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.     

人源抗A型肉毒毒素单链抗体的体外亲和筛选

以重组制备的A型肉毒毒素保护性抗原为配体,对人源噬菌体免疫抗体文库进行体外定向亲和筛选,获得特异结合子,其中与抗原高亲和力结合的抗体克隆B17基因全长750bp,可编码250个氨基酸,抗体可变区基因同源分析表明,分属VH4和κ chain Ⅱ家族,是一株人源特异单链抗体基因。人源单链抗体B17在大肠杆菌中获得了重组表达,表达产物可以竞争特异肉毒抗毒素马血清与抗原的结合,是国内首次获得的抗A型肉毒毒素保护性抗原的人源单链抗体,可以在肉毒毒素检测和治疗研究中发挥作用。     

IEF纯化重组A型肉毒毒素保护性抗原

在大肠杆菌中高效表达的重组A型肉毒毒素保护性抗原（rBoNTaH468),是以包涵体形式存在,将表达菌株发酵后,裂解菌体,制备包涵体,溶解后的包涵体溶液经样品处理,通过等地电聚焦制备型电泳纯化,纯化的重组A型肉毒毒素保护性抗原（rBoNTaH468)纯度高于90%,产量及回收率高,纯化的重组表达产物酶联检测具有结合活性,这为下一步A型肉毒毒素抗毒素的研制打下基础。     

A型肉毒毒素保护性抗原的基因修饰及高效表达

在A型肉毒毒素保护性抗原基因初步表达的基础上,为提高表达水平,依据EMBL的DNA数据库中A型肉毒毒素基因全序列,重新设计上游引物,通过修饰基因片段N端,保持氨基酸序列不变,从已获得的A型肉毒毒素与靶细胞起结合作用的重链C端基因中,扩增小的突变基因,克隆入pGEM-T载体进行测序,并以pBV220为表达载体构建重组表达质粒,在大肠杆菌中实现高效表达。结果表明,重组表达产物占全菌蛋白的40%,酶联检测重组表达产物具有特异结合活性。A型肉毒毒素保护性抗原基因的高效表达,为下一步基因工程抗毒素和疫苗的研制奠定了基础。     

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.     

A Simple,Rapid and Sensitive FRET Assay for Botulinum Neurotoxin Serotype B Detection

Botulinum neurotoxins (BoNTs), the most potent naturally-occurring neurotoxins known to humans, comprise seven distinct serotypes (BoNT/A-G), each of which exhibits unique substrate specificity. Many methods have been developed for BoNT detection, in particular for BoNT/A, with various complexity and sensitivity, while substrate based FRET assay is considered as the most widely used approach due to its simplicity and sensitivity. In this study, we designed a vesicle-associated membrane protein 2 (VAMP2) based FRET assay based on the understanding of the VAMP2 and light chain/B (LC/B) interactions in our previous studies. The current design constituted the shortest peptide, VAMP2 (63–85), with FRET dyes (EDAN and Dabcyl) labelled at position 76 and 85, respectively, which showed minimal effect on VAMP2 substrate catalysis by LC/B and therefore enhanced the sensitivity of the assay. The FRET peptide, designated as FVP-B, was specific to LC/B, with a detection sensitivity as low as ∼20 pM in 2 h. Importantly, FVP-B showed the potential to be scaled up and used in high throughput screening of LC/B inhibitor. The currently developed FRET assay is one of the most economic and rapid FRET assays for LC/B detection.     

Potent New Small-Molecule Inhibitor of Botulinum Neurotoxin Serotype A Endopeptidase Developed by Synthesis-Based Computer-Aided Molecular Design

