Two for the Price of One: Heterobivalent Ligand Design Targeting Two Binding Sites on Voltage-Gated Sodium Channels Slows Ligand Dissociation and Enhances Potency.

Voltage-gated sodium (NaV) channels are pore-forming transmembrane proteins that play essential roles in excitable cells, and they are key targets for antiepileptic, antiarrhythmic, and analgesic drugs. We implemented a heterobivalent design strategy to modulate the potency, selectivity, and binding kinetics of NaV channel ligands. We conjugated µ-conotoxin KIIIA, which occludes the pore of the NaV channels, to an analogue of huwentoxin-IV, a spider-venom peptide that allosterically modulates channel gating. Bioorthogonal hydrazide and copper-assisted azide-alkyne cycloaddition conjugation chemistries were employed to generate heterobivalent ligands using polyethylene glycol linkers spanning 40-120 Å. The ligand with an 80 Å linker had the most pronounced bivalent effects, with a significantly slower dissociation rate and 4-24-fold higher potency compared to those of the monovalent peptides for the human NaV1.4 channel. This study highlights the power of heterobivalent ligand design and expands the repertoire of pharmacological probes for exploring the function of NaV channels.

Low cost venom extractor based on Arduino(®) board for electrical venom extraction from arthropods and other small animals.

Extracting venom from small species is usually challenging. We describe here an affordable and versatile electrical venom extractor based on the Arduino(®) Mega 2560 Board, which is designed to extract venom from arthropods and other small animals. The device includes fine tuning of stimulation time and voltage. It was used to collect venom without apparent deleterious effects, and characterized for the first time the venom of Zoropsis spinimana, a common spider in French Mediterranean regions.

SVM-based prediction of propeptide cleavage sites in spider toxins identifies toxin innovation in an Australian tarantula.

Spider neurotoxins are commonly used as pharmacological tools and are a popular source of novel compounds with therapeutic and agrochemical potential. Since venom peptides are inherently toxic, the host spider must employ strategies to avoid adverse effects prior to venom use. It is partly for this reason that most spider toxins encode a protective proregion that upon enzymatic cleavage is excised from the mature peptide. In order to identify the mature toxin sequence directly from toxin transcripts, without resorting to protein sequencing, the propeptide cleavage site in the toxin precursor must be predicted bioinformatically. We evaluated different machine learning strategies (support vector machines, hidden Markov model and decision tree) and developed an algorithm (SpiderP) for prediction of propeptide cleavage sites in spider toxins. Our strategy uses a support vector machine (SVM) framework that combines both local and global sequence information. Our method is superior or comparable to current tools for prediction of propeptide sequences in spider toxins. Evaluation of the SVM method on an independent test set of known toxin sequences yielded 96% sensitivity and 100% specificity. Furthermore, we sequenced five novel peptides (not used to train the final predictor) from the venom of the Australian tarantula Selenotypus plumipes to test the accuracy of the predictor and found 80% sensitivity and 99.6% 8-mer specificity. Finally, we used the predictor together with homology information to predict and characterize seven groups of novel toxins from the deeply sequenced venom gland transcriptome of S. plumipes, which revealed structural complexity and innovations in the evolution of the toxins. The precursor prediction tool (SpiderP) is freely available on ArachnoServer (http://www.arachnoserver.org/spiderP.html), a web portal to a comprehensive relational database of spider toxins. All training data, test data, and scripts used are available from the SpiderP website.

Spatial structure and activity mechanism of a novel spider antimicrobial peptide.

Latarcins (Ltc), linear peptides (ca. 25 amino acid long) isolated from the venom of the Lachesana tarabaevi spider, exhibit a broad-spectrum antibacterial activity, most likely acting on the bacterial plasmatic membrane. We study the structure-activity relationships in the series of these compounds. At the first stage, we investigated the spatial structure of one of the peptides, Ltc2a, and its mode of membrane perturbation. This was done by a combination of experimental and theoretical methods. The approach includes (i) structural study of the peptide by CD spectroscopy in phospholipid liposomes and by (1)H NMR in detergent micelles, (ii) determination of the effect on the liposomes by a dye leakage fluorescent assay and (31)P NMR spectroscopy, (iii) refinement of the NMR-derived spatial structure via Monte Carlo simulations in an implicit water-octanol slab, and (iv) calculation of the molecular hydrophobicity potential. The molecule of Ltc2a was found to consist of two helical regions (residues 3-9 and 13-21) connected via a poorly ordered fragment. The effect of the peptide on the liposomes suggests the carpet mechanism of the membrane deterioration. This is also supported by the analysis of hydrophobic/hydrophilic characteristics of Ltc2a and homologous antimicrobial peptides. These peptides exhibiting a helix-hinge-helix structural motif are characterized by a distinct and feebly marked amphiphilicity of their N- and C-terminal helices, respectively, and by a hydrophobicity gradient along the peptide chain. The approach we suggested may be useful in studying not only other latarcins but also a wider class of membrane-active peptides.

Purification, cDNA cloning and function assessment of BmK abT, a unique component from the Old World scorpion species.

A new neurotoxic component named BmK abT was purified from the venom of Chinese scorpion Buthus martensi Karsch. The molecular weight of BmK abT was determined to be 7212 Da on a mass spectrum. The minimum lethal dose of BmK abT was tested to be about 1.5 microg per mouse by intracerebroventricular injection, and the dose induced significant paralysis effect on cockroach was about 5 microg by i.p. injection. The partial amino acid sequence indicated that it was a distinctive polypeptide in the scorpion neurotoxin family. Thereafter, the whole amino acid sequence of mature BmK abT was deduced from cDNA sequence by 5'- and 3'-rapid amplification of cDNA ends. Finally, it was defined to be composed of 63 residues with amidation at the C-terminal residue. By sequence comparison, BmK abT was found to be most similar to Ts VII, a beta-toxin from the New World scorpion. The patch-clamp recording on DRG neurons, unexpectedly, showed this toxin could prolong the action potential and increase the amplitude of the peak Na+ currents, which are the typical characters of alpha-toxin. These results suggested that BmK abT was a new toxic component found in the Old World scorpion species structurally similar to beta-toxins, but functionally similar to alpha-toxins.

Arthropod toxins as leads for novel insecticides: an assessment of polyamine amides as glutamate antagonists.

In the search for new toxins, preferably with new sites of action, the polyamine amides represent a new class of compounds with potential as insecticides and as pharmaceutical agents due to their antagonism of ligand-gated cation channels. In particular, they are potent antagonists of the L-glutamate receptors of insect skeletal muscle. In this paper, we report on synthetic studies to produce hybrid analogues based upon the argiotoxin spider toxins and philanthotoxin-433 which is obtained from a solitary, parasitic wasp. We speculate upon possible modes and sites of action for these antagonists and we discuss their potential as insecticides and in the possible treatment of ischaemic damage. The synthesis and characterization of 4-hydroxyphenylpropanoylspermine is reported and the locust muscle biological assay is described. Using this pharmacological screen, structure-activity relationships have been determined in our laboratories. These are reviewed in the light of the current literature. Voltage clamp studies of the synthetic analogue philanthotoxin-343 and the effects of this polyamine amide on glutamate receptors expressed in Xenopus oocytes are outlined. In conclusion, a description of our current ideas and understanding of the many sites and modes of action of the polyamine amides, based both upon our own studies and also upon those recently reported, is presented.