A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic neurotransmitter release. Botulinum neurotoxin serotype A (BoNT/A) is a zinc metalloprotease that blocks exocytosis of neurotransmitter by cleaving the SNAP-25 component of the SNARE complex. Currently there are no licensed medicines to treat BoNT/A poisoning after internalization of the toxin by motor neurons. The development of effective therapeutic measures to counter BoNT/A intoxication has been limited, due in part to the lack of robust high-throughput assays for screening small molecule libraries. Here we describe a high content imaging (HCI) assay with utility for identification of BoNT/A inhibitors. Initial optimization efforts focused on improving the reproducibility of inter-plate results across multiple, independent experiments. Automation of immunostaining, image acquisition, and image analysis were found to increase assay consistency and minimize variability while enabling the multiparameter evaluation of experimental compounds in a murine motor neuron system.     

Novel small molecule inhibitors of botulinum neurotoxin A metalloprotease activity

Botulinum neurotoxins (BoNTs) are among the most lethal biological substances to have been weaponized and are listed as biodefense category A agents. Currently, no small molecule (non-peptidic) therapeutics exist to counter this threat; hence, identifying and developing compounds that inhibit BoNTs is a high priority. In the present study, a high-throughput assay was used to identify small molecules that inhibit the metalloprotease activity of BoNT serotype A light chain (BoNT/A LC). All inhibitors were further verified using a HPLC-based assay. Conformational analyses of these compounds, in conjunction with molecular docking studies, were used to predict structural features that contribute to inhibitor binding and potency. Based on these results, a common pharmacophore for BoNT/A LC inhibitors is proposed. This is the first study to report small molecules (non-peptidics) that inhibit BoNT/A LC metalloprotease activity in the low microM range.     

Development of a delivery vehicle for intracellular transport of botulinum neurotoxin antagonists

A targeted delivery vehicle (DV) was developed for intracellular transport of emerging botulinum neurotoxin (BoNT) antagonists. The DV consisted of the isolated heavy chain (HC) of BoNT/A coupled to a 10-kDa amino dextran via the heterobifunctional linker 3-(2-pyridylthio)-propionyl hydrazide. The HC served to target BoNT-sensitive cells and promote internalization of the complex, while the dextran served as a platform to deliver model therapeutic molecules to the targeted cells. To determine the ability of this chimeric glycoprotein to enter neurons, dextran and HC were labeled independently with the fluorescent dyes Oregon green 488 and Cy3, respectively. Internalization of DV was monitored in primary cortical cells using laser confocal microscopy. Incubation of cells for 24 h with DV resulted in discrete punctate labeling of both soma and processes. The Cy3 and Oregon green 488 signals were generally co-localized, suggesting that the complex remained in the same intracellular compartment during the initial 24 h. The DV-associated fluorescence was reduced progressively by co-application of increasing concentrations of unlabeled BoNT/A holotoxin. The results suggest that the BoNT/A HC is able to mediate internalization of a coupled dextran, even though the latter bears no resemblance to the BoNT/A light chain (LC). The HC of BoNT/A thus offers promise as a selective carrier to deliver BoNT antagonists to the nerve terminal cytoplasm for inhibiting the proteolytic activity of internalized BoNT/A LC.     

Heterologous combinations of heavy and light chains from botulinum neurotoxin A and tetanus toxin inhibit neurotransmitter release in Aplysia.

The neuroparalytic activities of botulinum neurotoxin type A (BoNT A), tetanus toxin (TeTx), or homologous and heterologous combinations of their constituent polypeptides were examined at cholinergic and non-cholinergic synapses of Aplysia californica. When applied extracellularly, BoNT A or a mixture of its heavy (HC) and light (LC) chains were far more potent in blocking transmitter release at cholinergic than non-cholinergic synapses. The reverse was true for TeTx or a mixture its constituent chains. Such selectivity was assigned to differences in neuronal targetting and uptake of the neurotoxins since both exhibited similar potencies when injected directly into the cell body of either cell type. When bath-applied, heterologous combinations of the toxins' HC and LC appeared as effective as the parent neurotoxins from whence each HC was derived. Moreover, targetting/internalization was attributable to the analogous N-terminal moieties, H2 and beta 2, of the HC from BoNT A and TeTx. Thus, it may be postulated that the latter regions possess two functional domains, one being distinct and responsible for the divergent neuronal specificity, whereas the other serves a common role in translocating the LC of either toxin. Also, it was shown that the C-terminal portion of the HC of TeTx is unable to play the intracellular role of its counterpart in BoNT A.     

