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)

Zinc-dependent metalloprotease inhibitors phosphoramidon, captopril and a peptide hydroxamate were studied as potential pretreatment compounds by examining their ability to delay the onset or to prolong the time to 50% block of nerve-elicited muscle twitch tension in the mouse phrenic-nerve diaphragm (in vitro at 36 degrees C) after botulinum neurotoxin serotypes A and B (BoNT-A, BoNT-B). Addition of BoNT-A or BoNT-B (1 x 10(-10) M) produced 50% block of the twitch response at 56 +/- 9 min and 76 +/- 4 min, respectively. Preincubation (45 min) of muscles with phosphoramidon (0.2 mM) prolonged the time to 50% block by 15 min in BoNT-B-poisoned muscles with no effect on the time-course of paralysis in BoNT-A exposed muscles. When the same quantities of BoNT-A or BoNT-B (equivalent to 1 x 10(-10) M bath concentration) were preincubated for 2 hr with phosphoramidon (equivalent to 0.2 mM final bath concentration), and the incubation mixture was added to the muscle chamber, the times to 50% block were prolonged by 38 min and 18 min for BoNT-B and BoNT-A, respectively. Preincubation of diaphragms with captopril (up to 10 mM) or peptide hydroxamate (75 microM) failed to antagonize BoNT-A or BoNT-B-induced neuromuscular block. Among the three metalloprotease inhibitors examined here, only phosphoramidon showed a significant protection against both serotypes of BoNT. A search for better inhibitor compounds specifically tailored to match the active site on BoNT molecule deserves attention.
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PMID:A study of zinc-dependent metalloendopeptidase inhibitors as pharmacological antagonists in botulinum neurotoxin poisoning. 757 Jun 40

Tetanus toxin and botulinum neurotoxins are di-chain proteins of 150 kD molecular weight. They are produced by bacteria of the Clostridium genus. These toxins act on the nervous system by inhibiting neurotransmitter release (glycine and GABA in the case of tetanus toxin; acetylcholine in the case of botulinum neurotoxins) thus inducing the spastic or flaccid paralysis that characterizes tetanus and botulism, respectively. Their cellular mechanism of action involves three main steps, namely binding to the neurone membrane, internalization and intracellular blockade of the release mechanism for neurotransmitters. Membrane acceptors for these toxins are not yet fully identified; they would consist of membrane gangliosides and proteins. The internalization step would be achieved by endocytosis. Recent findings show that both binding and internalization are mediated only by the heavy chain of the toxins whereas the intracellular blockade of neurotransmitter release involves their light chain alone. The light chain has been identified as a zinc metalloprotease and its substrates would be proteins involved in the neurotransmitter release mechanism. The target of tetanus toxin and of botulinum neurotoxin type B is VAMP/synaptobrevin, a membrane protein of the synaptic vesicles of nerve cell terminals.
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PMID:[Molecular mechanism of action of tetanus toxin and botulinum neurotoxins]. 791 55

Type A botulinum neurotoxin catalyzed the hydrolysis of synthetic peptides based on the sequence of the 25-kD synaptosomal protein SNAP-25. In each peptide, the toxin cleaved at a single glutaminyl-arginine bond corresponding to residues 197 and 198 of SNAP-25, confirming earlier reports on the enzymatic specificity of the toxin in synaptosomal preparations. Metal chelators inhibited catalysis, consistent with a metalloprotease activity. In contrast to tetanus toxin and other botulinum toxin serotypes, type A toxin hydrolyzed relatively short, 17- to 20-residue peptides. In the substrates, SNAP-25 residue 202 and one or more of residues 187-191 were required for efficient hydrolysis, but residues 167-186 and 203-206 were not. The highest rates of hydrolysis were found when the C-terminal residues of the peptides were amidated.
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PMID:Proteolysis of synthetic peptides by type A botulinum neurotoxin. 874 31

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

Recombinant DNA techniques were used to develop an expression system for a 51-amino acid peptide fragment that encompasses residues 44-94 of human synaptobrevin 2. This protein is associated with secretory vesicles of nerve terminals and is a substrate for four of the seven serotypes of botulinum neurotoxin (BoNT). The DNA for the recombinant peptide was amplified by the polymerase chain reaction and cloned into the pTrxFus vector. The resulting synaptobrevin peptide was expressed as a thioredoxin fusion protein in E. coli and released into the medium by osmotic lysis. The 18.7-kDa thioredoxin-synaptobrevin protein, designated as TSB-51, is intended for use in a cell-free assay to test potential inhibitors of BoNT/B-mediated proteolysis of synaptobrevin with the ultimate aim of developing clinically effective therapeutic agents to counteract botulism. Incubation of TSB-51 with the purified light chain of BoNT/B resulted in proteolysis which was evident within 30 min and increased with time until completion (approximately 4 hr). Cleavage of TSB-51 appeared to be at the appropriate BoNT/B cleavage site as indicated by a reduced intensity of the 18.7-kDa band and the appearance of a band at 16.4 kDa on Tris-tricene polyacrylamide gradient gels. The concentration of free Zn2+ had a significant effect on the cleavage rate; low Zn2+ concentrations stimulated substrate cleavage, whereas high concentrations were inhibitory. Cleavage was not significantly depressed by the naturally occurring metalloprotease inhibitor phosphoramidon when tested at concentrations up to 5 mM. TSB-51 appears to be a useful substrate for studying BoNT/B and is expected to aid in the discovery of effective BoNT inhibitors.
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PMID:Production of an expression system for a synaptobrevin fragment to monitor cleavage by botulinum neurotoxin B. 971 40

