Probing the structure and function of the protease domain of botulinum neurotoxins using single-domain antibodies.

Botulinum neurotoxins (BoNTs) are among the deadliest of bacterial toxins. BoNT serotype A and B in particular pose the most serious threat to humans because of their high potency and persistence. To date, there is no effective treatment for late post-exposure therapy of botulism patients. Here, we aim to develop single-domain variable heavy-chain (VHH) antibodies targeting the protease domains (also known as the light chain, LC) of BoNT/A and BoNT/B as antidotes for post-intoxication treatments. Using a combination of X-ray crystallography and biochemical assays, we investigated the structures and inhibition mechanisms of a dozen unique VHHs that recognize four and three non-overlapping epitopes on the LC of BoNT/A and BoNT/B, respectively. We show that the VHHs that inhibit the LC activity occupy the extended substrate-recognition exosites or the cleavage pocket of LC/A or LC/B and thus block substrate binding. Notably, we identified several VHHs that recognize highly conserved epitopes across BoNT/A or BoNT/B subtypes, suggesting that these VHHs exhibit broad subtype efficacy. Further, we identify two novel conformations of the full-length LC/A, that could aid future development of inhibitors against BoNT/A. Our studies lay the foundation for structure-based engineering of protein- or peptide-based BoNT inhibitors with enhanced potencies and cross-subtypes properties.

Safety verification for polysorbate 20, pharmaceutical excipient for intramuscular administration, in Sprague-Dawley rats and New Zealand White rabbits.

Human serum albumin (HSA) has been widely used as a pharmaceutical excipient in Botulinum toxin serotype A (BoNT/A) products that are indicated for use in therapeutics and cosmetics. However, HSA as a human-derived material has some concerns, such as the potential risk of transmission of infectious agents, an insufficient supply, and difficulty in maintaining a certain quality. For those reasons, newly developed BoNT/A products (CORETOX®, Medytox, Inc., Republic of Korea) contained polysorbate 20, a non-human-derived excipient, to replace the HSA. However, most safety studies of polysorbate 20 have been conducted with non-invasive routes of administration, and thus there are a few studies on the safety of polysorbate 20 when administered intramuscularly. To secure the in vivo safety profile of polysorbate 20, a four-week repeated intramuscular dose toxicity study (0.02, 0.1, and 0.4 mg/kg, one injection every two weeks for a total of three injections) was conducted in 66 Sprague-Dawley (SD) rats. An intradermal irritation study was further conducted with 18 New Zealand White (NZW) rabbits. The toxicological evaluation of HSA (0.06 and 0.12 mg/kg) was also carried out as a comparative substance. Systemic and local toxicities were not observed in any of the SD rats or NZW rabbits based on clinical signs, body weight, hematology, clinical biochemistry, macroscopic findings on necropsy, histopathology of the injection site, and allergic reactions. The current study suggested that intramuscular administration of polysorbate 20 was considered to be safe at a level similar to that of HSA, which has an in vivo safety profile accumulated over the years. This provided the basis for the in vivo safety profile of polysorbate 20 administered intramuscularly and the scientific reliability of the use of polysorbate 20 as an alternative to HSA, which is used as an excipient for various pharmaceuticals in terms of its safety.

Two VHH Antibodies Neutralize Botulinum Neurotoxin E1 by Blocking Its Membrane Translocation in Host Cells.

Botulinum neurotoxin serotype E (BoNT/E) is one of the major causes of human botulism, which is a life-threatening disease caused by flaccid paralysis of muscles. After receptor-mediated toxin internalization into motor neurons, the translocation domain (HN) of BoNT/E transforms into a protein channel upon vesicle acidification in endosomes and delivers its protease domain (LC) across membrane to enter the neuronal cytosol. It is believed that the rapid onset of BoNT/E intoxication compared to other BoNT serotypes is related to its swift internalization and translocation. We recently identified two neutralizing single-domain camelid antibodies (VHHs) against BoNT/E1 termed JLE-E5 and JLE-E9. Here, we report the crystal structures of these two VHHs bound to the LCHN domain of BoNT/E1. The structures reveal that these VHHs recognize two distinct epitopes that are partially overlapping with the putative transmembrane regions on HN, and therefore could physically block membrane association of BoNT/E1. This is confirmed by our in vitro studies, which show that these VHHs inhibit the structural change of BoNT/E1 at acidic pH and interfere with BoNT/E1 association with lipid vesicles. Therefore, these two VHHs neutralize BoNT/E1 by preventing the transmembrane delivery of LC. Furthermore, structure-based sequence analyses show that the 3-dimensional epitopes of these two VHHs are largely conserved across many BoNT/E subtypes, suggesting a broad-spectrum protection against the BoNT/E family. In summary, this work improves our understanding of the membrane translocation mechanism of BoNT/E and paves the way for developing VHHs as diagnostics or therapeutics for the treatment of BoNT/E intoxication.