Monoclonal Antibodies Targeting the Alpha-Exosite of Botulinum Neurotoxin Serotype/A Inhibit Catalytic Activity

The paralytic disease botulism is caused by botulinum neurotoxins (BoNT), multi-domain proteins containing a zinc endopeptidase that cleaves the cognate SNARE protein, thereby blocking acetylcholine neurotransmitter release. Antitoxins currently used to treat botulism neutralize circulating BoNT but cannot enter, bind to or neutralize BoNT that has already entered the neuron. The light chain endopeptidase domain (LC) of BoNT serotype A (BoNT/A) was targeted for generation of monoclonal antibodies (mAbs) that could reverse paralysis resulting from intoxication by BoNT/A. Single-chain variable fragment (scFv) libraries from immunized humans and mice were displayed on the surface of yeast, and 19 BoNT/A LC-specific mAbs were isolated by using fluorescence-activated cell sorting (FACS). Affinities of the mAbs for BoNT/A LC ranged from a K_D value of 9.0×10⁻¹¹ M to 3.53×10⁻⁸ M (mean K_D 5.38×10⁻⁹ M and median K_D 1.53×10⁻⁹ M), as determined by flow cytometry analysis. Eleven mAbs inhibited BoNT/A LC catalytic activity with IC₅₀ values ranging from 8.3 ~73×10⁻⁹ M. The fine epitopes of selected mAbs were also mapped by alanine-scanning mutagenesis, revealing that the inhibitory mAbs bound the α-exosite region remote from the BoNT/A LC catalytic center. The results provide mAbs that could prove useful for intracellular reversal of paralysis post-intoxication and further define epitopes that could be targeted by small molecule inhibitors.     

Multiple domains of botulinum neurotoxin contribute to its inhibition of transmitter release in Aplysia neurons

The binding, internalization, and inhibition of transmitter release by botulinum neurotoxin (BoNT) was investigated using the intact toxin, its heavy (HC) or light (LC) chains, and a proteolytic fragment thereof. In Aplysia neurons, blockade of acetylcholine release upon external application of BoNT types A or E was prevented by reducing the temperature to 10 degrees C, due to arresting intoxication at the membrane binding step. At this low temperature, type A HC, H2 (comprised of the N-terminal of HC), or H2L (H2 disulfide-linked to LC) antagonized the neuroparalytic action of BoNT A or E, indicating that the latter bind saturably to common ecto-acceptor via the H2 region. In contrast, H2L was unable to counteract BoNT-induced paralysis at the murine neuromuscular junction. In accordance with this species difference, unlike native BoNT, saturable binding of 125I-labeled H2L could not be detected in mammalian peripheral or central nerve terminals. Possibly, more stringent structural requirements form the basis of the toxin's greater effectiveness in inhibiting neurotransmission at mouse nerve muscle synapses than Aplysia nerve terminals. In further identification of functional domains in the toxin, an unprocessed single-chain form of BoNT type E was found to be ineffective when applied extra- or intracellularly to Aplysia neurons. Notably, bath application of the latter to a neuron preinjected with HC, but not H2L or LC, resulted in a blockade of release. This shows that the single-chain species can become internalized and requires, not only LC, but also processed HC for its inhibitory action; consistently, the proteolyzed form of BoNT E was active.     

