Structures of Clostridium botulinum Neurotoxin Serotype A Light Chain complexed with small-molecule inhibitors highlight active-site flexibility

The potential for the use of Clostridial neurotoxins as bioweapons makes the development of small-molecule inhibitors of these deadly toxins a top priority. Recently, screening of a random hydroxamate library identified a small-molecule inhibitor of C. botulinum Neurotoxin Serotype A Light Chain (BoNT/A-LC), 4-chlorocinnamic hydroxamate, a derivative of which has been shown to have in vivo efficacy in mice and no toxicity. We describe the X-ray crystal structures of BoNT/A-LC in complexes with two potent small-molecule inhibitors. The structures of the enzyme with 4-chlorocinnamic hydroxamate or 2,4-dichlorocinnamic hydroxamate bound are compared to the structure of the enzyme complexed with L-arginine hydroxamate, an inhibitor with modest affinity. Taken together, this suite of structures provides surprising insights into the BoNT/A-LC active site, including unexpected conformational flexibility at the S1' site that changes the electrostatic environment of the binding pocket. Information gained from these structures will inform the design and optimization of more effective small-molecule inhibitors of BoNT/A-LC.     

Synthesis, characterization and development of a high-throughput methodology for the discovery of botulinum neurotoxin a inhibitors

Botulinum neurotoxins (BoNTs), etiological agents of the deadly food poisoning disease botulism, are the most toxic proteins currently known. Although only a few hundred cases of botulism are reported in the United States annually, there is growing interest in BoNTs attributable to their potential use as biological warfare agents. Neurotoxicity results from cleavage of the soluble NSF-attachment protein receptor complex proteins of the presynaptic vesicles by the BoNT light chain subunit, a Zn endopeptidase. Few effective inhibitors of BoNT/A LC (light chain) activity are known, and the discovery process is hampered by the lack of an efficient high-throughput assay for screening compound libraries. To alleviate this bottleneck, we have synthesized the peptide SNAPtide and have developed a robust assay for the high-throughput evaluation of BoNT/A LC inhibitors. Key aspects for the development of this optimized assay include the addition of a series of detergents, cosolvents, and salts, including 0.01% w/v Tween 20 to increase BoNT/A LC catalysis, stability, and ease of small molecule screening. To evaluate the effectiveness of the assay, a series of hydroxamate-based small molecules were synthesized and examined with BoNT/A LC. The methodology described is superior to other assays reported to date for the high-throughput identification of BoNT/A inhibitors.     

Structural insight into exosite binding and discovery of novel exosite inhibitors of botulinum neurotoxin serotype A through in silico screening

Botulinum neurotoxin serotype A (BoNT/A) is the most lethal toxin among the Tier 1 Select Agents. Development of potent and selective small molecule inhibitors against BoNT/A zinc metalloprotease remains a challenging problem due to its exceptionally large substrate binding surface and conformational plasticity. The exosites of the catalytic domain of BoNT/A are intriguing alternative sites for small molecule intervention, but their suitability for inhibitor design remains largely unexplored. In this study, we employed two recently identified exosite inhibitors, D-chicoric acid and lomofungin, to probe the structural features of the exosites and molecular mechanisms of synergistic inhibition. The results showed that D-chicoric acid favors binding at the α-exosite, whereas lomofungin preferentially binds at the β-exosite by mimicking the substrate β-sheet binding interaction. Molecular dynamics simulations and binding interaction analysis of the exosite inhibitors with BoNT/A revealed key elements and hotspots that likely contribute to the inhibitor binding and synergistic inhibition. Finally, we performed database virtual screening for novel inhibitors of BoNT/A targeting the exosites. Hits C1 and C2 showed non-competitive inhibition and likely target the α- and β-exosites, respectively. The identified exosite inhibitors may provide novel candidates for structure-based development of therapeutics against BoNT/A intoxication.     

Toward the discovery of potent inhibitors of botulinum neurotoxin A: development of a robust LC MS based assay operational from low to subnanomolar enzyme concentrations

The development of a sensitive, yet reliable assay for the analysis of botulinum neurotoxin A (BoNT/A) inhibitors is described; using this assay a new protease inhibitor was characterized and found to be one of the most potent inhibitors reported to date.     