Camelid VHH Antibodies that Neutralize Botulinum Neurotoxin Serotype E Intoxication or Protease Function.

Botulinum neurotoxin (BoNT) serotype E is one of three serotypes that cause the preponderance of human botulism cases and is a Tier 1 Select Agent. BoNT/E is unusual among BoNT serotypes for its rapid onset and short duration of intoxication. Here we report two large panels of unique, unrelated camelid single-domain antibodies (VHHs) that were selected for their ability to bind to BoNT/E holotoxin and/or to the BoNT/E light chain protease domain (LC/E). The 19 VHHs which bind to BoNT/E were characterized for their subunit specificity and 8 VHHs displayed the ability to neutralize BoNT/E intoxication of neurons. Heterodimer antitoxins consisting of two BoNT/E-neutralizing VHHs, including one heterodimer designed using structural information for simultaneous binding, were shown to protect mice against co-administered toxin challenges of up to 500 MIPLD50. The 22 unique VHHs which bind to LC/E were characterized for their binding properties and 9 displayed the ability to inhibit LC/E protease activity. Surprisingly, VHHs selected on plastic-coated LC/E were virtually unable to recognize soluble or captured LC/E while VHHs selected on captured LC/E were poorly able to recognize LC/E coated to a plastic surface. This panel of anti-LC/E VHHs offer insight into BoNT/E function, and some may have value as components of therapeutic antidotes that reverse paralysis following BoNT/E exposures.

Structural Insights into Rational Design of Single-Domain Antibody-Based Antitoxins against Botulinum Neurotoxins.

Botulinum neurotoxin (BoNT) is one of the most acutely lethal toxins known to humans, and effective treatment for BoNT intoxication is urgently needed. Single-domain antibodies (VHH) have been examined as a countermeasure for BoNT because of their high stability and ease of production. Here, we investigate the structures and the neutralization mechanisms for six unique VHHs targeting BoNT/A1 or BoNT/B1. These studies reveal diverse neutralizing mechanisms by which VHHs prevent host receptor binding or block transmembrane delivery of the BoNT protease domain. Guided by this knowledge, we design heterodimeric VHHs by connecting two neutralizing VHHs via a flexible spacer so they can bind simultaneously to the toxin. These bifunctional VHHs display much greater potency in a mouse co-intoxication model than similar heterodimers unable to bind simultaneously. Taken together, our studies offer insight into antibody neutralization of BoNTs and advance our ability to design multivalent anti-pathogen VHHs with improved therapeutic properties.

Therapeutic efficacy of equine botulism heptavalent antitoxin against all seven botulinum neurotoxins in symptomatic guinea pigs.

Botulism neurotoxins are highly toxic and are potential agents for bioterrorism. The development of effective therapy is essential to counter the possible use of these toxins in military and bioterrorism scenarios, and to provide treatment in cases of natural intoxication. Guinea pigs were intoxicated with a lethal dose of botulinum neurotoxin serotypes A, B, C, D, E, F or G, and at onset of the clinical disease intoxicated animals were treated with either BAT® [Botulism Antitoxin Heptavalent (A, B, C, D, E, F, G)-(Equine)] or placebo. BAT product treatment significantly (p<0.0001) enhanced survival compared to placebo for all botulinum neurotoxin serotypes and arrested or mitigated the progression of clinical signs of botulism intoxication. These results demonstrated the therapeutic efficacy of BAT product in guinea pigs and provided supporting evidence of effectiveness for licensure of BAT product under FDA 21 CFR Part 601 (Subpart H Animal Rule) as a therapeutic for botulism intoxication to serotypes A, B, C, D, E, F or G in adults and pediatric patients.