Time-dependent botulinum neurotoxin serotype A metalloprotease inhibitors

Botulinum neurotoxins (BoNTs) are the most lethal of biological substances, and are categorized as class A biothreat agents by the Centers for Disease Control and Prevention. There are currently no drugs to treat the deadly flaccid paralysis resulting from BoNT intoxication. Among the seven BoNT serotypes, the development of therapeutics to counter BoNT/A is a priority (due to its long half-life in the neuronal cytosol and its ease of production). In this regard, the BoNT/A enzyme light chain (LC) component, a zinc metalloprotease responsible for the intracellular cleavage of synaptosomal-associated protein of 25 kDa, is a desirable target for developing post-BoNT/A intoxication rescue therapeutics. In an earlier study, we reported the high throughput screening of a library containing 70,000 compounds, and uncovered a novel class of benzimidazole acrylonitrile-based BoNT/A LC inhibitors. Herein, we present both structure–activity relationships and a proposed mechanism of action for this novel inhibitor chemotype.     

Glycine insertion at protease cleavage site of SNAP25 resists cleavage but enhances affinity for botulinum neurotoxin serotype A

The light chain of botulinum neurotoxin A (BoNT/A‐LC) is a Zn‐dependent protease that specifically cleaves SNAP25 of the SNARE complex, thereby impairing vesicle fusion and neurotransmitter release at neuromuscular junctions. The C‐terminus of SNAP25 (residues 141–206) retains full activity for BoNT/A‐LC‐catalyzed cleavage at P1‐P1' (Gln197‐Arg198). Using the structure of a complex between the C‐terminus of SNAP25 and BoNT/A‐LC as a model to design SNAP25‐derived pseudosubstrate inhibitors (SNAPIs) that prevent presentation of the scissile bond to the active site, we introduced multiple His residues to replace Ala‐Asn‐Gln‐Arg (residues 195–198) at the substrate cleavage site, with the intent to identify possible side‐chain interactions with the active site Zn. We also introduced multiple Gly residues between the P1‐P1' residues to explore the spatial tolerance within the active‐site cleft. Using a FRET substrate YsCsY, we compared a series of SNAPIs for inhibition of BoNT/A‐LC. Among the SNAPIs tested, several known cleavage‐resistant, single‐point mutants of SNAP25 were poor inhibitors, with most of the mutants losing binding affinity. Replacement with His at the active site did not improve inhibition over wildtype substrate. In contrast, Gly‐insertion mutants were not only resistant to cleavage, but also surprisingly showed enhanced affinity for BoNT/A‐LC. Two of the Gly‐insertion mutants exhibited 10‐fold lower IC₅₀ values than the wildtype 66‐mer SNAP25 peptide. Our findings illustrate a scenario, where the induced fit between enzyme and bound pseudosubstrate fails to produce the strain and distortion required for catalysis to proceed.     

Conformational sampling of the botulinum neurotoxin serotype A light chain: implications for inhibitor binding

Botulinum neurotoxins (BoNTs) are the most potent of the known biological toxins, and consequently are listed as category A biowarfare agents. Currently, the only treatments against BoNTs include preventative antitoxins and long-term supportive care. Consequently, there is an urgent need for therapeutics to counter these enzymes--post exposure. In a previous study, we identified a number of small, nonpeptidic lead inhibitors of BoNT serotype A light chain (BoNT/A LC) metalloprotease activity, and we identified a common pharmacophore for these molecules. In this study, we have focused on how the dynamic movement of amino acid residues in and surrounding the substrate binding cleft of the BoNT/A LC might affect inhibitor binding modes. The X-ray crystal structures of two BoNT/A LCs (PDB refcodes=3BTA and 1E1H) were examined. Results from these analyses indicate that the core structural features of the examined BoNT/A LCs, including alpha-helices and beta-sheets, remained relatively unchanged during 1 ns dynamics trajectories. However, conformational flexibility was observed in surface loops bordering the substrate binding clefts in both examined structures. Our analyses indicate that these loops may possess the ability to decrease the solvent accessibility of the substrate binding cleft, while at the same time creating new residue contacts for the inhibitors. Loop movements and conformational/positional analyses of residues within the substrate binding cleft are discussed with respect to BoNT/A LC inhibitor binding and our common pharmacophore for inhibition. The results from these studies may aid in the future identification/development of more potent small molecule inhibitors that take advantage of new binding contacts in the BoNT/A LC.     