Small molecule inhibitors as countermeasures for botulinum neurotoxin intoxication

Botulinum neurotoxins (BoNTs) are the most potent of known toxins and are listed as category A biothreat agents by the U.S. CDC. The BoNT-mediated proteolysis of SNARE proteins inhibits the exocytosis of acetylcholine into neuromuscular junctions, leading to life-threatening flaccid paralysis. Currently, the only therapy for BoNT intoxication (which results in the disease state botulism) includes experimental preventative antibodies and long-term supportive care. Therefore, there is an urgent need to identify and develop inhibitors that will serve as both prophylactic agents and post-exposure 'rescue' therapeutics. This review focuses on recent progress to discover and develop small molecule inhibitors as therapeutic countermeasures for BoNT intoxication.     

Identification of a potent botulinum neurotoxin a protease inhibitor using in situ lead identification chemistry

[reaction: see text] Botulinum neurotoxins (BoNTs), etiological agents of the deadly food poisoning disease botulism, are the most toxic proteins currently known. By using in situ lead identification chemistry, we have uncovered the first class of inhibitors that displays nanomolar potency. From a 15 microM lead compound, structure-activity relationship studies were performed granting the most potent BoNT/A inhibitor reported to date that displays an inhibition constant of 300 nM.     

Llama-derived single-domain antibodies for the detection of botulinum A neurotoxin

Single-domain antibodies (sdAb) specific for botulinum neurotoxin serotype A (BoNT A) were selected from an immune llama phage display library derived from a llama that was immunized with BoNT A toxoid. The constructed phage library was panned using two methods: panning on plates coated with BoNT A toxoid (BoNT A Td) and BoNT A complex toxoid (BoNT Ac Td) and panning on microspheres coupled to BoNT A Td and BoNT A toxin (BoNT A Tx). Both panning methods selected for binders that had identical sequences, suggesting that panning on toxoided material may be as effective as panning on bead-immobilized toxin for isolating specific binders. All of the isolated binders tested were observed to recognize bead-immobilized BoNT A Tx in direct binding assays, and showed very little cross-reactivity towards other BoNT serotypes and unrelated protein. Sandwich assays that incorporated selected sdAb as capture and tracer elements demonstrated that all of the sdAb were able to recognize soluble (“live”) BoNT A Tx and BoNT Ac Tx with virtually no cross-reactivity with other BoNT serotypes. The isolated sdAb did not exhibit the high degree of thermal stability often associated with these reagents; after the first heating cycle most of the binding activity was lost, but the portion of the protein that did refold and recover antigen-binding activity showed only minimal loss on subsequent heating and cooling cycles. The binding kinetics of selected binders, assessed by both an equilibrium fluid array assay as well as surface plasmon resonance (SPR) using toxoided material, gave dissociation constants (K _D ) in the range 2.2 × 10⁻¹¹ to 1.6 × 10⁻¹⁰ M. These high-affinity binders may prove beneficial to the development of recombinant reagents for the rapid detection of BoNT A, particularly in field screening and monitoring applications.     

Epitope Targeting of Tertiary Protein Structure Enables Target‐Guided Synthesis of a Potent In‐Cell Inhibitor of Botulinum Neurotoxin

Botulinum neurotoxin (BoNT) serotype A is the most lethal known toxin and has an occluded structure, which prevents direct inhibition of its active site before it enters the cytosol. Target‐guided synthesis by in situ click chemistry is combined with synthetic epitope targeting to exploit the tertiary structure of the BoNT protein as a landscape for assembling a competitive inhibitor. A substrate‐mimicking peptide macrocycle is used as a direct inhibitor of BoNT. An epitope‐targeting in situ click screen is utilized to identify a second peptide macrocycle ligand that binds to an epitope that, in the folded BoNT structure, is active‐site‐adjacent. A second in situ click screen identifies a molecular bridge between the two macrocycles. The resulting divalent inhibitor exhibits an in vitro inhibition constant of 165 pM against the BoNT/A catalytic chain. The inhibitor is carried into cells by the intact holotoxin, and demonstrates protection and rescue of BoNT intoxication in a human neuron model.     