Targeted 8-hydroxyquinoline fragment based small molecule drug discovery against neglected botulinum neurotoxin type F.

OBJECTIVES: Botulinum neurotoxins are highly potent biological warfare agents. The unavailability of countermeasures against these neurotoxins has been a matter of extensive research. However, no clinical therapeutics has come to existence till date. The 8-hydroxyquinoline (8-HQ) scaffold is established privileged compound and its potential as drug candidate against BoNTs is recently being explored. METHODS: In present work, three course studies were performed involving in silico, in vitro and in vivo cascade to screen 8-HQ small molecule inhibitors against BoNT/F intoxication. ~800 molecules obtained from open repositories were screened in silico and commercially obtained twenty-four 8-HQ derived small molecule inhibitors were evaluated against rBoNT/F light chain through fluorescence thermal shift (FTS) assay. Selected compounds were further evaluated through endopeptidase assay. Further binding affinity analysis was done through surface plasmon resonance (SPR) based Proteon™ XPR 36 system. Finally, the in vivo efficacy of these compounds was evaluated in mice model. RESULTS: Three compounds NSC1011, NSC1014 and NSC84094 were found to be highly inhibitory after screening of 8-HQ compounds through FTS assay and endopeptidase assay. SPR based protein-small molecule interaction studies showed highest affinity binding of NSC1014 (KD: 5.58E-06) with BoNT/F-LC. NSC1011, NSC1014, and NSC84094 displayed IC50 of 30.47 ±â€¯6.24, 14.91 ±â€¯2.49 and 17.39 ±â€¯2.74 µM, respectively, in endopeptidase assay. NSC1011 and NSC1014 displayed marked extension of survival time in mice model. CONCLUSION: NSC1011 and NSC1014 have emerged as promising drug candidate against BoNT/F intoxication displaying higher potential than previously reported compounds.

Covalent Modifiers of Botulinum Neurotoxin Counteract Toxin Persistence.

Botulinum neurotoxins (BoNTs) are the most potent toxins known to man and a significant threat as weapons of bioterrorism. BoNTs contain a metalloprotease domain that blocks neurotransmitter release in nerve terminals, resulting in a descending, flaccid paralysis with a 5-10% mortality rate. Existing treatment options cannot access or neutralize the toxin following its endocytosis, so there is a clear need to develop novel therapies. Numerous substrate-based and zinc-chelating small-molecule inhibitors have been reported; however, none have progressed to the clinic. This is likely due to the difficulty that reversible inhibitors have in achieving sustained inhibition of the toxin, which has a half-life of months in vivo. An alternative strategy for mitigating BoNT persistence is covalent, irreversible inhibition of toxin function. However, few examples of covalent BoNT inhibitors have been reported. Here, we describe a competition-based screen to identify covalent modifiers of the conserved active-site-adjacent cysteine C165 in the BoNT/A serotype. We found that compounds containing cysteine-reactive electrophiles designed to target cysteine proteases failed to bind C165 while selenide compounds were efficient covalent binders of this cysteine. Importantly, covalent modification at C165 resulted in sustained, irreversible inhibition of BoNT/A protease activity. Covalent selenide inhibitors were nontoxic and protective in a neuronal assay of intoxication, making them promising new scaffolds for the study of the BoNT/A toxin as well as for the design of novel therapy agents.

An ambient temperature-stable antitoxin of nine co-formulated antibodies for botulism caused by serotypes A, B and E.