Holotoxin Activity of Botulinum Neurotoxin Subtype A4 Originating from a Nontoxigenic Clostridium botulinum Expression System

Clostridium botulinum subtype A4 neurotoxin (BoNT/A4) is naturally expressed in the dual-toxin-producing C. botulinum strain 657Ba at 100× lower titers than BoNT/B. In this study, we describe purification of recombinant BoNT/A4 (rBoNT/A4) expressed in a nonsporulating and nontoxigenic C. botulinum expression host strain. The rBoNT/A4 copurified with nontoxic toxin complex components provided in trans by the expression host and was proteolytically cleaved to the active dichain form. Activity of the recombinant BoNT/A4 in mice and in human neuronal cells was about 1,000-fold lower than that of BoNT/A1, and the recombinant BoNT/A4 was effectively neutralized by botulism heptavalent antitoxin. A previous report using recombinant truncated BoNT/A4 light chain (LC) expressed in Escherichia coli has indicated reduced stability and activity of BoNT/A4 LC compared to BoNT/A1 LC, which was surmounted by introduction of a single-amino-acid substitution, I264R. In order to determine whether this mutation would also affect the holotoxin activity of BoNT/A4, a recombinant full-length BoNT/A4 carrying this mutation as well as a second mutation predicted to increase solubility (L260F) was produced in the clostridial expression system. Comparative analyses of the in vitro, cellular, and in vivo activities of rBoNT/A4 and rBoNT/A4-L260F I264R showed 1,000-fold-lower activity than BoNT/A1 in both the mutated and nonmutated BoNT/A4. This indicates that these mutations do not alter the activity of BoNT/A4 holotoxin. In summary, a recombinant BoNT from a dual-toxin-producing strain was expressed and purified in an endogenous clostridial expression system, allowing analysis of this toxin.     

Iterative Structure-Based Peptide-Like Inhibitor Design against the Botulinum Neurotoxin Serotype A

The botulinum neurotoxin serotype A light chain (BoNT/A LC) protease is the catalytic component responsible for the neuroparalysis that is characteristic of the disease state botulism. Three related peptide-like molecules (PLMs) were designed using previous information from co-crystal structures, synthesized, and assayed for in vitro inhibition against BoNT/A LC. Our results indicate these PLMS are competitive inhibitors of the BoNT/A LC protease and their K_i values are in the nM-range. A co-crystal structure for one of these inhibitors was determined and reveals that the PLM, in accord with the goals of our design strategy, simultaneously involves both ionic interactions via its P1 residue and hydrophobic contacts by means of an aromatic group in the P2′ position. The PLM adopts a helical conformation similar to previously determined co-crystal structures of PLMs, although there are also major differences to these other structures such as contacts with specific BoNT/A LC residues. Our structure further demonstrates the remarkable plasticity of the substrate binding cleft of the BoNT/A LC protease and provides a paradigm for iterative structure-based design and development of BoNT/A LC inhibitors.     

Interaction of 125I-labeled botulinum neurotoxins with nerve terminals. II. Autoradiographic evidence for its uptake into motor nerves by acceptor-mediated endocytosis

Using pharmacological (Simpson, L.L., 1980, J. Pharmacol. Exp. Ther. 212:16-21) and autoradiographic techniques (Black, J.D., and J.O. Dolly, 1986, J. Cell Biol., 103:521-534), it has been shown that botulinum neurotoxin (BoNT) is translocated across the motor nerve terminal membrane to reach a postulated intraterminal target. In the present study, the nature of this uptake process was investigated using electron microscopic autoradiography. It was found that internalization is acceptor-mediated and that binding to specific cell surface acceptors involves the heavier chain of the toxin. In addition, uptake was shown to be energy and temperature-dependent and to be accelerated by nerve stimulation, a treatment which also shortens the time course of the toxin-induced neuroparalysis. These results, together with the observation that silver grains were often associated with endocytic structures within the nerve terminal, suggested that acceptor-mediated endocytosis is responsible for toxin uptake. This proposal is supported further by the fact that lysosomotropic agents, which are known to interfere with the endocytic pathway, retard the onset of BoNT-induced neuroparalysis and also affect the distribution of silver grains at nerve terminals treated with 125I-BoNT. Possible recycling of BoNT acceptors (an important aspect of acceptor-mediated endocytosis of toxins) at motor nerve terminals was indicated by comparing the extent of labeling in the presence and absence of metabolic inhibitors. On the basis of these collective results, it is concluded that BoNT is internalized by acceptor-mediated endocytosis and, hence, the data support the proposal that this toxin inhibits release of acetylcholine by interaction with an intracellular target.     