Botulinum neurotoxin serotype A (BoNTA) causes a life-threatening neuroparalytic disease known as botulism. Current treatment for post exposure of BoNTA uses antibodies that are effective in neutralizing the extracellular toxin to prevent further intoxication but generally cannot rescue already intoxicated neurons. Effective small-molecule inhibitors of BoNTA endopeptidase (BoNTAe) are desirable because such inhibitors potentially can neutralize the intracellular BoNTA and offer complementary treatment for botulism. Previously we reported a serotype-selective, small-molecule BoNTAe inhibitor with a K _i ^app value of 3.8±0.8 µM. This inhibitor was developed by lead identification using virtual screening followed by computer-aided optimization of a lead with an IC₅₀ value of 100 µM. However, it was difficult to further improve the lead from micromolar to even high nanomolar potency due to the unusually large enzyme-substrate interface of BoNTAe. The enzyme-substrate interface area of 4,840 Ų for BoNTAe is about four times larger than the typical protein-protein interface area of 750–1,500 Ų. Inhibitors must carry several functional groups to block the unusually large interface of BoNTAe, and syntheses of such inhibitors are therefore time-consuming and expensive. Herein we report the development of a serotype-selective, small-molecule, and competitive inhibitor of BoNTAe with a K _i value of 760±170 nM using synthesis-based computer-aided molecular design (SBCAMD). This new approach accounts the practicality and efficiency of inhibitor synthesis in addition to binding affinity and selectivity. We also report a three-dimensional model of BoNTAe in complex with the new inhibitor and the dynamics of the complex predicted by multiple molecular dynamics simulations, and discuss further structural optimization to achieve better in vivo efficacy in neutralizing BoNTA than those of our early micromolar leads. This work provides new insight into structural modification of known small-molecule BoNTAe inhibitors. It also demonstrates that SBCAMD is capable of improving potency of an inhibitor lead by nearly one order of magnitude, even for BoNTAe as one of the most challenging protein targets. The results are insightful for developing effective small-molecule inhibitors of protein targets with large active sites.     

Botulinum Neurotoxin Serotype C Associates with Dual Ganglioside Receptors to Facilitate Cell Entry

Botulinum neurotoxins (BoNTs) cleave SNARE proteins in motor neurons that inhibits synaptic vesicle (SV) exocytosis, resulting in flaccid paralysis. There are seven BoNT serotypes (A–G). In current models, BoNTs initially bind gangliosides on resting neurons and upon SV exocytosis associate with the luminal domains of SV-associated proteins as a second receptor. The entry of BoNT/C is less clear. Characterizing the heavy chain receptor binding domain (HCR), BoNT/C was shown to utilize gangliosides as dual host receptors. Crystallographic and biochemical studies showed that the two ganglioside binding sites, termed GBP2 and Sia-1, were independent and utilized unique mechanisms to bind complex gangliosides. The GBP2 binding site recognized gangliosides that contained a sia5 sialic acid, whereas the Sia-1 binding site recognized gangliosides that contained a sia7 sialic acid and sugars within the backbone of the ganglioside. Utilizing gangliosides that uniquely recognized the GBP2 and Sia-1 binding sites, HCR/C entry into Neuro-2A cells required both functional ganglioside binding sites. HCR/C entered cells differently than the HCR of tetanus toxin, which also utilizes dual gangliosides as host receptors. A point-mutated HCR/C that lacked GBP2 binding potential retained the ability to bind and enter Neuro-2A cells. This showed that ganglioside binding at the Sia-1 site was accessible on the plasma membrane, suggesting that SV exocytosis may not be required to expose BoNT/C receptors. These studies highlight the utility of BoNT HCRs as probes to study the role of gangliosides in neurotransmission.     

Botulinum Neurotoxin Serotype a Specific Cell-Based Potency Assay to Replace the Mouse Bioassay

Botulinum neurotoxin serotype A (BoNT/A), a potent therapeutic used to treat various disorders, inhibits vesicular neurotransmitter exocytosis by cleaving SNAP25. Development of cell-based potency assays (CBPAs) to assess the biological function of BoNT/A have been challenging because of its potency. CBPAs can evaluate the key steps of BoNT action: receptor binding, internalization-translocation, and catalytic activity; and therefore could replace the current mouse bioassay. Primary neurons possess appropriate sensitivity to develop potential replacement assays but those potency assays are difficult to perform and validate. This report describes a CBPA utilizing differentiated human neuroblastoma SiMa cells and a sandwich ELISA that measures BoNT/A-dependent intracellular increase of cleaved SNAP25. Assay sensitivity is similar to the mouse bioassay and measures neurotoxin biological activity in bulk drug substance and BOTOX® product (onabotulinumtoxinA). Validation of a version of this CBPA in a Quality Control laboratory has led to FDA, Health Canada, and European Union approval for potency testing of BOTOX®, BOTOX® Cosmetic, and Vistabel®. Moreover, we also developed and optimized a BoNT/A CBPA screening assay that can be used for the discovery of novel BoNT/A inhibitors to treat human disease.     

