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.     

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.     

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.     

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.     

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.     

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.     

The strange case of the botulinum neurotoxin: using chemistry and biology to modulate the most deadly poison

In the classic novella "The Strange Case of Dr. Jekyll and Mr. Hyde", Robert Louis Stevenson paints a stark picture of the duality of good and evil within a single man. Botulinum neurotoxin (BoNT), the most potent known toxin, possesses an analogous dichotomous nature: It shows a pronounced morbidity and mortality, but it is used with great effect in much lower doses in a wide range of clinical scenarios. Recently, tremendous strides have been made in the basic understanding of the structure and function of BoNT, which have translated into widespread efforts towards the discovery of biomacromolecules and small molecules that specifically modulate BoNT activity. Particular emphasis has been placed on the identification of inhibitors that can counteract BoNT exposure in the event of a bioterrorist attack. This Review summarizes the current advances in the development of therapeutics, including vaccines, peptides, and small-molecule inhibitors, for the prevention and treatment of botulism.     

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.     

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.     

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.     

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.     

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.

     

Determination of nonsteroidal anti-inflammatory drugs in human specimens by capillary zone electrophoresis and micellar electrokinetic chromatography

Therapeutic drug monitoring of anti-inflammatory drugs is necessary for the identification of the agents that cause toxic events and for the decision on the treatment for intoxication. Recently, capillary electrophoresis (CE) has been developed for the simple and rapid analyses of a variety of chemical agents. Micellar electrokinetic chromatography (MEKC) can separate acidic, neutral and basic anti-inflammatory drugs in serum. Furthermore, serum samples are directly applied to the CE system without any pretreatments, and some anti-inflammatory drugs can be separated from serum albumin in the MEKC analysis. On the other hand, capillary zone electrophoresis (CZE) enables us to determine a few microg/mL levels of acidic anti-inflammatory drugs with simple running buffer and stacking technique. A rapid and simultaneous determination of several analgesic anti-inflammatory agents, including ibuprofen, acetaminophen, indomethacin, and salicylic acid in human serum has been developed by using CZE. Therefore, the CZE and MEKC analysis may become a potentially useful alternative to high-performance liquid chromatography (HPLC) and fluorescence polarization immunoassay (FPIA) for therapeutic drug monitoring, particularly in serum of patients suffering from intoxication by overdosage of nonsteroidal anti-inflammatory agents.     

Expression Optimization and Characterization of the Catalytic Domain of Human MT3-MMP

Introduction Matrixmetalloproteinases(MMPs)areafamilyof calciumandzincrequiringendoproteinasesthattogether candegradeallthemaincomponentsoftheextra cellu larmatrixandbasementmembranes[1].MMPsarein volvedinawiderangeofproteolyticevents,innormal andpatholog…     

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.     

Generation of oxamic acid libraries: antimalarials and inhibitors of Plasmodium falciparum lactate dehydrogenase

Lactate dehydrogenase (LDH) is a key enzyme in the glycolytic pathway of Plasmodium falciparum (pf) and has several unique amino acids, related to other LDHs, at the active site, making it an attractive target for antimalarial agents. Oxamate, a competitive inhibitor, shows high substrate affinity for pfLDH. This class of compounds has been viewed as potential antimalarial agents. Thus, we have developed an effective automated synthetic strategy for the rapid synthesis of oxamic acid and ester libraries to screen for potential lead inhibitors. One hundred sixty-seven oxamic acids were synthesized using a "catch and release" method with overall yields of 20-70%. Most of the compounds synthesized had some inhibitory effects, but compounds 5 and 6 were the most active against both chloroquine- and mefloquine-resistant strains with IC50 values of 15.4 and 9.41 microM and 20.4 and 8.40 microM, respectively. Some oxamic acids showed activities against pfLDH and mammalian LDH (mLDH) at the micromolar range. These oxamic acids selectively inhibited pfLDH 2-5 fold over mLDH. Oxamic acid 21 was the most active against pfLDH at IC50 = 14 and mLDH at IC50 = 25 microM, suggesting that oxamic acid derivatives are potential inhibitors of pfLDH and that further study is required to develop selective inhibitors of pfLDH over mLDH.     