Spectroscopic analysis of pH-induced changes in the molecular features of type A botulinum neurotoxin light chain

Clostridial botulinum neurotoxins (BoNTs) cause neuroparalysis by blocking neurotransmitter release at the neuromuscular junctions. While the toxin's heavy chain (HC) is involved in binding and internalization, the light chain (LC) acts as a unique Zn(2+)-endopeptidase against a target protein in the exocytotic docking/fusion machinery. During the translocation of the LC to the cytosol, it is exposed to the endosomal low pH. Low pH showed a dramatic change in the BoNT/A LC polypeptide folding as indicated by differential heat denaturation. Furthermore, binding of 1-anilinonaphthalenesulfonate (ANS) revealed exposure of hydrophobic domains of BoNT/A LC at low pH. Low-pH-induced structural (and by implication the endopeptidase activity) changes were completely reversible. Exposure of BoNT/A LC to low pH (4.7) did not, however, evoke the loss of Zn(2+) bound to its active site. Implications of these observations to the delivery of active BoNT/A LC to the nerve cell are discussed. We further analyzed the nature of low-pH-induced change in the polypeptide folding of BoNT/A LC by Trp fluorescence measurements. The Trp fluorescence peak was observed at 322 nm, and the two fluorescence lifetime components estimated at 2.1 ns (88%) and 0.6 ns (12%) did not change much at low pH. These observations suggested that the two Trp residues are buried and constrained in a hydrophobic environment, and it is likely that the core of the BoNT/A LC protein matrix does not participate in the low-pH-induced structural alteration. This conclusion was further supported by the near-UV circular dichroism spectra under two pH conditions.     

Inhibition of neurotransmitter release by botulinum neurotoxins and tetanus toxin at Aplysia synapses: role of the constituent chains

1. The effects on the release of transmitter by botulinum neurotoxins (BoNT; types A, B, E), tetanus toxin (TeTx), constituent chains or fragments were studied on identified cholinergic and non-cholinergic synapses in Aplysia. 2. Cholinergic synapses in the buccal ganglion were found to be greater than 100 fold more sensitive to extracellular application of BoNT than to TeTx whereas in non-cholinergic synapses of the cerebral ganglion the potencies of the toxins were reversed. When intracellularly applied TeTx and BoNT were found nearly equipotent. This disparity in the susceptibilities of BoNT and TeTx to inhibit transmission was attributed to differences in the toxin's acceptors or uptake systems in the two neurone types. 3. Micro-injection into cholinergic neurones of the isolated renatured toxins' chains showed that both light and heavy chains of BoNT are intracellularly required whereas the light chain of TeTx alone is sufficient. 4. The heavy chain of BoNT as well as that of TeTx were found to mediate internalization of active moieties via its amino-terminal half. Furthermore the heavy chain of one toxin could internalize the light chain of the other.     