Safe and effective antitoxins to treat and prevent botulism are needed for biodefense. We have developed recombinant antibody-based therapeutics for botulinum neurotoxin (BoNT) serotypes A, B, and E. The mechanism of action of this antitoxin requires that three mAbs bind one toxin molecule to achieve clearance. Here we present a co-formulation of an antitoxin to the three most important serotypes. Combining these antibodies obviates the need to identify the serotype causing intoxication prior to drug administration, which would facilitate administration. The lyophilized powder formulation contains nine mAbs, three mAbs for each of the three serotypes (A, B, E). The formulation was stored as a liquid and lyophilized powder for up to one year, and characterized by binding affinity and multiple physicochemical methods. No significant increase in soluble higher order aggregates, cleavage products, or change in charge isoforms was measured after storage as a lyophilized powder at 50°C for one year. Furthermore, toxin-domain binding ELISA data indicated that each of the individual antibodies in the lyophilized drug product showed essentially full binding capability to their respective toxin domains after being stored at 50°C for one year. Physicochemical characterization of the formulation demonstrated the nine individual mAbs were remarkably stable. This work demonstrates feasibility of lyophilized, oligoclonal antibody therapies for biodefense with ambient temperature stability, that would facilitate stockpiling, distribution, and administration.

Challenges in searching for therapeutics against Botulinum Neurotoxins.

INTRODUCTION: Botulinum neurotoxins (BoNTs) are the most potent toxins known. BoNTs are responsible for botulism, a deadly neuroparalytic syndrome caused by the inactivation of neurotransmitter release at peripheral nerve terminals. Thanks to their specificity and potency, BoNTs are both considered potential bio-weapons and therapeutics of choice for a variety of medical syndromes. Several variants of BoNTs have been identified with individual biological properties and little antigenic relation. This expands greatly the potential of BoNTs as therapeutics but poses a major safety problem, increasing the need for finding appropriate antidotes. Areas covered: The authors describe the multi-step molecular mechanism through which BoNTs enter nerve terminals and discuss the many levels at which the toxins can be inhibited. They review the outcomes of the different strategies adopted to limit neurotoxicity and counter intoxication. Potential new targets arising from the last discoveries of the mechanism of action and the approaches to promote neuromuscular junction recovery are also discussed. Expert opinion: Current drug discovery efforts have mainly focused on BoNT type A and addressed primarily light chain proteolytic activity. Development of pan-BoNT inhibitors acting independently of BoNT immunological properties and targeting a common step of the intoxication process should be encouraged.

In-Vivo Neutralization of Botulinum Neurotoxin Serotype E Using Rabbit Polyclonal Antibody Developed against BoNT/E Light Chain.

BACKGROUND: Clostridium botulinum is an obligate anaerobic, Gram positive bacterium that secretes extremely toxic substances known as botulinum neurotoxins (BoNTs) that cause serious paralytic illness called botulism. Based upon the serological properties, these neurotoxin have been classified into seven serotypes designated from A to G. Due to extreme toxicity of BoNTs, these neurotoxins have been designated as category A biowarfare agents. There is no commercial neutralizing antibody available for the treatment of botulism. Hence there is an urgent need to develop therapeutic intervention for prevention and cure of botulism within short period. BoNT antiserum injection is still the effective treatment. METHOD: In the present study, the recombinant light chain of BoNT/E was successfully purified in soluble form. The purified rBoNT/E LC was used for the generation of polyclonal antibody in rabbit. In order to find out the neutralizing capacity of generated antisera, rabbit antiserum was incubated with 20 LD50 of botulinum neurotoxin type E for 1 hour at 37°C and then injected intraperitoneally (IP) into mice. Further in another set of experiments antiserum was administered in different ways that included administration of - antiserum and BoNT/E toxin simultaneously without preincubation, one after another at the same and different time points for its therapeutic ability. To find out cross neutralization capacity, rBoNT/E LC antiserum was pre-incubated with 5 LD50 of BoNT/A, BoNT/B, BoNT/F and then injected (IP) into mice. In all the cases mice were observed continuously for 96 hours. RESULT: The results clearly indicate that developed polyclonal rabbit antiserum showed serotype specific neutralization of BoNT/E toxin only but not of BoNT/A, BoNT/B and BoNT/F. CONCLUSION: The developed antibodies will be used for preventive and therapeutic intervention of type 'E' botulism.

Neutralization of Botulinum Neurotoxin Type E by a Humanized Antibody.

