Spider Venom Peptide Research Articles

Using a conditioned place preference assay in fruit flies to examine effects of insecticidal compounds on contextual memory

Insecticides are vital for safeguarding agricultural crops against pests, albeit many lack selectivity towards pest species and are poorly bio-degradable. This leads to targeting of beneficial organisms like pollinators and widespread environmental contamination of soil and water. Exposure to insecticides such as neonicotinoids causes insect paralysis and mortality at higher doses, while sublethal doses can disrupt other functions that are crucial for survival such as learning and memory performance. Potent and selective arachnid venom peptides affecting a variety of molecular targets are being explored as bioinsecticide candidates. However, their effect on insect learning is poorly understood. We therefore established a sucrose-induced conditioned place preference (CPP) assay using Drosophila melanogaster fruit flies to provide a means of evaluating how various classes of insecticidal compounds interact with insect memory to assess their broader ecological consequences. Our results confirmed the adverse effect of a sublethal dose of the neonicotinoid insecticide imidacloprid (20 pg/fly) on fly CPP formation upon daily injection during the conditioning phase. However, imidacloprid did not affect CPP retrieval when applied after the conditioning phase. Sublethal doses of the two insecticidal spider venom peptides μ-DGTX-Dc1a (Dc1a; 70 pg/fly) and U1-AGTX-Ta1a (Ta1a; 125 pg/fly) had no effect on either CPP formation or retrieval, underlining their potential as novel and safe bioinsecticide candidates.

A novel spider venom peptide from the predatory mite Neoseiulus barkeri shows lethal effect on phytophagous pests

The long-term use of pesticides in the field, and the high fertility and adaptability of phytophagous mites have led to resistance problems; consequently, novel safe and efficient active substances are necessary to broaden the tools of pest mite control. Natural enemies of arthropods typically secrete substances with paralytic or lethal effects on their prey, and those substances are a resource for future biopesticides. In this study, two putative venom peptide genes were identified in a parasitic mite Neoseiulus barkeri transcriptome. Recombinant venom NbSP2 peptide injected into Tetranychus cinnabarinus mites was significantly more lethal than recombinant NBSP1. NbSP2 was also lethal to Spodoptera litura when injected but not when fed to third instar larvae. The interaction proteins of NbSP2 in T. cinnabarinus and S. litura were identified by affinity chromatography. Among these proteins, ATP synthase subunit β (ATP SSβ) was deduced as a potential target. Four binding sites were predicted between NBSP2 and ATP SSβ of T. cinnabarinus and S. litura. In conclusion, we identified a venom peptide with activity against T. cinnabarinus and S. litura. This study provides a novel component for development of a new biological pesticide.

Genetically encoded libraries and spider venoms as emerging sources for crop protective peptides.

Agricultural crops are targeted by various pathogens (fungi, bacteria, and viruses) and pests (herbivorous arthropods). Antimicrobial and insecticidal peptides are increasingly recognized as eco-friendly tools for crop protection due to their low propensity for resistance development and the fact that they are fully biodegradable. However, historical challenges have hindered their development, including poor stability, limited availability, reproducibility issues, high production costs, and unwanted toxicity. Toxicity is a primary concern because crop-protective peptides interact with various organisms of environmental and economic significance. This review focuses on the potential of genetically encoded peptide libraries like the use of two-hybrid-based methods for antimicrobial peptides identification and insecticidal spider venom peptides as two main approaches for targeting plant pathogens and pests. We discuss some key findings and challenges regarding the practical application of each strategy. We conclude that genetically encoded peptide library- and spider venom-derived crop protective peptides offer a sustainable and environmentally responsible approach for addressing modern crop protection needs in the agricultural sector.