Neuronal targeting, internalization, and biological activity of a recombinant atoxic derivative of botulinum neurotoxin A

Non-toxic derivatives of botulinum neurotoxin A (BoNT/A) have potential use as neuron-targeting delivery vehicles, and as reagents to study intracellular trafficking. We have designed and expressed an atoxic derivative of BoNT/A (BoNT/A ad) as a full-length 150 kDa molecule consisting of a 50 kDa light chain (LC) and a 100 kDa heavy chain (HC) joined by a disulfide bond and rendered atoxic through the introduction of metalloprotease-inactivating point mutations in the light chain. Studies in neuronal cultures demonstrated that BoNT/A ad cannot cleave synaptosomal-associated protein 25 (SNAP25), the substrate of wt BoNT/A, and that it effectively competes with wt BoNT/A for binding to endogenous neuronal receptors. In vitro and in vivo studies indicate accumulation of BoNT/A ad at the neuromuscular junction of the mouse diaphragm. Immunoprecipitation studies indicate that the LC of BoNT/A ad forms a complex with SNAP25 present in the neuronal cytosolic fraction, demonstrating that the atoxic LC retains the SNAP25 binding capability of the wt toxin. Toxicity of BoNT/A ad was found to be reduced approximately 100,000-fold relative to wt BoNT/A.     

Endopeptidase Activities of Botulinum Neurotoxin Type B Complex,Holotoxin, and Light Chain

Botulinum neurotoxin (BoNT) serotype B (BoNT/B) is one of the serotypes of BoNT that causes deadly human botulism, though it is used clinically for treatment of many neuromuscular diseases. BoNT/B is produced by Clostridium botulinum, and it is secreted along with a group of neurotoxin-associated proteins (NAPs) in the form of a BoNT/B complex. The complex dissociates into a 150-kDa holotoxin and NAPs at alkaline pHs. The 150-kDa BoNT/B holotoxin can be nicked to produce a 50-kDa domain referred to as the light chain (LC) and a 100-kDa heavy chain, with the former possessing a unique endopeptidase activity. The two chains remain linked through a disulfide bond that can be reduced to separate the two chains. The endopeptidase activity is present in all three forms of the toxin (complex, purified BoNT/B holotoxin, and separated light chain), which are used by different researchers to develop detection methods and screen for inhibitors. In this research, the endopeptidase activities of the three forms, for the first time, were compared under the same conditions. The results show that enzyme activities of the three forms differ significantly and are largely dependent on nicking and disulfide reduction conditions. Under the conditions used, LC had the highest level of activity, and the complex had the lowest. The activity was enhanced by nicking of BoNT/B holotoxin and was enhanced even more by dithiothreitol (DTT) reduction after nicking. This information is useful for understanding the properties of BoNT endopeptidases and for comparing the efficacies of different inhibitors when they are tested with different forms of BoNT endopeptidase.Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the most toxic substances known to humans and block the release of neurotransmitters, resulting in flaccid muscle paralysis. There are seven serotypes of BoNT, designated A to G, which are serologically distinct. An antitoxin against one serotype does not work on other serotypes. Different BoNT serotypes differ in their amino acid sequences, their substrates, or cleavage sites on the same substrate. Of the seven serotypes, BoNT type A (BoNT/A), BoNT/B, BoNT/E, and BoNT/F are known to cause human botulism (9). The extreme lethality of BoNTs makes them potent bioterror agents. BoNT/A and BoNT/B are two serotypes which have been approved by the Food and Drug Administration (FDA) for cosmetic purposes and for treatment of a wide range of neuromuscular diseases, including cervical dystonia (3).Like other BoNT serotypes, BoNT/B is secreted by the bacteria as a complex of the holotoxin and several nontoxic proteins called neurotoxin-associated proteins (NAPs). The NAPs protect the holotoxin from harsh environmental conditions, such as the high temperature, low pH, and multiple proteases present in the gastrointestinal tract (14, 17). The holotoxin, of about 150 kDa, can be obtained by removing the non-covalently bound accessory proteins with ion-exchange chromatography. The 150-kDa polypeptide chain consists of a 100-kDa heavy chain (HC) and a 50-kDa light chain (LC), which are synthesized as a single polypeptide chain but nicked by endogenous or exogenous proteases and remain linked through a disulfide bond (Fig. ​(Fig.1).1). The HC binds the receptors on neuronal cells and helps translocate the LC into the cell. The BoNT/B LC cleaves the vesicle-associated membrane protein (VAMP), also called synaptobrevin. VAMP is necessary for the docking and fusion of synaptic vesicles to plasma membrane at the neuromuscular junctions for neurotransmitter release. Once the VAMP is cleaved, the neurotransmitters in synaptic vesicles cannot be released, resulting in flaccid paralysis that can be fatal.Open in a separate windowFIG. 1.Schematic diagram of BoNT/B pure toxin. Dark gray, light chain; light gray, heavy chain; hatch-marked box, the active site of the toxin. The 50-kDa light chain and 100-kDa heavy chain are linked through a disulfide bridge as well as a covalent bond. The latter is partially nicked by bacterial proteases before the toxin is secreted.Strains producing BoNT/B can be nonproteolytic or proteolytic (4). BoNT/B from nonproteolytic strains occurs as a single polypeptide chain of 150 kDa. BoNT/B secreted by proteolytic strains is a mixture of the single polypeptide chain and a dichain in which the peptide bond linking the HC and LC has been nicked by proteases produced by the bacteria (Fig. ​(Fig.1).1). The single polypeptide chain in both nonproteolytic and proteolytic cultures can be converted to the dichain form through in vitro trypsinization. The HC and LC in the dichain can be further separated by breaking the disulfide bond with a reducing agent such as dithiothreitol (DTT) and treating it with chaotropic reagents such as urea (10).The complex, holotoxin, and LC are three different forms of BoNT/B with endopeptidase activity, although LC is the only active unit in all three forms. The complex is the native form of the toxin, which causes botulism. It is also the main component of the only licensed drug with BoNT/B currently available (2). The complex, holotoxin, and LC of BoNT/B have all been extensively used to develop methods to detect this serotype or to screen for inhibitors against the toxin (1, 5, 7, 8, 13, 15, 16). Since different forms of the toxin were used by different researchers, it is difficult to compare the sensitivities of different detection methods or the efficacies of different inhibitors. Therefore, in this study, the activities of BoNT/B complex, holotoxin, and LC were compared under the same conditions for the first time. The results suggest that the endopeptidase activity with a peptide substrate varies substantially depending on whether BoNT/B is used in its native complex form, its isolated holotoxin form, or a separated LC form. The LC form was the most active form of the endopeptidase under the conditions used.     