Botulinum neurotoxins (BoNTs) cause botulism and are the deadliest naturally-occurring substances known to humans. BoNTs have been classified as one of the category A agents by the Centers for Disease Control and Prevention, indicating their potential use as bioweapons. To counter bio-threat and naturally-occurring botulism cases, well-tolerated antibodies by humans that neutralize BoNTs are relevant. In our previous work, we showed the neutralizing potential of macaque (Macaca fascicularis)-derived scFv-Fc (scFv-Fc ELC18) by in vitro endopeptidase immunoassay and ex vivo mouse phrenic nerve-hemidiaphragm assay by targeting the light chain of the botulinum neurotoxin type E (BoNT/E). In the present study, we germline-humanized scFv-Fc ELC18 into a full IgG hu8ELC18 to increase its immunotolerance by humans. We demonstrated the protection and prophylaxis capacity of hu8ELC18 against BoNT/E in a mouse model. A concentration of 2.5 ng/mouse of hu8ELC18 protected against 5 mouse lethal dose (MLD) in a mouse protection assay and complete neutralization of 1 LD50 of pure BoNT/E toxin was achieved with 8 ng of hu8ELC18 in mouse paralysis assay. Furthermore, hu8ELC18 protected mice from 5 MLD if injected up to 14 days prior to intraperitoneal BoNT/E administration. This newly-developed humanized IgG is expected to have high tolerance in humans.

Identification of small molecule inhibitors of botulinum neurotoxin serotype E via footprint similarity.

Botulinum neurotoxins (BoNT) are among the most poisonous substances known, and of the 7 serotypes (A-G) identified thus far at least 4 can cause death in humans. The goal of this work was identification of inhibitors that specifically target the light chain catalytic site of the highly pathogenic but lesser-studied E serotype (BoNT/E). Large-scale computational screening, employing the program DOCK, was used to perform atomic-level docking of 1.4 million small molecules to prioritize those making favorable interactions with the BoNT/E site. In particular, 'footprint similarity' (FPS) scoring was used to identify compounds that could potentially mimic features on the known substrate tetrapeptide RIME. Among 92 compounds purchased and experimentally tested, compound C562-1101 emerged as the most promising hit with an apparent IC50 value three-fold more potent than that of the first reported BoNT/E small molecule inhibitor NSC-77053. Additional analysis showed the predicted binding pose of C562-1101 was geometrically and energetically stable over an ensemble of structures generated by molecular dynamic simulations and that many of the intended interactions seen with RIME were maintained. Several analogs were also computationally designed and predicted to have further molecular mimicry thereby demonstrating the potential utility of footprint-based scoring protocols to help guide hit refinement.

Chloroquine Analog Interaction with C2- and Iota-Toxin in Vitro and in Living Cells.

C2-toxin from Clostridium botulinum and Iota-toxin from Clostridium perfringens belong both to the binary A-B-type of toxins consisting of two separately secreted components, an enzymatic subunit A and a binding component B that facilitates the entry of the corresponding enzymatic subunit into the target cells. The enzymatic subunits are in both cases actin ADP-ribosyltransferases that modify R177 of globular actin finally leading to cell death. Following their binding to host cells' receptors and internalization, the two binding components form heptameric channels in endosomal membranes which mediate the translocation of the enzymatic components Iota a and C2I from endosomes into the cytosol of the target cells. The binding components form ion-permeable channels in artificial and biological membranes. Chloroquine and related 4-aminoquinolines were able to block channel formation in vitro and intoxication of living cells. In this study, we extended our previous work to the use of different chloroquine analogs and demonstrate that positively charged aminoquinolinium salts are able to block channels formed in lipid bilayer membranes by the binding components of C2- and Iota-toxin. Similarly, these molecules protect cultured mammalian cells from intoxication with C2- and Iota-toxin. The aminoquinolinium salts did presumably not interfere with actin ADP-ribosylation or receptor binding but blocked the pores formed by C2IIa and Iota b in living cells and in vitro. The blocking efficiency of pores formed by Iota b and C2IIa by the chloroquine analogs showed interesting differences indicating structural variations between the types of protein-conducting nanochannels formed by Iota b and C2IIa.

Small molecule non-peptide inhibitors of botulinum neurotoxin serotype E: Structure-activity relationship and a pharmacophore model.

