Cupiennius spiders (Trechaleidae) from southern Mexico: DNA barcoding, venomics, and biological effect.

Background: Members of the genus Cupiennius Simon, 1891 are categorized as wandering spiders and are part of the family Trechaleidae. The genomics and proteomics of Cupiennius spiders from North America remain uncharacterized. The present study explores for the first time molecular data from the endemic species Cupiennius chiapanensis Medina, 2006, and also presents new data for Cupiennius salei (Keyserling, 1878), both collected in southern Mexico. Methods: In total, 88 Cupiennius specimens were collected from southern Mexico and morphologically identified. DNA was extracted and the mitochondrial COI fragment was amplified. COI sequences were analyzed, and a phylogenetic tree was inferred for species from the Americas. Genetic diversity was analyzed using haplotype networks and gene distances. Venom was obtained from C. chiapanensis and C. salei by electrostimulation. The venom was separated by HPLC, visualized using SDS-PAGE, and quantified for use in toxicity bioassays in mice and insects. Results: Analysis of COI sequences from C. chiapanensis showed 94% identity with C. salei, while C. salei exhibited 94-97% identity with sequences from Central and South American conspecifics. The venom from C. chiapanensis exhibited toxic activity against crickets. Venoms from C. chiapanensis and C. salei caused death in Anastrepha obliqua flies. Analysis of venom fractions from C. salei and C. chiapanensis revealed molecular masses of a similar size as some previously reported toxins and neurotoxic components. We determined the amino acid sequences of ChiaTx1 and ChiaTx2, toxins that are reported here for the first time and which showed toxicity against mice and insects. Conclusion: Our work is the first to report COI-based DNA barcoding sequences from southern Mexican Cupiennius spiders. Compounds with toxic activity were identified in venom from both species.

Cupiennius spiders (Trechaleidae) from southern Mexico: DNA barcoding, venomics, and biological effect

Background: Members of the genus Cupiennius Simon, 1891 are categorized as wandering spiders and are part of the family Trechaleidae. The genomics and proteomics of Cupiennius spiders from North America remain uncharacterized. The present study explores for the first time molecular data from the endemic species Cupiennius chiapanensis Medina, 2006, and also presents new data for Cupiennius salei (Keyserling, 1878), both collected in southern Mexico. Methods: In total, 88 Cupiennius specimens were collected from southern Mexico and morphologically identified. DNA was extracted and the mitochondrial COI fragment was amplified. COI sequences were analyzed, and a phylogenetic tree was inferred for species from the Americas. Genetic diversity was analyzed using haplotype networks and gene distances. Venom was obtained from C. chiapanensis and C. salei by electrostimulation. The venom was separated by HPLC, visualized using SDS-PAGE, and quantified for use in toxicity bioassays in mice and insects. Results: Analysis of COI sequences from C. chiapanensis showed 94% identity with C. salei, while C. salei exhibited 94-97% identity with sequences from Central and South American conspecifics. The venom from C. chiapanensis exhibited toxic activity against crickets. Venoms from C. chiapanensis and C. salei caused death in Anastrepha obliqua flies. Analysis of venom fractions from C. salei and C. chiapanensis revealed molecular masses of a similar size as some previously reported toxins and neurotoxic components. We determined the amino acid sequences of ChiaTx1 and ChiaTx2, toxins that are reported here for the first time and which showed toxicity against mice and insects. Conclusion: Our work is the first to report COI-based DNA barcoding sequences from southern Mexican Cupiennius spiders. Compounds with toxic activity were identified in venom from both species.(AU)

The Identification of a Novel Spider Toxin Peptide, Lycotoxin-Pa2a, with Antibacterial and Anti-Inflammatory Activities.