Screening for inhibitors of the hepatitis C virus internal ribosome entry site RNA

The highly conserved internal ribosome entry site (IRES) of hepatitis C virus (HCV) regulates translation of the viral RNA genome and is essential for the expression of HCV proteins in infected host cells. The structured subdomain IIa of the IRES element is the target site of recently discovered benzimidazole inhibitors that selectively block viral translation through capture of an extended conformation of an RNA internal loop. Here, we describe the development of a FRET-based screening assay for similarly acting HCV translation inhibitors. The assay relies on monitoring fluorescence changes that indicate rearrangement of the RNA target conformation upon ligand binding. Screening of a small pilot set of potential RNA binders identified a benzoxazole scaffold as a ligand that bound selectively to IIa IRES target and was confirmed as an inhibitor of in vitro viral translation. The screening approach outlined here provides an efficient method to discover HCV translation inhibitors that may provide leads for the development of novel antiviral therapies directed at the highly conserved IRES RNA.     

Differential roles of developmentally distinct SNAP-25 isoforms in the neurotransmitter release process

The role of SNAP-25 (synaptosomal associated protein of 25 kDa) isotypes in the neurotransmitter release process was examined by varying their relative abundance during PC12 cell differentiation induced by nerve growth factor (NGF). Norepinephrine release by NGF-differentiated PC12 cells is more sensitive to type A botulinum toxin (BoNT/A) than by nondifferentiated cells, while both differentiated and nondifferentiated PC12 cells are equally sensitive to type E botulinum toxin (BoNT/E). The differential sensitivity to BoNT/A corresponds to an altered susceptibility of SNAP-25 isotypes to BoNT/A cleavage in vitro, whereas both isotypes are equally vulnerable to cleavage by BoNT/E. Using recombinant SNAP-25 preparations, we show that BoNT/A cleaves SNAP-25b (present in differentiated cells) 2-fold more readily than SNAP-25a (present in both differentiated and nondifferentiated cells). Structural studies using far-ultraviolet circular dichroism (UV--CD) and thermal denaturation suggest a difference in the polypeptide folding as the underlying molecular basis for the differential sensitivity of SNAP-25b and SNAP-25a to BoNT/A cleavage. We propose differential roles for SNAP-25b and SNAP-25a in the neurotransmitter release process since our results suggest that BoNT/A inhibits neurotransmitter release by primarily cleaving SNAP-25b.     