Superactivation of the botulinum neurotoxin serotype A light chain metalloprotease: a new wrinkle in botulinum neurotoxin

Small molecules based upon a 2-acylguanidine-5-phenyl thiophene scaffold that can activate the light chain metalloprotease of botulinum neurotoxin serotype A (BoNT LC/A) by an apparent reduction in Km are reported. On the basis of structure-activity relationships and the activation profile, one or more molecules of activator specifically bind to a defined site on the toxin, causing the observed rate enhancement. With the ever-growing clinical uses of BoNT, compounds such as those reported here may provide a method for combating the emerging adaptive immune responses to BoNT.     

De novo subtype and strain identification of botulinum neurotoxin type B through toxin proteomics

Botulinum neurotoxins (BoNTs) cause the disease botulism, which can be lethal if untreated. There are seven known serotypes of BoNT, A-G, defined by their response to antisera. Many serotypes are distinguished into differing subtypes based on amino acid sequence, and many subtypes are further differentiated into toxin variants. Previous work in our laboratory described the use of a proteomics approach to distinguish subtype BoNT/A1 from BoNT/A2 where BoNT identities were confirmed after searching data against a database containing protein sequences of all known BoNT/A subtypes. We now describe here a similar approach to differentiate subtypes BoNT/B1, /B2, /B3, /B4, and /B5. Additionally, to identify new subtypes or hitherto unpublished amino acid substitutions, we created an amino acid substitution database covering every possible amino acid change. We used this database to differentiate multiple toxin variants within subtypes of BoNT/B1 and B2. More importantly, with our amino acid substitution database, we were able to identify a novel BoNT/B subtype, designated here as BoNT/B7. These techniques allow for subtype and strain level identification of both known and unknown BoNT/B rapidly with no DNA required.     

Bead-based microfluidic toxin sensor integrating evaporative signal amplification

We have devised a microfluidic platform that incorporates substrate-laden silica beads for sensing the proteolytic activity of botulinum neurotoxin type A (BoNT/A)-one of the most poisonous substances known and a significant biological threat. The sensor relies on toxin-mediated cleavage of a fluorophore-tagged peptide substrate specific for only BoNT/A. Peptide immobilized on beads is recognized and cleaved by the toxin, releasing fluorescent fragments into solution that can be concentrated at an isolated port via evaporation and detected using microscopy. Evaporative concentration in combination with a specific channel geometry provides up to a 3-fold signal amplification in 35 min, allowing for detection of low levels of fluorophore-labeled peptide-a task not easily accomplished using traditional channel designs. Our bead-based microfluidic platform can sense BoNT/A down to 10 pg of toxin per mL buffer solution in 3.5 h and can be adapted to sensing other toxins that operate via enzymatic cleavage of a known substrate.     

Generation of a Recombinant Full-Length Human Antibody Binding to Botulinum Neurotoxin A

In order to develop a recombinant full-length human anti-botulinum neurotoxin A (BoNT/A) antibody, human peripheral blood mononuclear cells (PBMC) were collected from three healthy volunteers and induced for BoNT/A-specific immune response by in vitro immunization. The genes encoding human Fd fragment, consisting of antibody heavy chain variable region and constant region 1 with the genes encoding antibody light chain, were cloned from the immunized PBMC. Afterwards, one combinatory human antigen-binding fragment (Fab) library was constructed using a lambda phage vector system. The size of the constructed library was approximately 10⁵ Escherichia coli transformants. After screening the library by BoNT/A antigen using a plaque lifting with immunostaining approach, 55 clones were identified as positive. The Fab gene of the most reactive clone exhibiting particularly strong BoNT/A binding signal was further subcloned into a full-length human IgG1 antibody gene template in an adenoviral expression vector, in which the heavy and light chains were linked by a foot-and-mouth-disease virus-derived 2A self-cleavage peptide under a single promoter. After the full-length human IgG1 was expressed in mammalian cells and purified with protein L column, sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the heavy and light chains of the antibody were cleaved completely. The affinity expressed as the dissociation constant (K _d) for the recombinant human antibody to bind to BoNT/A was determined by indirect enzyme-linked immunosorbent assay and results confirmed that the recombinant full-length human antibody retained BoNT/A-binding specificity with K _d value of 10⁻⁷ M.     

