Abstract Background: Temperature regulation is essentially important for survival of poikilotherms such as snakes. Body temperature is regulated by snakes through behavioral and physiological responses. The global-warming crisis, combined with the need to house large population of snakes in limited spaces, increases the likelihood of exposing snakes to high ambient temperature (HTa), requiring it reliance on physiological responses. This study aimed to study the effect of HTa exposure on physiological responses and venom production, which have rarely been studied. Methods: Eleven adult monocled cobras (Naja kaouthia Lesson, 1831) were divided into two groups. The concurrent control group was housed in a temperature-controlled room, and the heat exposed group was housed in the same room with gradually increasing temperatures (25°C-35°C) for 4 h on four consecutive days. Data were collected 3 days before the experiment as the baseline and then compared with day 1 and day 4 after HTa exposure data representing immediate and prolonged effects. Body temperature, body weight, water intake, heart rate, hematology, plasma biochemistry, body-fluid compartments, hormonal response, heat shock protein expression and venom production were measured. Results: In response to HTa exposure, body temperature and heart rate increased, plasma volume significantly decreased, but water intake increased. Hematocrit and plasma protein progressively decreased in the latter stages of experimentation, but HTa diminished this effect. HTa only increased plasma corticosterone on day 1. Exposure to HTa increased venom protein concentration on day 4 and diminished the decreased proportion effect of frequent venom collection on phospholipase A2 component. Conclusion: Increased heart rate and fluid shift from the intravascular compartment appeared to be the underlying mechanism for heat dissipation during HTa exposure. Under the study condition, HTa caused heat stress, but the snake could adapt after continued exposure. Additionally, HTa increased venom protein concentration in N. kaouthia, particularly phospholipase A2 component.

Abstract Background: Chagas disease (CD), caused by Trypanosoma cruzi, affects approximately seven million individuals worldwide, with the highest number of cases in Latin America. CD has two phases, of which the chronic phase is characterized by reduced efficacy in drug therapies. This and other factors make developing new strategies that aim to identify molecules capable of becoming alternatives to or complement current chemotherapy vitally important. Methods: Cruzain and AsCystatin were obtained recombinantly through expression in E. coli. Bioinformatic assays were conducted with both molecules, followed by in vitro enzyme inhibition assays. Subsequently, in silico studies allowed for the design of peptides, which were then assessed for molecular interactions with cruzain. The designed peptides were synthesized, and their inhibitory potential on cruzain and their trypanocidal and cytotoxic effects in vitro were finally assessed. Results: AsCystatin, a potential inhibitor of cysteine proteases, was identified from previously published scientific literature. In silico assays suggested that AsCystatin interacts with key regions of cruzain, and was subsequently produced through heterologous expression, obtaining a protein with a high degree of purity. Next, the inhibition of AsCystatin on the activity of cruzain was assessed, observing that approximately 20 µM of cystatin could inhibit 50% of the catalytic activity of the recombinant enzyme. Based on the in-silico analysis performed previously, original, and modified peptides were designed and tested, which allowed for identifying four peptides with inhibitory capacity on the enzymatic activity of cruzain. Finally, three of these peptides showed trypanocidal activity on epimastigote forms of T. cruzi in in vitro models. Conclusion: It was possible to identify AsCystatin and four peptides derived from this protein with inhibitory activity on cruzain, highlighting the trypanocidal effect of these peptides observed in in vitro assays.