The funnel-web spider venom derived single knot peptide Hc3a modulates acid-sensing ion channel 1a desensitisation

Acid-sensing ion channel 1a (ASIC1a) is a proton-gated channel involved in synaptic transmission, pain signalling, and several ischemia-associated pathological conditions. The spider venom-derived peptides PcTx1 and Hi1a are two of the most potent ASIC1a inhibitors known and have been instrumental in furthering our understanding of the structure, function, and biological roles of ASICs. To date, homologous spider peptides with different pharmacological profiles at ASIC1a have yet to be discovered. Here we report the characterisation of Hc3a, a single inhibitor cystine knot peptide from the Australian funnel-web spider Hadronyche cerberea with sequence similarity to PcTx1. We show that Hc3a has complex pharmacology and binds different ASIC1a conformational states (closed, open, and desensitised) with different affinities, with the most prominent effect on desensitisation. Hc3a slows the desensitisation kinetics of proton-activated ASIC1a currents across multiple application pHs, and when bound directly to ASIC1a in the desensitised conformation promotes current inhibition. The solution structure of Hc3a was solved, and the peptide-channel interaction examined via mutagenesis studies to highlight how small differences in sequence between Hc3a and PcTx1 can lead to peptides with distinct pharmacology. The discovery of Hc3a expands the pharmacological diversity of spider venom peptides targeting ASIC1a and adds to the toolbox of compounds to study the intricacies of ASIC1 gating.

Identification of Peptides in Spider Venom Using Mass Spectrometry.

Spider venoms are composed of hundreds of proteins and peptides. Several of these venom toxins are cysteine-rich peptides in the mass range of 3-9kDa. Small peptides (<3kDa) can be fully characterized by mass spectrometry analysis, while proteins are generally identified by the bottom-up approach in which proteins are first digested with trypsin to generate shorter peptides for MS/MS characterization. In general, it is sufficient for protein identification to sequence two or more peptides, but for venom peptidomics it is desirable to completely elucidate peptide sequences and the number of disulfide bonds in the molecules. In this chapter, we describe a methodology to completely sequence and determine the number of disulfide bonds of spider venom peptides in the mass range of 3-9kDa by multiple enzyme digestion, mass spectrometry of native and digested peptides, de novo analysis, and sequence overlap alignment.

Spider-Venom Peptides: Structure, Bioactivity, Strategy, and Research Applications.

Spiders (Araneae), having thrived for over 300 million years, exhibit remarkable diversity, with 47,000 described species and an estimated 150,000 species in existence. Evolving with intricate venom, spiders are nature's skilled predators. While only a small fraction of spiders pose a threat to humans, their venoms contain complex compounds, holding promise as drug leads. Spider venoms primarily serve to immobilize prey, achieved through neurotoxins targeting ion channels. Peptides constitute a major part of these venoms, displaying diverse pharmacological activities, and making them appealing for drug development. Moreover, spider-venom peptides have emerged as valuable tools for exploring human disease mechanisms. This review focuses on the roles of spider-venom peptides in spider survival strategies and their dual significance as pharmaceutical research tools. By integrating recent discoveries, it provides a comprehensive overview of these peptides, their targets, bioactivities, and their relevance in spider survival and medical research.

Examination of the mechanism of Piezo ion channel in 5-HT synthesis in the enterochromaffin cell and its association with gut motility.

In the gastrointestinal tract, serotonin (5-hydroxytryptamine, 5-HT) is an important monoamine that regulates intestinal dynamics. QGP-1 cells are human-derived enterochromaffin cells that secrete 5-HT and functionally express Piezo ion channels associated with cellular mechanosensation. Piezo ion channels can be blocked by Grammostola spatulata mechanotoxin 4 (GsMTx4), a spider venom peptide that inhibits cationic mechanosensitive channels. The primary aim of this study was to explore the effects of GsMTx4 on 5-HT secretion in QGP-1 cells in vitro. We investigated the transcript and protein levels of the Piezo1/2 ion channel, tryptophan hydroxylase 1 (TPH1), and mitogen-activated protein kinase signaling pathways. In addition, we observed that GsMTx4 affected mouse intestinal motility in vivo. Furthermore, GsMTx4 blocked the response of QGP-1 cells to ultrasound, a mechanical stimulus.The prolonged presence of GsMTx4 increased the 5-HT levels in the QGP-1 cell culture system, whereas Piezo1/2 expression decreased, and TPH1 expression increased. This effect was accompanied by the increased phosphorylation of the p38 protein. GsMTx4 increased the entire intestinal passage time of carmine without altering intestinal inflammation. Taken together, inhibition of Piezo1/2 can mediate an increase in 5-HT, which is associated with TPH1, a key enzyme for 5-HT synthesis. It is also accompanied by the activation of the p38 signaling pathway. Inhibitors of Piezo1/2 can modulate 5-HT secretion and influence intestinal motility.