Cleavage of SNAP25 and Its Shorter Versions by the Protease Domain of Serotype A Botulinum Neurotoxin

Various substrates, catalysts, and assay methods are currently used to screen inhibitors for their effect on the proteolytic activity of botulinum neurotoxin. As a result, significant variation exists in the reported results. Recently, we found that one source of variation was the use of various catalysts, and have therefore evaluated its three forms. In this paper, we characterize three substrates under near uniform reaction conditions using the most active catalytic form of the toxin. Bovine serum albumin at varying optimum concentrations stimulated enzymatic activity with all three substrates. Sodium chloride had a stimulating effect on the full length synaptosomal-associated protein of 25 kDa (SNAP25) and its 66-mer substrates but had an inhibitory effect on the 17-mer substrate. We found that under optimum conditions, full length SNAP25 was a better substrate than its shorter 66-mer or 17-mer forms both in terms of k_cat, K_m, and catalytic efficiency k_cat/K_m. Assay times greater than 15 min introduced large variations and significantly reduced the catalytic efficiency. In addition to characterizing the three substrates, our results identify potential sources of variations in previous published results, and underscore the importance of using well-defined reaction components and assay conditions.     

Structural and Functional Interactions between Transient Receptor Potential Vanilloid Subfamily 1 and Botulinum Neurotoxin Serotype A

Background

Botulinum neurotoxins are produced by Clostridium botulinum bacteria. There are eight serologically distinct botulinum neurotoxin isoforms (serotypes A–H). Currently, botulinum neurotoxin serotype A (BoNT⁄A) is commonly used for the treatment of many disorders, such as hyperactive musculoskeletal disorders, dystonia, and pain. However, the effectiveness of BoNT⁄A for pain alleviation and the mechanisms that mediate the analgesic effects of BoNT⁄A remain unclear. To define the antinociceptive mechanisms by which BoNT/A functions, the interactions between BoNT⁄A and the transient receptor potential vanilloid subfamily 1 (TRPV1) were investigated using immunofluorescence, co-immunoprecipitation, and western blot analysis in primary mouse embryonic dorsal root ganglion neuronal cultures.

Results

1) Three-week-old cultured dorsal root ganglion neurons highly expressed transient TRPV1, synaptic vesicle 2A (SV2A) and synaptosomal-associated protein 25 (SNAP-25). SV2A and SNAP-25 are the binding receptor and target protein, respectively, of BoNT⁄A. 2) TRPV1 colocalized with both BoNT⁄A and cleaved SNAP-25 when BoNT⁄A was added to dorsal root ganglia neuronal cultures. 3) After 24 hours of BoNT⁄A treatment (1 nmol⁄l), both TRPV1 and BoNT⁄A positive bands were detected in western blots of immunoprecipitated pellets. 4) Blocking TRPV1 with a specific antibody decreased the cleavage of SNAP-25 by BoNT⁄A.

Conclusion

BoNT/A interacts with TRPV1 both structurally and functionally in cultured mouse embryonic dorsal root ganglion neurons. These results suggest that an alternative mechanism is used by BoNT⁄A to mediate pain relief.     

B型肉毒毒素保护性抗原Hc在大肠杆菌中的可溶性高表达

目的：通过序列优化及表达条件改进,在大肠杆菌中高效可溶性表达B型肉毒毒素保护性抗原He（Bont／B-He）。方法：对Bont／B-He基因片段优化,用大肠杆菌常用密码子替换稀有密码子,并将（C＋C）含量由76．2％降至56-3％;人工合成多条具有重叠互补序列的寡核苷酸片段,采用重叠延伸PCR方法获得了Bont／B-He的全长基因,并构建了原核可溶性表达载体;将经酶切和测序鉴定正确的重组质粒转化大肠杆菌B121（DE3）感受态细胞,用IPTC诱导Bont／B-He的表达并进行纯化及Western印迹鉴定。结果和结论：目的蛋白在大肠杆菌B121（DE3）中获得了可溶性高表达,占菌体裂解液上清总蛋白的26．7％,表达量达到30mg／L,是目前国内外已知表达的最高水平;经Ni柱一步纯化后,目的蛋白纯度可达到80.3％,为肉毒毒素中和抗体的制备及亚单位疫苗的研究奠定了基础。     

Botulinum Neurotoxin Type A is Internalized and Translocated from Small Synaptic Vesicles at the Neuromuscular Junction

Botulinum neurotoxin type A (BoNT/A) is the most frequent cause of human botulism and, at the same time, is largely used in human therapy. Some evidence indicates that it enters inside nerve terminals via endocytosis of synaptic vesicles, though this has not been directly proven. The metalloprotease L chain of the neurotoxin then reaches the cytosol in a process driven by low pH, but the acidic compartment wherefrom it translocates has not been identified. Using immunoelectron microscope, we show that BoNT/A does indeed enter inside synaptic vesicles and that each vesicle contains either one or two toxin molecules. This finding indicates that it is the BoNT/A protein receptor synaptic vesicle protein 2, and not its polysialoganglioside receptor that determines the number of toxin molecules taken up by a single vesicle. In addition, by rapid quenching the vesicle trans-membrane pH gradient, we show that the neurotoxin translocation into the cytosol is a fast process. Taken together, these results strongly indicate that translocation of BoNT/A takes place from synaptic vesicles, and not from endosomal compartments, and that the translocation machinery is operated by no more than two neurotoxin molecules.     