Abstract Background: Non-small cell lung cancers (NSCLC) represent the primary cause of cancer-related deaths worldwide. Rhopalurus junceus venom has been shown to exert cytotoxic effects against a panel of epithelial cancer cells in vitro and suggested that NSCLC was the subtype most susceptible to the treatment. Methods: This study evaluated the effect of Rhopalurus junceus scorpion venom on cell viability, in non-cancerous (MRC-5, lung; CHO-K1, ovary) and NSCLC (A549; NCI-H460) cell lines. The effects on cell cycle, apoptosis, and cell signaling-related proteins were determined by flow cytometry and WB. Protein fractions responsible for the observed effect were identified using HPLC. Results: Scorpion venom was more effective against NSCLC than non-cancerous cells. Emax values were 20.0 ± 5.8% and 22.47 ± 6.02% in A549 and NCI-H460 cancer cells, respectively, as compared to 50 ± 8.1% in MRC-5 and 54.99 ± 7.39% in CHO-K1 cells. It arrested NSCLC cells in the G2/M phase, while non-cancerous cells were arrested in the S (MRC-5) or G0/G1 (CHO-K1) phases. No changes were observed in the Bax/Bcl-2 or the cleaved-caspase 3/Total caspase 3 ratios in cells treated with venom. Likewise, the scorpion venom treatment did not affect p-ERK, p-AKT, or p-38MAPK protein levels. In contrast, scorpion venom treatment increased the cytosolic apoptosis-inducing factor (AIF) in A549 cells, indicating caspase-independent apoptosis. Additionally, combined etoposide/venom exposure provoked G2/M arrest and apoptosis in NSCLC more strongly than either substance alone. Furthermore, upon crude venom fractioning through RP-HPLC, we found two soluble fractions with high cytotoxic effects. Conclusion: The present study concludes that a specific fraction of Rhopalurus junceus venom reduces cell viability of NSCLC cells. The AIF protein plays a key role in mediating caspase-independent apoptotic cell death. These findings suggest that Rhopalurus junceus venom enhances the anticancer effect of etoposide in vitro by causing cell cycle arrest and caspase-independent apoptosis.

Abstract Background: This study examines the impact of Phα1β, a spider peptide derived from the venom of Phoneutria nigriventer, on the Kv11.1 potassium channel in HEK293 cells transfected with the human ERG potassium channel. Phα1β inhibits high-voltage calcium channels and acts as an antagonist of the TRPA1 receptor, both of which play crucial roles in pain transduction pathways. Over the past 15 years, our research has demonstrated the potential of Phα1β, in both its native and recombinant forms, as a promising analgesic drug through preclinical tests conducted on rodent pain models. Regulatory agencies require the evaluation of new drugs on human ERG channels. Methods: To assess hERG potassium channel inhibition, we utilized the FLIPR® Potassium Assay, a commercially available kit. The assay involved testing the effects of Phα1β alongside the well-established hERG potassium channel blocker dofetilide, which served as a positive control. The viability of HEK-293 cells was assessed using the colorimetric MTT reduction test (3-(4, dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), whereby viable cells reduce the MTT salt, forming a formazan complex within their mitochondria, as previously described. Results: Phα1β was tested at concentrations of 56, 225, 450, and 900 pMol, resulting in a discreet inhibition of hERG potassium channel activity at higher concentrations, approximately 13.47%, with an IC50 value exceeding 900 pMol. Dofetilide, administered at concentrations ranging from 0.0001 to 10 µM, displayed a concentration-dependent inhibition of the hERG potassium channel, with a mean IC50 value of 0.1642 µM (0.1189-0.2282 µM). To evaluate cytotoxicity, HEK293-hERG cells were exposed to Phα1β concentrations of 56/900 pMol for 24 hours, resulting in no significant alteration in cell viability. Conclusion: Our findings indicate that even at high concentrations, Phα1β does not impede the functionality of the hERG potassium channel nor affect cell viability.

