescent amino acid analog, L-pyrenylalanine (Pya), substituted for the normal Phe88 of synaptobrevin-2. Cleavage by BoNT light chain yielded fragments of 38 and 11 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 microM ICD 1578, cleavage of [Pya88] S 39-88 was progressively reduced (IC50 = 27.6 microM), and 100 microM 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.
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PMID:Efficacy of a novel metalloprotease inhibitor on botulinum neurotoxin B activity. 966 24

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.
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PMID:Evaluation of phosphoramidon and three synthetic phosphonates for inhibition of botulinum neurotoxin B catalytic activity. 1059 92

The binding activity of a rabbit polyclonal antiserum raised against a 51-residue peptide (P51) homologous to human VAMP2 (residues 44-94) was examined. Human VAMP2 is an 18-kDa protein located on the external membrane of small synaptic vesicles and is targeted by four of the seven botulinum neurotoxin (BoNT) serotypes (B, D, F and G). The antiserum, designated anti-P51, recognized P51 but exhibited little cross-reactivity with the two cleavage products that result from BoNT/B-mediated proteolysis of P51. The larger of these fragments, designated as P33 (residues 44-76), exhibited a weak but measurable interaction with the antiserum. The smaller cleavage product, designated as P18 (residues 77-94), was not recognized by the antiserum. Anti-P51 was used to monitor BoNT/B light chain (LC)-mediated cleavage of P51 using an indirect ELISA. The serine protease inhibitor phenylmethylsulfonyl fluoride did not inhibit BoNT/B activity, but the zinc chelator N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN) and the elastase inhibitor 7- N -phenylcarbamoylamino-4-chloro-3-propyloxyisocoumarin (ICD 1578) produced complete blockade of BoNT/B LC action. Under ideal conditions, it will be possible to evaluate up to seven candidate anti-BoNT/B drugs in triplicate at four concentrations using a single 96-well microtiter plate. These findings indicate that the ELISA will be suitable for rapid screening of BoNT/B inhibitors.
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PMID:Rapid microplate assay for monitoring botulinum neurotoxin B catalytic activity. 1059 93