Development of highly sensitive chemiluminescence enzyme immunoassay based on the anti-recombinant HC subunit of botulinum neurotoxin type A monoclonal antibodies

Botulinum neurotoxins (BoNTs) are the most poisonous substances ever known. The early detection of these toxins could bear more time for appropriate medical intervention. The standard method for detecting BoNTs is the mouse bioassay, which is time consuming (up to 4 days) and requires a large number of laboratory animals. The immunologic detection methods could detect the toxins within a day, but most of these methods are less sensitive compared with the mouse bioassay due to the lack of high-affinity antibodies. Recently, the recombinant H_C subunit of botulinum neurotoxin type A (rAH_C) was expressed as an effective vaccine against botulism, indicating that the rAH_C could be an effective immunogen that raises the monoclonal antibody (mAb) for detecting BoNT/A. After immunized BALB/c mice with rAH_C, 56 mAbs were generated. Two of these mAbs were selected to establish a highly sensitive sandwich chemiluminescence enzyme immunoassay (CLEIA), in which FMMU-BTA-49 and FMMU-BTA-22 were used as capture antibody and detection antibody, respectively. The calculated limit of detection (LOD) based on molecular weight of rAH_C and BoNT/A reached 0.45 pg mL⁻¹. This CLEIA can be used in the detection of BoNT/A in matrices such as milk and beef extract. This method has 20–40 fold lower LOD than that of the mouse bioassay and takes only 3 h to complete the detection, indicating that it can be used as a valuable method to detect and quantify BoNT/A.     

Enzyme-amplified protein microarray and a fluidic renewable surface fluorescence immunoassay for botulinum neurotoxin detection using high-affinity recombinant antibodies

Two immunoassay platforms were developed for either the sensitive or rapid detection of botulinum neurotoxin A (BoNT/A), using high-affinity recombinant monoclonal antibodies against the receptor binding domain of the heavy chain of BoNT/A. These antibodies also bind the same epitopes of the receptor binding domain present on a nontoxic recombinant heavy chain fragment used for assay development and testing in the current study. An enzyme-linked immunosorbent assay (ELISA) microarray using tyramide amplification for localized labeling was developed for the specific and sensitive detection of BoNT. This assay has the sensitivity to detect BoNT in buffer and blood plasma samples down to 14 fM (1.4 pg mL⁻¹). Three capture antibodies and one antibody combination were compared in the development of this assay. Using a selected pair from the same set of recombinant monoclonal antibodies, a renewable surface microcolumn sensor was developed for the rapid detection of BoNT/A in an automated fluidic system. The ELISA microarray assay, because of its sensitivity, offers a screening test with detection limits comparable to the mouse bioassay, with results available in hours instead of days. The renewable surface assay is less sensitive but much faster, providing results in less than 10 min.     

Micellar electrokinetic chromatography and laser induced fluorescence detection of botulinum neurotoxin type A activity using a dual-labelled substrate

The activity of Botulinum neurotoxin type A (BoNT A) can be measured by monitoring the toxin's endopeptidase reaction with its peptide substrate. In this report, a sensitive and simple capillary electrophoresis (CE) method for analysing BoNT A activity was developed using a peptide substrate labelled with Fluorescein isothiocynate (FITC) at the N-terminal and biotin at the C-terminal. This dual labelling enables not only highly sensitive laser induced fluorescence (LIF) detection of the reaction product, but also good analytical separation of the product from the peptide substrate by Micellar Electrokinetic Chromatography (MEKC). The separation between the product peak and the substrate peak was approximately 5 min using the dual-labelled substrate, while just about 1 min using the FITC-labelled substrate without biotinylation. Using the current assay method, BoNT A with concentration as low as 0.1 ng ml^?1 (3.6 U mL^?1 in mouse LD₅₀) in water was detected with a S:N ratio of 3 (RSD <19%) and a linear range of four orders of magnitude. With CE's advantages of very small sample volume needed, this method may find particular applications as in assays of BoNT A activity in water samples and kinetic analyses of toxin activity.     