Abstract Background: Fish venoms have been poorly characterized and the available information about their composition suggests they are uncomplicated secretions that, combined with epidermal mucus, could induce an inflammatory reaction, excruciating pain, and, in some cases, local tissue injuries. Methods: In this study, we characterized the 24-hour histopathological effects of lionfish venom in a mouse experimental model by testing the main fractions obtained by size exclusion-HPLC. By partial proteomics analysis, we also correlated these in vivo effects with the presence of some potentially toxic venom components. Results: We observed a strong lesion on the skin and evident necrosis in the skeletal muscle. None of the tissue-damaging effects were induced by the fraction containing cytolysins, membrane pore-forming toxins ubiquitously present in species of scorpionfish, stonefish, and lionfish, among others. On the contrary, injuries were associated with the presence of other components, which have remained practically ignored so far. This is the case of an abundant protein, present in venom, with homology to a Golgi-associated plant pathogenic protein 1-like (GAPR1), which belongs to the same protein superfamily as venom CRISPs and insect allergens. Conclusion: This GAPR1-like protein and the hyaluronidase are probably responsible for the hemostasis impairment and hemorrhagic lesions observed in mouse skin, whereas muscle injuries can be indirectly caused by a combination of inflammatory and hemorrhagic events. More information is required to establish the components accountable for the myonecrotic effect.

Abstract Background: Leishmaniasis is a neglected disease that mainly affects impoverished populations and receives limited attention from governments and research institutions. Current treatments are based on antimonial therapies, which present high toxicity and cause significant side effects, such as cardiotoxicity and hepatotoxicity. This study proposes using crotapotin, isolated from Crotalus durissus terrificus venom, as a potential inhibitor of the enzyme trypanothione reductase from Leishmania braziliensis (LbTR). Methods: In silico assays were conducted to evaluate the interaction of crotapotin with LbTR using molecular docking and molecular dynamics techniques. Recombinant LbTR was expressed in E. coli, and its enzymatic activity was confirmed. The inhibitory action of crotapotin on LbTR was then tested in enzymatic assays. Results: The stability of these interactions was confirmed over 200 ns molecular dynamics simulations, with a clustering analysis using the GROMACS method revealing a total of 12 distinct clusters. The five most representative clusters showed low RMSD values, indicating high structural stability of the LbTR-crotapotin complex. In particular, cluster 1, with 3,398 frames and an average RMSD of 0.189 nm from the centroid, suggests a dominant stable conformation of the complex. Additional clusters maintained average RMSD values between 0.173 nm and 0.193 nm, further reinforcing the robustness of the complex under physiological conditions. Recombinant LbTR expression was successful, yielding 4.8 mg/L with high purity, as verified by SDS-PAGE. In the enzymatic assays, crotapotin partially inhibited LbTR activity, with an IC50 of 223.4 μM. Conclusion: The in silico findings suggest a stable and structured interaction between crotapotin and LbTR, with low structural fluctuation, although the inhibition observed in in vitro assays was moderate. These results indicate the potential of crotapotin as a promising basis for developing specific LbTR inhibitors, contributing to the bioprospecting of new antiparasitic agents.

Abstract Background: Osteosarcoma is the most common primary bone tumor in humans. It is a locally aggressive tumor at the primary site, with metastasis being the main cause of death in patients. Studies on dogs have gained prominence in oncology, as they are valuable spontaneous models of osteosarcoma. In the context of natural compounds, biotoxins are attracting increasing research interest as new therapeutic agents against cancer, such as melittin, that represents 40 to 50% of the dry weight of bee venom, and studies have already shown its antitumor effects. Methods: We analyzed the anti-migratory and anti-invasive potential of melittin, with the wound healing and Transwell tests, apoptosis with Annexin V/IP and cell viability with the MTT test in 2D and 3D models. Results: Melittin had a cytotoxic effect on osteosarcoma cell lines, with an IC50 between 1.5 and 2.5 µg/mL. In the wound healing test and Transwell test, melittin prevented cell migration and invasion, resulting in cell death due to iodide propidium marking in canine, murine and human cell lines. Melittin exhibited cytotoxicity in a 3D model of osteospheres, with a significantly higher IC50 in this type of culture, with values between 3.5 and 4.0 µg/mL. Conclusion: We conclude that melittin has antitumor and antimetastatic properties in canine, murine and human osteosarcoma cell lines. Consequently, we believe that further research on this promising compound will facilitate its application in the development of therapeutic agents for osteosarcoma, ultimately contributing to improved survival outcomes for cancer patients.