With the increasing challenge of controlling infectious diseases due to the emergence of antibiotic-resistant strains, the importance of discovering new antimicrobial agents is rapidly increasing. Animal venoms contain a variety of functional peptides, making them a promising platform for pharmaceutical development. In this study, a novel toxin peptide with antibacterial and anti-inflammatory activities was discovered from the spider venom gland transcriptome by implementing computational approaches. Lycotoxin-Pa2a (Lytx-Pa2a) showed homology to known-spider toxin, where functional prediction indicated the potential of both antibacterial and anti-inflammatory peptides without hemolytic activity. The colony-forming assay and minimum inhibitory concentration test showed that Lytx-Pa2a exhibited comparable or stronger antibacterial activity against pathogenic strains than melittin. Following mechanistic studies revealed that Lytx-Pa2a disrupts both cytoplasmic and outer membranes of bacteria while simultaneously inducing the accumulation of reactive oxygen species. The peptide exerted no significant toxicity when treated to human primary cells, murine macrophages, and bovine red blood cells. Moreover, Lytx-Pa2a alleviated lipopolysaccharide-induced inflammation in mouse macrophages by suppressing the expression of inflammatory mediators. These findings not only suggested that Lytx-Pa2a with dual activity can be utilized as a new antimicrobial agent for infectious diseases but also demonstrated the implementation of in silico methods for discovering a novel functional peptide, which may enhance the future utilization of biological resources.

Peptide NCTX15 derived from spider toxin gland effectively relieves hyperuricemia in mice.

Hyperuricemia is a clinical disease characterized by a continuous increase in uric acid (UA) due to purine metabolism disorder. As current drug treatments are limited, it is imperative to explore new drugs that offer better safety and efficacy. In this study, Nephila clavata toxin gland homogenates were isolated and purified by exclusion chromatography and high-performance liquid chromatography, resulting in the identification and isolation of a short peptide (NCTX15) with the sequence 'QSGHTFK'. Analysis showed that NCTX15 exhibited no cytotoxicity in mouse macrophages or toxic and hemolytic activity in mice. Notably, NCTX15 inhibited UA production by down-regulating urate transporter 1 and glucose transporter 9 and up-regulating organic anion transporter 1, thus promoting UA excretion. In addition, NCTX15 alleviated the inflammatory response and renal injury by inhibiting the expression of inflammatory factors interleukin-6, interleukin-1ß, tumor necrosis factor alpha, NLR family, pyrin domain-containing 3, and pyroptosis-related factor gasdermin D. These results indicate that NCTX15 displayed urate-lowering, anti-inflammatory, and analgesic effects. As the first urate-reducing short peptide isolated from a spider toxin gland homogenate, NCTX15 exhibits considerable potential as a novel drug molecule for anti-gout and hyperuricemia treatment.

Structure-function and rational design of a spider toxin Ssp1a at human voltage-gated sodium channel subtypes.

The structure-function and optimization studies of NaV-inhibiting spider toxins have focused on developing selective inhibitors for peripheral pain-sensing NaV1.7. With several NaV subtypes emerging as potential therapeutic targets, structure-function analysis of NaV-inhibiting spider toxins at such subtypes is warranted. Using the recently discovered spider toxin Ssp1a, this study extends the structure-function relationships of NaV-inhibiting spider toxins beyond NaV1.7 to include the epilepsy target NaV1.2 and the pain target NaV1.3. Based on these results and docking studies, we designed analogues for improved potency and/or subtype-selectivity, with S7R-E18K-rSsp1a and N14D-P27R-rSsp1a identified as promising leads. S7R-E18K-rSsp1a increased the rSsp1a potency at these three NaV subtypes, especially at NaV1.3 (∼10-fold), while N14D-P27R-rSsp1a enhanced NaV1.2/1.7 selectivity over NaV1.3. This study highlights the challenge of developing subtype-selective spider toxin inhibitors across multiple NaV subtypes that might offer a more effective therapeutic approach. The findings of this study provide a basis for further rational design of Ssp1a and related NaSpTx1 homologs targeting NaV1.2, NaV1.3 and/or NaV1.7 as research tools and therapeutic leads.

Insecticidal toxicity of ω-Atypitoxin-Cs1a and its inhibitory effects on insect voltage-gated calcium channels.