Type A botulinum neurotoxin (botox A) is a zinc metalloprotease that cleaves only one peptide bond in the synaptosomal protein, SNAP-25. Single-residue changes in a 17-residue substrate peptide were used to develop the first specific, competitive inhibitors of its proteolytic activity. Substrate analog peptides with P4, P3, P2' or P3' cysteine were readily hydrolyzed by the toxin, but those with P1 or P2 cysteine were not cleaved and were inhibitors. Peptides with either D- or L-cysteine as the N-terminus, followed by the last six residues of the substrate, were the most effective inhibitors, each with a Ki value of 2 microM. Elimination of the cysteine sulfhydryl group yielded much less effective inhibitors, suggesting that inhibition was primarily due to binding of the active-site zinc by the sulfhydryl group. Botox A displayed an unusual requirement for arginine as the P1' inhibitor residue, demonstrating that the S1' binding subsite of botox A is dissimilar to those of most other zinc metalloproteases. This characteristic is an important element in shaping the substrate specificity of botox A.
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PMID:Type A botulinum neurotoxin proteolytic activity: development of competitive inhibitors and implications for substrate specificity at the S1' binding subsite. 975 59

Clostridium neurotoxins produce inhibition of both basal and K(+)-evoked serotonin release in rat brain synaptosomes. To produce these effects, tetanus toxin (TeTx), as well as botulinum neurotoxin type A (BoNT/A), added to brain synaptosomes, must be incubated at 37 degrees C over a long interval (hours). This serotonin exocytosis inhibition was abolished with previous treatment with specific Zn2(+)-metalloprotease inhibitors. Nevertheless, a short incubation time produces different behavior of the indicated neurotoxins: TeTx significantly blocks the sodium-dependent, high-affinity serotonin uptake, whereas a small increase of this uptake was found with BoNT/A. Both Zn2(+)-metalloprotease active fragments, light chains of TeTx and BoNT/A, are unable to reproduce the block of the serotonin uptake, whereas the C-terminal portion of the TeTx heavy chain (Hc-TeTx), which binds specifically to the target tissue, inhibited the serotonin uptake in a dose-dependent manner. The IC50 of Hc-TeTx ranges from 0.62 to 2.08 nM. Binding of [3H]imipramine and [3H]serotonin did not change after toxin treatments, which indicates that these clostridium neurotoxins do not act on the serotonin high-affinity site at the serotonin transporter or at other serotonin high-affinity sites. These results could indicate that TeTx and Hc-TeTx bind to different targets than BoNT/A in the plasma membrane.
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PMID:Clostridium neurotoxins influence serotonin uptake and release differently in rat brain synaptosomes. 1021 76

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

Clostridium botulinum neurotoxins are zinc endopeptidase proteins responsible for cleaving specific peptide bonds of proteins of neuroexocytosis apparatus. The ability of drugs to interfere with toxin's catalytic activity is being evaluated with zinc chelators and metalloprotease inhibitors. It is important to develop effective pharmacological treatment for the intact holotoxin before the catalytic domain separates and enters the cytosol. We present here evidence for a novel mechanism of an inhibitor binding to the holotoxin and for the chelation of zinc from our structural studies on Clostridium botulinum neurotoxin type B in complex with a potential metalloprotease inhibitor, bis(5-amidino-2-benzimidazolyl)methane, and provide snapshots of the reaction as it progresses. The binding and inhibition mechanism of this inhibitor to the neurotoxin seems to be unique for intact botulinum neurotoxins. The environment of the active site rearranges in the presence of the inhibitor, and the zinc ion is gradually removed from the active site and transported to a different site in the protein, probably causing loss of catalytic activity.
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PMID:A novel mechanism for Clostridium botulinum neurotoxin inhibition. 1214 45

Tetanus and botulinum neurotoxins (TeNT and BoNTs) block neuroexocytosis via specific cleavage and inactivation of SNARE proteins. Such activity is exerted by the N-terminal 50 kDa light chain (L) domain, which is a zinc-dependent endopeptidase. TeNT, BoNT/B, /D, /F and /G cleave vesicle associated membrane protein (VAMP), a protein of the neurotransmitter-containing small synaptic vesicles, at different single peptide bonds. Since the proteolytic activity of these metalloproteases is higher on native VAMP inserted in synaptic vesicles than on recombinant VAMP, we have investigated the influence of liposomes of different lipid composition on this activity. We found that the rate of VAMP cleavage with all neurotoxins tested here is strongly enhanced by negatively charged lipid mixtures. This effect is at least partially due to the binding of the metalloprotease to the lipid membranes, with electrostatic interactions playing an important role.
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PMID:VAMP/synaptobrevin cleavage by tetanus and botulinum neurotoxins is strongly enhanced by acidic liposomes. 1272 12


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