Two Novel Mosquitocidal Peptides Isolated from the Venom of the Bahia Scarlet Tarantula (Lasiodora klugi).

Effective control of diseases transmitted by Aedes aegypti is primarily achieved through vector control by chemical insecticides. However, the emergence of insecticide resistance in A. aegypti undermines current control efforts. Arachnid venoms are rich in toxins with activity against dipteran insects and we therefore employed a panel of 41 spider and 9 scorpion venoms to screen for mosquitocidal toxins. Using an assay-guided fractionation approach, we isolated two peptides from the venom of the tarantula Lasiodora klugi with activity against adult A. aegypti. The isolated peptides were named U-TRTX-Lk1a and U-TRTX-Lk2a and comprised 41 and 49 residues with monoisotopic masses of 4687.02 Da and 5718.88 Da, respectively. U-TRTX-Lk1a exhibited an LD50 of 38.3 pmol/g when injected into A. aegypti and its modeled structure conformed to the inhibitor cystine knot motif. U-TRTX-Lk2a has an LD50 of 45.4 pmol/g against adult A. aegypti and its predicted structure conforms to the disulfide-directed β-hairpin motif. These spider-venom peptides represent potential leads for the development of novel control agents for A. aegypti.

Screening of Spider Venom Peptides and Molecular Docking of FLT3 and LCK

The drawbacks of traditional chemotherapy include its inability to dissolve in water, lack of selectivity, and multidrug resistance. The use of anticancer peptides is a unique therapeutic approach against cancer cells. In this In-silico work, the kinase inhibition activity for both chosen target molecules (Flt3 and Lck protein) was evaluated in order to find a possible anti-leukemic spider venom peptide. Out of the 11 spider venom peptides, Lycosin-I peptide (from Lycosa singoriensis) for Lck and Latarcin 2a peptide (from Lachesana tarabaevi) for Flt3 were suggested as the best lead peptides for the creation of anti-leukemic drugs.

Evaluation of Peptide Ligation Strategies for the Synthesis of the Bivalent Acid-Sensing Ion Channel Inhibitor Hi1a.

Hi1a is a naturally occurring bivalent spider-venom peptide that is being investigated as a promising molecule for limiting ischemic damage in strokes, myocardial infarction, and organ transplantation. However, the challenges associated with the synthesis and production of the peptide in large quantities have slowed the progress in this area; hence, access to synthetic Hi1a is an essential milestone for the development of Hi1a as a pharmacological tool and potential therapeutic.

Towards a Sustainable Management of the Spotted-Wing Drosophila: Disclosing the Effects of Two Spider Venom Peptides on Drosophila suzukii.

The spotted-wing drosophila (Drosophila suzukii) is a polyphagous pest that causes severe damage and economic losses to soft-skinned fruit production. Current control methods are dominated by inefficient cultural practices and broad-spectrum insecticides that, in addition to having toxic effects on non-target organisms, are becoming less effective due to acquired resistance. The increasing awareness of the real impact of insecticides on health and the environment has promoted the exploration of new insecticidal compounds, addressing novel molecular targets. This study explores the efficacy of two orally delivered spider venom peptides (SVPs), J-atracotoxin-Hv1c (Hv1c) and µ-theraphotoxin-Hhn2b (TRTX), to manage D. suzukii, through survival assays and the evaluation of gene expression associated with detoxification pathways. Treatment with TRTX at 111.5 µM for 48 h enhanced fly longevity compared with the control group. Gene expression analysis suggests that detoxification and stress-related mechanisms, such as expression of P450 proteins and apoptotic stimuli signaling, are triggered in D. suzukii flies in response to these treatments. Our results highlight the potential interest of SVPs to control this pest, shedding light on how to ultimately develop improved target-specific formulations.

Ion Channels-related Neuroprotection and Analgesia Mediated by Spider Venom Peptides.