A Photoactivatable Botulinum Neurotoxin for Inducible Control of Neurotransmission

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Intranasal Delivery of Botulinum Neurotoxin A Protects against Hippocampal Neuron Death in the Lithium-Pilocarpine Rat Model

Neurochemical Research - Botulinum neurotoxins (BoNTs) block the release of a series of neurotransmitters, which are pivotal for neuron action. Intrahippocampal administration of BoNTs inhibits...     

Factors Affecting Autocatalysis of Botulinum A Neurotoxin Light Chain*

The light chain of botulinum neurotoxin serotype A undergoes autocatalytic fragmentation into two major peptides during purification and storage (Ahmed S. A. et al. 2001, J. Protein Chem. 20:221–231) by both intermolecular and intramolecular mechanisms (Ahmed S. A. etal. 2003, Biochemistry 42:12539–12549). In this study, we investigated the effects of buffers and salts on this autocatalytic reaction in the presence and absence of zinc chloride. In the presence of zinc chloride, the fragmentation reaction was enhanced in each of acetate, MES, HEPES and phosphate buffers with maximum occurring in acetate when compared to those in the absence of zinc chloride. Adding sodium chloride in phosphate buffer in the presence of zinc chloride increased the extent of proteolysis. Irrespective of the presence of zinc chloride, adding sodium chloride or potassium chloride in phosphate buffer elicited an additional proteolytic reaction. Higher concentrations of sodium phosphate buffer enhanced the autocatalytic reaction in the absence of zinc chloride. In contrast, in the presence of zinc chloride, higher concentrations of sodium phosphate decreased the autocatalytic reaction. Optimum pH of autocatalysis was not affected significantly by the absence or presence of zinc chloride. Like zinc chloride, other chlorides of divalent metals, such as magnesium, cobalt, iron and calcium also enhanced the autocatalytic reaction. Polyols such as ethylene glycol protected the light chain from fragmentation. Exposure of light chain to UV radiation led to enhanced fragmentation. In order to avoid fragmentation, the protein should be stored frozen in a low concentration buffer of neutral or higher pH devoid of any metal. Our results provide a choice of buffers and salts for isolation, purification and storage of intact botulinum neurotoxin serotype A light chain.     

A型肉毒毒素抑制剂的分子全息定量构效关系研究

目的:建立A型肉毒毒素抑制剂的定量构效关系模型。方法:应用分子全息定量构效关系(HQSAR)技术,研究了14种A型肉毒毒素抑制剂的抑制活性与其二维分子结构之间的关系,讨论了碎片区分参数及碎片长度对模型质量的影响。结果:最佳全息条件下产生的模型相关系数r2为0.780,交叉验证相关系数q2LOO为0.583。所建模型具有良好的拟和效果和较高的预测能力,HQSAR模型贡献图显示抑制剂分子中的噻吩环及羟胺对活性有较大贡献。结论:本研究对新抑制剂的设计具有一定的指导作用。     

重组肉毒神经毒素A轻链(BoNT/A LC)突变体的构建、表达与活性分析

A型肉毒神经毒素的轻链(BoNT/A LC)是一种锌依赖性的金属内肽酶.通过X射线分析其结构并结合一些文献报道表明,轻链上的Arg362和Tyr365直接参与了酶的催化作用,而Glu350则处于其活性位点的中心位置.采用定点突变技术,对编码这3个关键性的氨基酸位点的碱基进行突变(Arg362Ala、Tyr365Phe、Glu350Ala),获得了BoNT/A LC突变体.突变体蛋白与BoNT/A的底物蛋白SNAP-25进行切割反应,结果表明,未经突变的BoNT/A轻链蛋白能够特异性地识别SNAP-25蛋白上的Q197-R198位点,而突变体蛋白则完全无法识别该位点,不具有金属内肽酶活性,成功地去除了肉毒神经毒素的毒力,为下一步的全长肉毒神经毒素重组疫苗的研究打下了基础.