BACKGROUND: Excessive use of chemical insecticides raises concerns about insecticide resistance, urging the development of novel insecticides. Peptide neurotoxins from spider venom are an incredibly rich source of ion channel modulators with potent insecticidal activity. A neurotoxin U1-Atypitoxin-Cs1a from the spider Calommata signata was annotated previously. It was of interest to investigate its insecticidal activity and potential molecular targets. RESULTS: Cs1a was heterologously expressed, purified and pharmacologically characterized here. The recombinant neurotoxin inhibited high-voltage-activated calcium channel currents with an median inhibitory concentration (IC50 ) value of 0.182 ± 0.026 µm on cockroach DUM neurons and thus was designated as ω-Atypitoxin-Cs1a. The recombinant Cs1a was toxic to three insect pests of agricultural importance, Nilaparvata lugens, Spodoptera frugiperda and Plutella xylostella with median lethal concentration (LD50 ) values of 0.121, 0.172 and 0.356 nmol g-1 , respectively, at 24 h postinjection. Cs1a was equivalently toxic to both insecticide-susceptible and -resistant insects. Cs1a exhibited low toxicity to Danio rerio with an LD50 of 2.316 nmol g-1 . CONCLUSION: Our results suggest that ω-Atypitoxin-Cs1a is a potent CaV channel inhibitor and an attractive candidate reagent for pest control and resistance management. © 2023 Society of Chemical Industry.

Recombinant Production, NMR Solution Structure, and Membrane Interaction of the Phα1ß Toxin, a TRPA1 Modulator from the Brazilian Armed Spider Phoneutria nigriventer.

Phα1ß (PnTx3-6) is a neurotoxin from the spider Phoneutria nigriventer venom, originally identified as an antagonist of two ion channels involved in nociception: N-type voltage-gated calcium channel (CaV2.2) and TRPA1. In animal models, Phα1ß administration reduces both acute and chronic pain. Here, we report the efficient bacterial expression system for the recombinant production of Phα1ß and its 15N-labeled analogue. Spatial structure and dynamics of Phα1ß were determined via NMR spectroscopy. The N-terminal domain (Ala1-Ala40) contains the inhibitor cystine knot (ICK or knottin) motif, which is common to spider neurotoxins. The C-terminal α-helix (Asn41-Cys52) stapled to ICK by two disulfides exhibits the µs-ms time-scale fluctuations. The Phα1ß structure with the disulfide bond patterns Cys1-5, Cys2-7, Cys3-12, Cys4-10, Cys6-11, Cys8-9 is the first spider knottin with six disulfide bridges in one ICK domain, and is a good reference to other toxins from the ctenitoxin family. Phα1ß has a large hydrophobic region on its surface and demonstrates a moderate affinity for partially anionic lipid vesicles at low salt conditions. Surprisingly, 10 µM Phα1ß significantly increases the amplitude of diclofenac-evoked currents and does not affect the allyl isothiocyanate (AITC)-evoked currents through the rat TRPA1 channel expressed in Xenopus oocytes. Targeting several unrelated ion channels, membrane binding, and the modulation of TRPA1 channel activity allow for considering Phα1ß as a gating modifier toxin, probably interacting with S1-S4 gating domains from a membrane-bound state.

Efficient synthesis and anticancer evaluation of spider toxin peptide LVTX-8-based analogues with enhanced stability.

Cytotoxic peptides derived from spider venoms have been considered as promising candidates for anticancer treatment. The novel cell penetrating peptide LVTX-8, which is a 25-residue amphipathic α-helical peptide isolated from spider Lycosa vittata, exhibited potent cytotoxicity and is a potential precursor for further anticancer drug development. Nevertheless, LVTX-8 may be easily degraded by multiple proteases, inducing the proteolytic stability problem and short half-life. In this study, ten LVTX-8-based analogs were rationally designed and the efficient manual synthetic method was established by the DIC/Oxyma based condensation system. The cytotoxicity of synthetic peptides was systematically evaluated against seven cancer cell lines. Seven of the derived peptides exhibited high cytotoxicity towards tested cancer in vitro, which was better than or comparable to that of natural LVTX-8. In particular, both N-acetyl and C-hydrazide modified LVTX-8 (825) and the conjugate methotrexate (MTX)-GFLG-LVTX-8 (827) possessed more durable anticancer efficiency, higher proteolytic stability, as well as lower hemolysis. Finally, we confirmed that LVTX-8 could disrupt the integrity of cell membrane, target the mitochondria and reduce the mitochondrial membrane potential to induce the cell death. Taken together, the structural modifications were conducted on LVTX-8 for the first time and the stability significantly improved derivatives 825 and 827 may provide useful references for the modifications of cytotoxic peptides.