TCEP treatment reduces proteolytic activity of BoNT/B in human neuronal SHSY‐5Y cells

The light chain (LC) of botulinum neurotoxin B (BoNT/B) is unable to enter target neuronal cells by itself. It is brought into the cell in association with the BoNT/B heavy chain (HC) through endocytosis. The BoNT HC‐LC subunits are held together by a single disulfide bond. Intracellular reduction of this bond and separation of the two subunits activates the endopeptidase activity of the LC. This requirement suggests a strategy to prevent uptake by prophylactic reduction to disrupt the disulfide bond prior to endocytosis of the complex. We examined the utility of tris‐(2‐carboxyethyl)‐phosphine hydrochloride (TCEP), a relatively non‐toxic, non‐sulfur containing disulfide bond reducing agent that lacks the undesirable properties of mercapto‐containing reducing agents. We found that TCEP was as effective as DTT with maximal LC endopeptidase activation occurring at 1 mM, a concentration not toxic to the human neuronal cell line, SHSY‐5Y. In these cells, 1 mM TCEP maximally protected against BoNT/B inhibition of [³H]‐NA release, achieving 72% of the release from un‐intoxicated controls. This effect appears to be due to the sparing of SNARE proteins as the levels of VAMP‐2, the specific target of BoNT/B, were protected. These results show that TCEP disrupts the structure of BoNT/B by reduction of the LC and HC bridging disulfide bond and prevents neuronal intoxication. Since disulfide bond coupling between toxin subunits is a general motif for many toxins, e.g., ricin, snake venom, and all BoNT serotypes, this suggests that TCEP is a promising means to protect against these toxins by preventing cell penetration. J. Cell. Biochem. 107: 1021–1030, 2009. Published 2009 Wiley‐Liss, Inc.     

Light chain of botulinum neurotoxin serotype A: structural resolution of a catalytic intermediate

Botulinum neurotoxin serotype A (BoNT/A, 1296 residues) is a zinc metalloprotease that cleaves SNAP25 to inhibit the fusion of neurotransmitter-carrying vesicles to the plasma membrane of peripheral neurons. BoNT/A is a disulfide-linked di-chain protein composed of an N-terminal, thermolysin-like metalloprotease light chain domain (LC/A, 448 residues) and a C-terminal heavy chain domain (848 residues) that can be divided into two subdomains, a translocation subdomain and a receptor binding subdomain. LC/A cleaves SNAP25 between residues Gln197-Arg198 and, unlike thermolysin, recognizes an extended region of SNAP25 for cleavage. The structure of a recombinant LC/A (1-425) treated with EDTA (No-Zn LC/A) was determined. The overall structure of No-Zn LC/A is similar to that reported for the holotoxin, except that it lacks the Zn ion, indicating that the role of Zn is catalytic not structural. In addition, structures of a noncatalytic mutant LC/A (Arg362Ala/Tyr365Phe) complexed with and without an inhibitor, ArgHX, were determined. The overall structure and the active site conformation for the mutant are the same as wild type. When the inhibitor binds to the active site, the carbonyl and N-hydroxyl groups form a bidentate ligand to the Zn ion and the arginine moiety binds to Asp369, suggesting that the inhibitor-bound structure mimics a catalytic intermediate with the Arg moiety binding at the P1' site. Consistent with this model, mutation of Asp369 to Ala decreases the catalytic activity of LC/A by approximately 600-fold, and the residual activity is not inhibited by ArgHX. These results provide new information on the reaction mechanism and insight into the development of strategies for small molecule inhibitors of BoNTs.