RP-HPLC Fractionation of Tarantula Venom Yields Neuroactive Compounds with Therapeutic Potential

Spider venoms have been found to be rich natural sources of bioactive chemicals, ranging from low-molecular-mass compounds to larger molecules such as low molecular mass peptides, proteins, and enzymes. Some of these compounds have been reported to exhibit neuroactivity and show potential as therapeutic agents against neurological disorders. Thus, this study analyzed the neurobehavioral effects of selected venom fractions from Philippine tarantula species compared to FDA-approved drugs targeting neuroreceptors, ion channels, and enzymes. Venom was collected from the tarantula by electrostimulation and fractionated by reverse-phase high-performance liquid chromatography (RP-HPLC). A paper based on their efforts was published in the Journal of Venomous Animals and Toxins including Tropical Diseases.¹

Production of venom is an evolutionary adaptation which serves both offensive (the enabling of prey capture and digestion) and defensive protection from predators) roles.² Animal venoms are complex mixtures of bioactive molecules which act on a variety of molecular targets, such as ion channels, receptors, and enzymes.^3,4 Many of these compounds, especially those from arthropod venoms (such as spiders and scorpions), are neuroactive, and can modulate both the central and peripheral nervous systems.⁵Due to their chemical diversity, arthropod venoms are of special interest; they contain peptide inhibitors which modulate neuronal activity through the blocking or alteration of potassium channel gating.^6,7 Furthermore, neurotoxins from venoms have been shown to hold the possibility for use in treatment for inflammatory and neurodegenerative diseases, both for their effects on neural signaling as well as their antimicrobial properties.^8,9

Nine of the eleven fractions resulting from the RP-HPLC process used in this study were subjected to neurobehavioral analysis using zebrafish (Danio rerio) as the animal model. The fractions were administered intraperitoneally, and their neurobehavioral effects were examined using the novel tank test, fear response, social interaction, and mirror biting tests. Donepezil, lidocaine, and diazepam were used as positive controls, and normal saline solution (NSS) as the negative control of the study.¹

Preliminary screening of all the fractions revealed that Fraction 1 with 0.1 µg/µL exhibited donepezil-like behavior based on similar rapid-swimming movement from 0 to 31 time intervals, Fraction 4 with 0.1 µg/µL concentration exhibited diazepam-like behavior due to non-significant differences in its time spent on top of the tank ranging from 20 to 40 min, and Fraction 8 with 0.1 µg/µL concentration exhibited lidocaine-like behavior based on both rapid swimming movement and time spent on top of the tank. Fractions 1, 4, and 8 were further evaluated by determining their dose-dependent response, which follows the effect of their corresponding positive control. Analysis of Fraction 1 resulted in the annotation of several non-peptidic components 4-OH-PhLac434 and its isomer using VenoMS and isopimaric acid, palmitamide, 9-octadecenamide, and 13-docosenamide as putative compounds present in this spider venom using GNPS.¹

“Overall,” write the authors of the study,¹ “the fractions of venom from the Orphnaecus tarantula species appear to induce distinct neurobehavioral effects, which may include hyperactivity, anxiolytic-like responses, and potential antinociceptive properties.”

The researchers believe that future studies should focus on the purification and structural characterization of the active compounds, followed by functional validation using receptor-binding assays and electrophysiological techniques. Furthermore, there will be additional behavioral assays and structure-activity relationship (SAR) analyses conducted to obtain a better understanding of the pharmacological profiles and therapeutic potential of these venom-derived molecules.¹

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References

1. Bautista, J. L. C.; Abellanosa, E. A. M.; Molino, R. E. J. et al. A Venom Fraction from the Philippine tarantula (Orphnaecus sp.) Reveals Low-Molecular-Weight Compounds that Potentiate Drug-Like Neurobehavioral Responses in Danio rerio. J Venom Anim Toxins Incl Trop Dis.2026,32, e20240063. DOI: 10.1590/1678-9199-JVATITD-2024-0063
2. de Souza, J.; Goncalves, B.; Gomez, M. et al. Animal Toxins as Therapeutic Tools to Treat Neurodegenerative Diseases. Front Pharmacol. 2018, 9, 145. DOI: 10.3389/fphar.2018.00145
3. Utkin, Y. Animal Venom Studies: Current Benefits and Future Developments World J Biol Chem. 2015, 6 (2), 28-33. DOI: 10.4331/wjbc.v6.i2.28
4. Chen, N.; Xu, S.; Zhang, Y. et al. Animal Protein Toxins: Origins and Therapeutic Applications. Biophys Rep. 2018, 4 (5), 233-242. DOI:10.1007/s41048-018-0067-x
5. Kachel, H.; Buckingham, S.; Sattelle, D. Insect Toxins: Selective Pharmacological Tools and Drug/Chemical Leads. Curr Opin Insect Sci. 2018, 30. DOI: 10.1016/j.cois.2018.10.001
6. Rádis-Baptista, G.; Konno, K. Arthropod Venom Components and Their Potential Usage. Toxins 2020, 12 (2), 82. DOI: 10.3390/toxins12020082
7. Gati, C.; Mortari, M.; Schwartz, E. Towards Therapeutic Applications of Arthropod Venom K+-Channel Blockers in CNS Neurologic Diseases Involving Memory Acquisition and Storage. J Toxicol. 2002, 1-21. DOI: 10.1155/2012/756358
8. Carniglia, L.; Ramirez, D.; Durand, D. et al. Neuropeptides and Microglial Activation in Inflammation, Pain, and Neurodegenerative Diseases. Mediators Inflamm. 2017, 5048616. DOI: 10.1155/2017/5048616.2017
9. de Araujo Boleti, A.; de Oliveira Flores, T.; Moreno, S. et al. Neuroinflammation: An Overview of Neurodegenerative and Metabolic Diseases and of Biotechnological Studies, J. Biotechnol. Neurochem Int. 2020, 136, 104714. DOI: 10.1016/j.neuint.2020.104714