Ion channels play critical roles in generating and propagating action potentials and in neurotransmitter release at a subset of excitatory and inhibitory synapses. Dysfunction of these channels has been linked to various health conditions, such as neurodegenerative diseases and chronic pain. Neurodegeneration is one of the underlying causes of a range of neurological pathologies, such as Alzheimer's disease (AD), Parkinson's disease (PD), cerebral ischemia, brain injury, and retinal ischemia. Pain is a symptom that can serve as an index of the severity and activity of a disease condition, a prognostic indicator, and a criterion of treatment efficacy. Neurological disorders and pain are conditions that undeniably impact a patient's survival, health, and quality of life, with possible financial consequences. Venoms are the best-known natural source of ion channel modulators. Venom peptides are increasingly recognized as potential therapeutic tools due to their high selectivity and potency gained through millions of years of evolutionary selection pressure. Spiders have been evolving complex and diverse repertoires of peptides in their venoms with vast pharmacological activities for more than 300 million years. These include peptides that potently and selectively modulate a range of targets, such as enzymes, receptors, and ion channels. Thus, components of spider venoms hold considerable capacity as drug candidates for alleviating or reducing neurodegeneration and pain. This review aims to summarize what is known about spider toxins acting upon ion channels, providing neuroprotective and analgesic effects.

Emerging cures for cancer: peptides from scorpion and spider venom

Animal toxins have shown applicability in treatments of various diseases, here some investigations of scorpion and spider venom peptides as cancer treatments have been presented. Scorpion peptides are believed to have antitumor and analgesic effects and may present the potential to be applied in human medicine as a drug for cancer. Similarly, some spider peptides either directly or indirectly are also proved to regulate tumour cell growth and death. Mechanism involved includes promoting cell apoptosis to prevent translocation of cancer cells thus control tumour growth. Such effects make these peptides promising drug candidates for cancer treatment. All five of scorpion venom peptide drugs being approved by FDA for clinical treatment, from which remarkable treating outcomes have been observed when treating cancers. In this paper, general aspects of different scorpion venoms as well as their anticancer mechanism have been thoroughly analysed, from which the successful application of Buthus martensii Karsch analgesic peptide in treating carcinoma was elected as the representative case.

Enhancing the oral and topical insecticidal efficacy of a commercialized spider venom peptide biopesticide via fusion to the carrier snowdrop lectin (Galanthus nivalis agglutinin).

Spear®-T sold as a contact foliar spray for the control of glasshouse pests such as aphids, thrips, spider mites and whiteflies, contains the recombinant spider venom peptide GS-ω/κ-HxTx-Hv1h (named as GS-ω/κ-HxTx-Hv1a by Vestaron) as the active ingredient. Here we investigate whether fusion of the peptide to snowdrop lectin, (Galanthus nivalis agglutinin; GNA) enhances the efficacy of this venom peptide towards aphid pests. Recombinant GS-ω/κ-HxTx-Hv1h (HxTx-Hv1h) and an HxTx-Hv1h/GNA fusion protein were produced using the yeast Pichia pastoris. Purified proteins showed comparable toxicity when injected into lepidopteran (Mamestra brassicae) larvae, but significant differences in oral and contact activity towards aphids. HxTx-Hv1h had comparable acute oral toxicity to pea (Acyrthosiphon pisum) and peach potato (Myzus persicae) aphids with respective Day (2) median lethal concentration (LC50 ) values of 111 and 108 μm derived from diet assays. The fusion protein also showed comparable oral toxicity to both species but D2 LC50 values were >3-fold lower (35 and 33 μm for pea and peach potato aphids, respectively) as compared to HxTx-Hv1h. Topically applied toxin and fusion protein, but not GNA, caused significant reductions in pea aphid survival. Contact effects on mortality were significantly greater for aphids exposed to fusion protein as compared to toxin alone. Whole aphid fluorescence microscopy and immunoblotting suggest that improved efficacy is due to enhanced persistence of HxTx-Hv1h when fused to GNA following internalisation of ingested or topically applied proteins. This is the first study to report on the insecticidal activity of HxTx-Hv1h towards aphids and results suggest that a fusion protein-based approach offers opportunities to significantly enhance oral and contact efficacy of naturally derived toxins, such as HxTx-Hv1h, towards crop pests. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

µ-Theraphotoxin Pn3a inhibition of CaV3.3 channels reveals a novel isoform-selective drug binding site.

