Scorpion venoms have long captivated scientific researchers, primarily due to the potency and specificity of the mechanism of action of their derived components. Among other molecules, these venoms contain highly active compounds, including antimicrobial peptides (AMPs) and ion channel-specific components that selectively target biological receptors with remarkable affinity. Some of these receptors have emerged as prime therapeutic targets for addressing various human pathologies, including cancer and infectious diseases, and have served as models for designing novel drugs. Consequently, extensive biochemical and proteomic investigations have focused on characterizing scorpion venoms. This review provides a comprehensive overview of the key methodologies used in the extraction, purification, analysis, and characterization of AMPs and other bioactive molecules present in scorpion venoms. Noteworthy techniques such as gel electrophoresis, reverse-phase high-performance liquid chromatography, size exclusion chromatography, and “omics” approaches are explored, along with various combinations of methods that enable bioassay-guided venom fractionation. Furthermore, this review presents four adapted proteomic workflows that lead to the comprehensive dissection of the scorpion venom proteome, with an emphasis on AMPs. These workflows differ based on whether the venom is pre-fractionated using separation techniques or is proteolytically digested directly before further proteomic analyses. Since the composition and functionality of scorpion venoms are species-specific, the selection and sequence of the techniques for venom analyses, including these workflows, should be tailored to the specific parameters of the study.

The deleterious consequences of snake envenomation are due to the extreme protein complexity of snake venoms. Therefore, the identification of their components is crucial for understanding the clinical manifestations of envenomation pathophysiology and for the development of effective antivenoms. In addition, snake venoms are considered as libraries of bioactive molecules that can be used to develop innovative drugs. Numerous separation and analytical techniques are combined to study snake venom composition including chromatographic techniques such as size exclusion and RP-HPLC and electrophoretic techniques. Herein, we present in detail these existing techniques and their applications in snake venom research. In the first part, we discuss the different possible technical combinations that could be used to isolate and purify SV proteins using what is known as bioassay-guided fractionation. In the second part, we describe four different proteomic strategies that could be applied for venomics studies to evaluate whole venom composition, including the mostly used technique: RP-HPLC. Eventually, we show that to date, there is no standard technique used for the separation of all snake venoms. Thus, different combinations might be developed, taking into consideration the main objective of the study, the available resources, and the properties of the target molecules to be isolated.

The complications following snake bite envenoming are due to the venom’s biological activities, which can act on different systems of the prey. These activities arise from the fact that snake venoms are rich in bioactive molecules, which are also of interest for designing drugs. The venom of Montivipera bornmuelleri, known as the Lebanon viper, has been shown to exert antibacterial, anticancer, and immunomodulatory effects. However, the venom’s activity on the nervous system has not yet been studied, and its effect on the cardiovascular system needs further investigation. Because zebrafish is a convenient model to study tissue alterations induced by toxic agents, we challenged it with the venom of Montivipera bornmuelleri. We show that this venom leads to developmental toxicity but not teratogenicity in zebrafish embryos. The venom also induces neurotoxic effects and disrupts the zebrafish cardiovascular system, leading to heartbeat rate reduction and hemorrhage. Our findings demonstrate the potential neurotoxicity and cardiotoxicity of M. bornmuelleri’s venom, suggesting a multitarget strategy during envenomation.

The vegetal alkaloid toxin veratridine (VTD) is a selective voltage-gated Na⁺ (Na_V) channel activator, widely used as a pharmacological tool in vascular physiology. We have previously shown that Na_V channels, expressed in arteries, contribute to vascular tone in mouse mesenteric arteries (MAs). Here, we aimed to better characterize the mechanisms of action of VTD using mouse cecocolic arteries (CAs), a model of resistance artery. Using wire myography, we found that VTD induced vasorelaxation in mouse CAs. This VTD-induced relaxation was insensitive to prazosin, an α1-adrenergic receptor antagonist, but abolished by atropine, a muscarinic receptor antagonist. Indeed, VTD–vasorelaxant effect was totally inhibited by the Na_V channel blocker tetrodotoxin (0.3 µM), the NO synthase inhibitor L-NNA (20 µM), and low extracellular Na⁺ concentration (14.9 mM) and was partially blocked by the NCX1 antagonist SEA0400 (45.4% at 1 µM). Thus, we assumed that the VTD-induced vasorelaxation in CAs was due to acetylcholine release by parasympathetic neurons, which induced NO synthase activation mediated by the NCX1-Ca²⁺ entry mode in endothelial cells (ECs). We demonstrated NCX1 expression in ECs by RT-qPCR and immunohisto- and western immunolabelling. VTD did not induce an increase in intracellular Ca²⁺ ([Ca²⁺]i), while SEA0400 partially blocked acetylcholine-triggered [Ca²⁺]i elevations in Mile Sven 1 ECs. Altogether, these results illustrate that VTD activates Na_V channels in parasympathetic neurons and then vasorelaxation in resistance arteries, which could explain arterial hypotension after VTD intoxication.

Caractérisation de substances naturelles actives sur la fonction cardio-vasculaire à partir de venin de la vipère libanaise, Montivipera bornmuelleri et d’une chimiothèque d’alcaloïdes de plantes : Identification d’un nouveau peptide vasorelaxant issu du venin de Montivipera bornmuelleri: L’hypertension, principale cause de maladies cardiovasculaires et de décès prématurés, reste mal contrôlée, soulignant le besoin urgent de nouveaux traitements. Les morsures de vipères provoquent une hypotension, les molécules de leurs venins induisant une vasorelaxation, réduisant ainsi la pression artérielle. Les vipères du genre Montivipera, endémiques du Proche et Moyen-Orient, ont des venins peu étudiés, et leurs effets vasorelaxants n’ont pas été explorés. De plus, les analyses protéomiques des venins de Montivipera sont souvent incomplètes. Notre étude visait à caractériser pharmacologiquement les effets vasorelaxants des venins de Montivipera avec la myographie sur fil et à isoler les composants vasorelaxants du venin de M. bornmuelleri par fractionnement bio-guidé. Nous avons également effectué une analyse protéomique des venins de M. bornmuelleri, M. bulgardaghica, M. albizona, M. xanthina et M. raddei. Nos résultats ont révélé que les venins induisaient une vasorelaxation des artères mésentériques via différentes voies endothéliales, suggérant des molécules à mécanismes variés. Nous avons isolé une PLA2 vasorelaxante de M. bornmuelleri, n'agissant que par des voies endothéliales. Mb-PLA2, une protéine de 121 acides aminés et de 13 658,98 Da, a été caractérisée. Sa structure 3D, modélisée, révèle des similitudes avec d’autres PLA2. Son effet vasorelaxant implique les voies NO et EDHF, avec des activités catalytiques et non catalytiques. L’analyse protéomique a permis d’identifier entre 129 et 179 protéines, réparties en 17 à 19 familles, incluant des SVMP, SVSP, PLA2 et svVEGF. Cette étude enrichit la compréhension des venins de Montivipera et guide le développement d'anti-venins spécifiques. En conclusion, l’étude met en évidence la diversité des molécules vasorelaxantes et propose Mb-PLA2 comme nouvelle cible pour des traitements antihypertenseurs.