Botulinum neurotoxins (BoNTs) are the most poisonous biological substance known to humans. They cause flaccid paralysis by blocking the release of acetylcholine at the neuromuscular junction. Here, we report a number of small molecule non-peptide inhibitors of BoNT serotype E. The structure-activity relationship and a pharmacophore model are presented. Although non-peptidic in nature, these inhibitors mimic key features of the uncleavable substrate peptide Arg-Ile-Met-Glu (RIME) of the SNAP-25 protein. Among the compounds tested, most of the potent inhibitors bear a zinc-chelating moiety connected to a hydrophobic and aromatic moiety through a carboxyl or amide linker. All of them show low micromolar IC50 values.

High-Throughput Screening Uncovers Novel Botulinum Neurotoxin Inhibitor Chemotypes.

Botulism is caused by potent and specific bacterial neurotoxins that infect host neurons and block neurotransmitter release. Treatment for botulism is limited to administration of an antitoxin within a short time window, before the toxin enters neurons. Alternatively, current botulism drug development targets the toxin light chain, which is a zinc-dependent metalloprotease that is delivered into neurons and mediates long-term pathology. Several groups have identified inhibitory small molecules, peptides, or aptamers, although no molecule has advanced to the clinic due to a lack of efficacy in advanced models. Here we used a homogeneous high-throughput enzyme assay to screen three libraries of drug-like small molecules for new chemotypes that modulate recombinant botulinum neurotoxin light chain activity. High-throughput screening of 97088 compounds identified numerous small molecules that activate or inhibit metalloprotease activity. We describe four major classes of inhibitory compounds identified, detail their structure-activity relationships, and assess their relative inhibitory potency. A previously unreported chemotype in any context of enzyme inhibition is described with potent submicromolar inhibition (Ki = 200-300 nM). Additional detailed kinetic analyses and cellular cytotoxicity assays indicate the best compound from this series is a competitive inhibitor with cytotoxicity values around 4-5 µM. Given the potency and drug-like character of these lead compounds, further studies, including cellular activity assays and DMPK analysis, are justified.

Searching for Therapeutics Against Botulinum Neurotoxins: A True Challenge for Drug Discovery.

Botulinum neurotoxins (BoNTs), the most potent known toxins, cause severe muscle paralysis and death at nanogram exposures and are considered biothreat agents. BoNTs target the neuromuscular junction where they release smaller zinc metalloprotease light chains (LCs) into the neuron cytosol that selectively cleave SNARE proteins and thus block the exocytosis of acetylcholine neurotransmitters necessary for skeletal muscle contraction. The majority of efforts to develop post-symptomatic therapeutics for botulism poisoning have focused on inhibiting the LC and tremendous strides have been made in understanding how the LC binds to the SNARE proteins via X-ray crystallography. Subsequent homology modeling and structure based drug design have led to the discovery of multiple small molecule BoNT/A inhibitors in the 0.05 ~10 µΜ range, but to date none have shown significant post-symptomatic efficacy in an animal model of botulinum intoxication. With the lack of reported pharmacokinetic data, we have analyzed the BoNT/A inhibitor lead chemical matter from a physicochemical property point of view and have attempted to understand if bioavailability of drug at the neuromuscular junction is the root cause of this apparent in vitro/in vivo disconnect in the field.

A Novel Inhibitor Prevents the Peripheral Neuroparalysis of Botulinum Neurotoxins.

Botulinum neurotoxins (BoNTs) form a large class of potent and deadly neurotoxins. Given their growing number, it is of paramount importance to discover novel inhibitors targeting common steps of their intoxication process. Recently, EGA was shown to inhibit the action of bacterial toxins and viruses exhibiting a pH-dependent translocation step in mammalian cells, by interfering with their entry route. As BoNTs act in the cytosol of nerve terminals, the entry into an appropriate compartment wherefrom they translocate the catalytic moiety is essential for toxicity. Herein we propose an optimized procedure to synthesize EGA and we show that, in vitro, it prevents the neurotoxicity of different BoNT serotypes by interfering with their trafficking. Furthermore, in mice, EGA mitigates botulism symptoms induced by BoNT/A and significantly decreases the lethality of BoNT/B and BoNT/D. This opens the possibility of using EGA as a lead compound to develop novel inhibitors of botulinum neurotoxins.