Novel small-molecule inhibitors of arylamine N-acetyltransferases: drug discovery by high-throughput screening

Arylamine N-acetyltransferases (NATs) are a family of enzymes found in eukaryotes and prokaryotes. While the precise endogenous function of NAT remains unknown for most organisms, recent evidence has shown that the expression of human NAT1 is up-regulated in estrogen receptor positive breast cancer. Additionally, NAT in mycobacteria is required for mycobacterial cell wall biosynthesis and survival of the organisms within macrophage. It is therefore important to develop small molecule inhibitors of NATs as molecular tools to study the function of NATs in various organisms. Such inhibitors may also prove useful in future drug design, for example in the development of anti tubercular agents. We describe a high-throughput screen of a proprietary library of 5016 drug-like compounds against three prokaryotic NAT enzymes and two eukaryotic NAT enzymes.     

基于分子描述符和机器学习方法预测和虚拟筛选MMP-13对MMP-1的选择性抑制剂

基质金属蛋白酶-13 (MMP-13)为预防和治疗骨关节炎(OA)提供了充满希望的靶标. 通过抑制剂来阻断MMP-13的活性将会对治疗OA疾病产生潜在的作用. 然而,宽谱抑制剂同样抑制MMP家族的其它成员,特别是MMP-1,这将会导致肌与骨的综合症. 因此,设计和发现潜在的MMP-13 相对于MMP-1 的高效选择性抑制剂,在对治疗OA新型药物的研发中具有相当重要的现实意义. 本研究通过两种机器学习方法(ML)：支持向量机(SVM)和随机森林(RF)来建立分类模型,用于预测不同结构的MMP-13 对MMP-1 的选择性抑制剂. 所建这些模型的预测效果都已经达到了令人满意的精度. 在这两种ML模型中,RF对于MMP-13选择性抑制剂和非抑制剂的精度分别达到97.58%和100%. 同时,与MMP-13对MMP-1的选择性抑制最相关的分子描述符也基于不同的特征选择方法被两种模型挑选出来. 最后,用预测效果最好的RF模型虚拟筛选了ZINC数据库的“fragment-like”子集,从而得到了一系列潜在的候选药物. 研究表明,机器学习方法,特别是RF方法,对于发现潜在的MMP-13选择性抑制剂十分有效. 同时还得到了一些与MMP-13的选择性抑制相关的分子描述符.     

Life at the End of the Chromosome: Telomeres and Telomerase

Telomerase, the enzyme that replicates the ends of linear chromosomes, is implicated in cellular aging and in cancer. The molecular components that form the catalytic core of this ribonucleoprotein enzyme (a section of the active site with bound substrates is depicted) have recently been identified in multiple organisms, including humans. The stage is now set for chemists to develop telomerase inhibitors, which hold promise as cancer chemotherapeutic agents.     

Highlights on Steroidal Arylidene Derivatives as a Source of Pharmacologically Active Compounds: A Review

Steroids constitute a unique class of chemical compounds, playing an important role in physiopathological processes, and have high pharmacological interest. Additionally, steroids have been associated with a relatively low toxicity and high bioavailability. Nowadays, multiple steroidal derivatives are clinically available for the treatment of numerous diseases. Moreover, different structural modifications on their skeleton have been explored, aiming to develop compounds with new and improved pharmacological properties. Thus, steroidal arylidene derivatives emerged as a relevant example of these modifications. This family of compounds has been mainly described as 17β-hydroxysteroid dehydrogenase type 1 and aromatase inhibitors, as well as neuroprotective and anticancer agents. Besides, due to their straightforward preparation and intrinsic chemical reactivity, steroidal arylidene derivatives are important synthetic intermediates for the preparation of other compounds, particularly bearing heterocyclic systems. In fact, starting from arylidenesteroids, it was possible to develop bioactive steroidal pyrazolines, pyrazoles, pyrimidines, pyridines, spiro-pyrrolidines, amongst others. Most of these products have also been studied as anti-inflammatory and anticancer agents, as well as 5α-reductase and aromatase inhibitors. This work aims to provide a comprehensive overview of steroidal arylidene derivatives described in the literature, highlighting their bioactivities and importance as synthetic intermediates for other pharmacologically active compounds.