Low voltage-activated calcium currents are mediated by T-type calcium channels CaV3.1, CaV3.2, and CaV3.3, which modulate a variety of physiological processes including sleep, cardiac pace-making, pain, and epilepsy. CaV3 isoforms' biophysical properties, overlapping expression, and lack of subtype-selective pharmacology hinder the determination of their specific physiological roles in health and disease. We have identified μ-theraphotoxin Pn3a as the first subtype-selective spider venom peptide inhibitor of CaV3.3, with >100-fold lower potency against the other T-type isoforms. Pn3a modifies CaV3.3 gating through a depolarizing shift in the voltage dependence of activation thus decreasing CaV3.3-mediated currents in the normal range of activation potentials. Paddle chimeras of KV1.7 channels bearing voltage sensor sequences from all four CaV3.3 domains revealed preferential binding of Pn3a to the S3-S4 region of domain II (CaV3.3DII). This novel T-type channel pharmacological site was explored through computational docking simulations of Pn3a, site-directed mutagenesis, and full domain II swaps between CaV3 channels highlighting it as a subtype-specific pharmacophore. This research expands our understanding of T-type calcium channel pharmacology and supports the suitability of Pn3a as a molecular tool in the study of the physiological roles of CaV3.3 channels.

Preparation and performance characteristics of spider venom peptide nanocapsules

ω-hexatoxin-Hvn1b is an insecticidal toxin produced by the Tasmanian funnel-web spider (Hadronyche venenata), that can be exploited for development of novel bioinsecticides. Due to its larger size and low membrane permeability, this toxin usually has a slower mode of action compared to conventional small molecule insecticides. Nanoscale materials have unique optical, electrical, mechanical and biological properties, and show great application prospects for pesticide delivery. The physical and chemical properties of nanocapsules were characterized using transmission electron microscopy, laser particle size analysis, Fourier transform infrared spectroscopy, contact angle testing and with a fluorescence spectrophotometer. The results indicated that the nanocapsules were spherical, with an average particle size of 197.70 nm, the encapsulation efficiency rate was 75.82% and the Zeta potential was -32.90 mV. Penetration experiments showed that the nanocapsules could promote protein passage through the intestinal tract of Spodoptera litura and reach the body fluid. Then we expressed ω-hexatoxin-Hvn1b by prokaryotic expression. Bioassay results showed that the oral toxicity of ω-hexatoxin-Hvn1b nanocapsules to S.litura was higher than that of the ω-hexatoxin-Hvn1b. In this paper, we reported a construction method of spider venom peptide nanocapsules based on polylactic-co-glycolic acid by multiple emulsion for delivery of protein to improve the insecticidal effect and oral activity of ω-hexatoxin-Hv1a. © 2022 Society of Chemical Industry.

Viral vector-mediated expressions of venom peptides as novel gene therapy for anxiety and depression

The venoms of arachnids, cone snails, and snakes consist of many peptides that have various physiological activities, e.g., modulation of ion channels. We have recently prepared lentiviral vectors that express spider venom peptides and observed their successful expression and secretion by the transduced cells themselves. We expect that this expression method can be applied to mental disorders as a new type of gene therapy. Since most psychiatric symptoms are caused by dysregulations of neuronal activities and/or synaptic transmission, a single injection of a viral vector that expresses venom peptides would modulate ion channels in the brain and thereby compensate for the dysregulations, resulting in long-lasting therapeutic potential. Here, we discuss the details of the application methods and which venom peptide would exert therapeutic effect for anxiety and depression and how.

Therapeutic potential of viral vectors that express venom peptides for neurological diseases

The venoms of arachnids, cone snails, and snakes consist of many peptides that have varieties of physiological activities, e.g., modulation of ion channels. We have recently prepared lentiviral vectors that express spider venom peptides and successfully observed their expression and secretion by the transduced cells. We expect that this expression method can be applied to neurological diseases. Because most neurological and psychiatric symptoms are caused by the dysregulations of neuronal activities and/or synaptic transmission, a single injection of a viral vector that expresses venom peptides would compensate for the dysregulations and thereby exert a therapeutic effect. Here, we discuss the details of application methods, and how and what venom peptide would exert therapeutic potential for a given disorder, e.g., chronic pain, epilepsy, and Parkinson’s and Huntington’s diseases. We also discuss the potential obstacles, i.e., lack of genetic information, side effects, and immune response, and methods to circumvent them.