Molecular mechanism of the spider toxin κ-LhTx-I acting on the bacterial voltage-gated sodium channel NaChBac.

The bacterial sodium channel NaChBac is the prokaryotic prototype for the eukaryotic NaV and CaV channels, which could be used as a relatively simple model to study their structure-function relationships. However, few modulators of NaChBac have been reported thus far, and the pharmacology of NaChBac remains to be investigated. In the present study, we show that the spider toxin κ-LhTx-1, an antagonist of the KV4 family potassium channels, potently inhibits NaChBac with an IC50 of 491.0 ± 61.7 nM. Kinetics analysis revealed that κ-LhTx-1 inhibits NaChBac by impeding the voltage-sensor activation. Site-directed mutagenesis confirmed that phenylalanine-103 (F103) in the S3-S4 extracellular loop of NaChBac was critical for interacting with κ-LhTx-1. Molecular docking predicts the binding interface between κ-LhTx-1 and NaChBac and highlights a dominant hydrophobic interaction between W27 in κ-LhTx-1 and F103 in NaChBac that stabilizes the interface. In contrast, κ-LhTx-1 showed weak activity on the mammalian NaV channels, with 10 µM toxin slightly inhibiting the peak currents of NaV1.2-1.9 subtypes. Taken together, our study shows that κ-LhTx-1 inhibits the bacterial sodium channel, NaChBac, using a voltage-sensor trapping mechanism similar to mammalian NaV site 4 toxins. κ-LhTx-1 could be used as a ligand to study the toxin-channel interactions in the native membrane environments, given that the NaChBac structure was successfully resolved in a nanodisc.

Neonatal NaV 1.5 channels: pharmacological distinctiveness of a cancer-related voltage-gated sodium channel splice variant.

BACKGROUND AND PURPOSE: Voltage-gated sodium (NaV ) channels are expressed de novo in carcinomas where their activity promotes invasiveness. Breast and colon cancer cells express the neonatal splice variant of NaV 1.5 (nNaV 1.5), which has several amino acid substitutions in the domain I voltage-sensor compared with its adult counterpart (aNaV 1.5). This study aimed to determine whether nNaV 1.5 channels could be distinguished pharmacologically from aNaV 1.5 channels. EXPERIMENTAL APPROACH: Cells expressing either nNaV 1.5 or aNaV 1.5 channels were exposed to low MW inhibitors, an antibody or natural toxins, and changes in electrophysiological parameters were measured. Stable expression in EBNA cells and transient expression in Xenopus laevis oocytes were used. Currents were recorded by whole-cell patch clamp and two-electrode voltage-clamp, respectively. KEY RESULTS: Several clinically used blockers of NaV channels (lidocaine, procaine, phenytoin, mexiletine, ranolazine, and riluzole) could not distinguish between nNaV 1.5 or aNaV 1.5 channels. However, two tarantula toxins (HaTx and ProTx-II) and a polyclonal antibody (NESOpAb) preferentially inhibited currents elicited by either nNaV 1.5 or aNaV 1.5 channels by binding to the spliced region of the channel. Furthermore, the amino acid residue at position 211 (aspartate in aNaV 1.5/lysine in nNaV 1.5), that is, the charge reversal in the spliced region of the channel, played a key role in the selectivity, especially in antibody binding. CONCLUSION AND IMPLICATIONS: We conclude that the cancer-related nNaV 1.5 channel can be distinguished pharmacologically from its nearest neighbour, aNaV 1.5 channels. Thus, it may be possible to design low MW compounds as antimetastatic drugs for non-toxic therapy of nNaV 1.5-expressing carcinomas.