Development of inhibitors of the aspartyl protease renin for the treatment of hypertension

Renin is the rate-limiting enzyme in the renin-angiotensin-aldosterone system (RAS) which controls blood pressure and volume. The biological function of renin is to cleave the N-terminus of angiotensinogen releasing the decapeptide, angiotensin I (ANGI). Subsequently, angiotensin I is further processed by the angiotensin converting enzyme (ACE) to produce angiotensin II (ANGII). The RAS cascade is a major target for the clinical management of hypertension. Current clinical treatments include angiotensin converting enzyme inhibitors (ACEi) and ANGII receptor blockers (ARBs). As the rate-limiting enzyme in ANGII production, renin inhibitors have been pursued as an additional class of anti-hypertensives. Clinical studies conducted with renin inhibitors have shown them to be as effective as ACE inhibitors in lowering blood pressure. Most importantly, inhibitors of renin may have a number of potential advantages over ACEi and ARBs. Renin is specific for angiotensinogen and will not carry the ancillary pharmacology associated with ACEi or ARBs. To date, no renin inhibitors have made it to market. The development of these inhibitors has been hindered by poor bioavailability and complex synthesis. However, despite the pharmacokinetic challenges of designing renin inhibitors, the enzyme remains a promising target for the development of novel treatments for hypertension. This review will consist of an overview of renin biology, the pharmacology of renin and RAS and focus in on renin as a target for blood pressure regulation. We also cover the evaluation of renin inhibitors in animal models and clinical studies. Presently a number of new generation inhibitors of renin are in development with at least one in the clinic and these will be discussed. Finally we will discuss what might distinguish renin inhibitors from current therapeutic options and discuss other therapeutic indications renin inhibitors might have.     

Sensitive detection of type G botulinum neurotoxin through Endopep-MS peptide substrate optimization

Clostridium botulinum produces botulinum neurotoxins (BoNTs) that are one of the most poisonous substances. In order to respond to public health emergencies, there is a need to develop sensitive and specific methods for detecting botulinum toxin in various clinical matrices. Our laboratory has developed a mass spectrometry-based Endopep-MS assay that is able to rapidly detect and differentiate BoNT serotypes A–G by immunoaffinity capture of toxins and detection of unique cleavage products of peptide substrates. To improve the sensitivity of the Endopep-MS assay for the detection of BoNT serotype G, we report here the optimization of synthetic peptide substrates through systematic substitution, deletion, and incorporation of unnatural amino acids. Our data show that the resulting optimized peptides produced a significant improvement (two orders of magnitude) in assay sensitivity and allowed the detection of 0.01 mouseLD₅₀ toxin present in buffer solution.

     

Inhibition of Calcium Naphthenate. Experimental Methods to Study the Effect of Commercially Available Naphthenate Inhibitors

In this study, different methods have been developed for studying naphthenate inhibitors. One first method is based on interfacial activity of systems with naphthenic acids, inhibitors, and calcium viewing reaction mechanisms at interfaces of the inhibitors. A second method is based on gravimetrical determination of interfacial layer in a two phase system and gives a thermodynamic approach to naphthenate formation, and a third method utilizes a ultraviolet-active model tetraacid to directly determine the depletion in bulk phase concentration during a two-phase reaction. The results indicate that inhibitors may act through several mechanisms, and depending on the total system different mechanisms may have the highest efficiency also by combining several mechanisms.     

Synthesis of a number of derivatives of alkaloids and of nitrogen-containing heterocycles and their anticholinesterase activities

N-Methyl- and N-phenylcarbamates based on a number of alkaloids and nitrogen-containing heterocycles have been synthesized, and they have proved to be weak irreversible inhibitors of acetylcholinesterase and butyrylcholinesterase. It has been shown that the choline fragments of the above-mentioned carbamates and their β-methylcholine analogs are reversible inhibitors of both cholinesterases and make a substantial contribution to the anticholinesterase activity. Selective inhibitors of ACE and BuCE have been found among the compounds synthesized.