Engineering of a Spider Peptide via Conserved Structure-Function Traits Optimizes Sodium Channel Inhibition In Vitro and Anti-Nociception In Vivo.

Venom peptides are potent and selective modulators of voltage-gated ion channels that regulate neuronal function both in health and in disease. We previously identified the spider venom peptide Tap1a from the Venezuelan tarantula Theraphosa apophysis that targeted multiple voltage-gated sodium and calcium channels in visceral pain pathways and inhibited visceral mechano-sensing neurons contributing to irritable bowel syndrome. In this work, alanine scanning and domain activity analysis revealed Tap1a inhibited sodium channels by binding with nanomolar affinity to the voltage-sensor domain II utilising conserved structure-function features characteristic of spider peptides belonging to family NaSpTx1. In order to speed up the development of optimized NaV-targeting peptides with greater inhibitory potency and enhanced in vivo activity, we tested the hypothesis that incorporating residues identified from other optimized NaSpTx1 peptides into Tap1a could also optimize its potency for NaVs. Applying this approach, we designed the peptides Tap1a-OPT1 and Tap1a-OPT2 exhibiting significant increased potency for NaV1.1, NaV1.2, NaV1.3, NaV1.6 and NaV1.7 involved in several neurological disorders including acute and chronic pain, motor neuron disease and epilepsy. Tap1a-OPT1 showed increased potency for the off-target NaV1.4, while this off-target activity was absent in Tap1a-OPT2. This enhanced potency arose through a slowed off-rate mechanism. Optimized inhibition of NaV channels observed in vitro translated in vivo, with reversal of nocifensive behaviours in a murine model of NaV-mediated pain also enhanced by Tap1a-OPT. Molecular docking studies suggested that improved interactions within loops 3 and 4, and C-terminal of Tap1a-OPT and the NaV channel voltage-sensor domain II were the main drivers of potency optimization. Overall, the rationally designed peptide Tap1a-OPT displayed new and refined structure-function features which are likely the major contributors to its enhanced bioactive properties observed in vivo. This work contributes to the rapid engineering and optimization of potent spider peptides multi-targeting NaV channels, and the research into novel drugs to treat neurological diseases.

Can a Veterinary Drug be Repurposed for Human Cancers?: Cytotoxic Effect of Tarantula cubensis Venom on Human Cancer Cells

Tarantula cubensis is known as Cuban tarantula having a venom that contains a diverse mixture of potent compounds with various biological activities. These peptides have been shown to have antitumor activities, therefore features of spider-venom peptides prompted scientists to test them as a potential anticancer drug. The purpose of the study was to investigate the potential cytotoxic effect of Tarantula cubensis venom (Logoplex®) on human cancer cells including prostate (PC-3), lung (H69), breast (MDA-MB-231), and ovarian (OVCAR-3). Moreover, non-tumorigenic MCF-10A cells were used to evaluate the possible cancer cell-specific effect of the extract. The increasing concentrations of Logoplex® were applied for 24, 48 and 72 h. MTT assay was used to assess cell viability. Concentration-response curves and the IC50 values were determined via Graphpad Prism software. Logoplex® caused a time- and concentration-dependent cytotoxic effect in MDA-MB-231, PC-3, OVCAR-3 and MCF-10A cells and the highest cytotoxicity was achieved at 72h. However, in H69 cells, there was a concentration-dependent cytotoxic effect and the highest cytotoxicity was achieved at 24h. IC50 values of Logoplex® in MDA-MB-231, OVCAR-3, PC-3, H69 and MCF-10A cells were determined as 159.3±2.1, 48.9±1.8, 40.2±1.2, 498.3±1.2 and 217.8±2.0 µg/mL, respectively. Logoplex® showed a lower cytotoxic effect against normal cells than the cancer cells suggesting a cancer cell-specific effect. According to the preliminary results of this study, although Logoplex® is a veterinary drug, its cytotoxic effect on human cancer cells suggests that it should be re-evaluated as a potential cytotoxic agent. Analyses to identify functional compounds of Tarantula cubensis venom, and future studies addressing its mechanism of action on cancer cells are recommended.