Abstract Background: Spiders of medical importance in the Amazon region belong to the genera Phoneutria, Loxosceles and Latrodectus. Natural history data show that Phoneutria spp. occur in both periodically flooded forest areas (igapós) and non-flooded areas (terra firme), as well as in commercial plantations in the Amazon. Negative interactions with wandering spiders (Phoneutria spp.) can occur along forest trails, leading to homes, schools and workplaces. Harmful species, such as Loxosceles amazonica and Latrodectus aff. curacaviensis, are mainly associated with accidents in rural settings. Methods: To understand the dynamics of spider accidents in the Brazilian Amazon, we conducted a search for scientific articles in five databases (Google Scholar, PubMed/MEDLINE, Scopus by Elsevier and SciELO). In addition, we analyzed the content of four reference books on the ecological aspects of Amazonian spiders. All told, we identified 64 eligible studies, including six regional surveys published between 1996 and 2016. Results: From 2015 to 2022, a total of 25 human lives were lost to spider envenomation in the Brazilian Amazon. An analysis of the data revealed that many riverside families engage in agricultural practices that expose them to venomous animals. Hospital data reveal that most patients bitten by spiders come from impoverished rural communities, which rely on public hospitals of Brazil’s Unified Health System (SUS) for medical treatment. The results indicate that spider bites in the Amazon represent a neglected public health problem, especially in locations far from capital cities. Conclusion: Amerindian and non-Amerindian communities living in areas at high risk of venomous animal attacks do not receive adequate attention in health policies. Given the wide dispersion of rural populations vulnerable to venomous animal incidents in the Amazon, the establishment of new referral medical centers is an essential strategy, especially for riverside communities with limited access to health services.

Abstract Background: Scorpion envenomation poses a significant health threat in endemic regions, eliciting complex immune responses in affected individuals. Recent research suggests that the timing of envenomation - whether it occurs during the day or night - may influence the host inflammatory response and subsequent organ damage. This study investigates the impact of envenomation timing on host inflammatory and oxidative responses using an experimental scorpion envenomation model. Methods: Mice were divided into two groups, corresponding to their resting phase (day) and activity phase (night), and were monitored for twenty-four hours post-envenomation. We analyzed systemic inflammatory markers, hormonal changes within the hypothalamic-pituitary-adrenal (HPA) axis, and assessed liver toxicity. Results: Our findings reveal that the release of the myeloperoxidase enzyme, along with the pro-inflammatory cytokines IL-6 and IL-17, varied significantly based on the timing of envenomation. Notably, envenomation occurring during the nighttime resulted in elevated levels of these mediators. We also observed a pronounced imbalance in oxidative stress, characterized by a higher presence of prooxidant species during the daytime and enhanced antioxidant activities during the nighttime. This diurnal variation highlights the dynamic nature of the inflammatory and oxidative processes. Importantly, our analysis points to the probable involvement of corticosterone, the final effector of the HPA axis, in modulating these variations in the inflammatory response. By influencing both the intensity of the immune response and the degree of oxidative stress, corticosterone appears to play a pivotal role in the overall pathophysiology of scorpion envenomation. Conclusion: This study provides valuable insights into how the timing of scorpion envenomation influences inflammatory responses and organ-specific toxicity, offering potential implications for the treatment and management of envenomation cases.

Abstract Background: Deinagkistrodon acutus, or the hundred-pace snake, poses severe health risks due to its venom. Envenomation by this snake leads to complications such as hemorrhage, edema, and coagulopathy. Traditional antivenoms are limited by venom variability and often contain non-neutralizing antibodies, highlighting the need for more precise and effective immunogens. Methods: This study utilized epitope-based antibody technology to develop a targeted sera against venom metalloproteinases (MPs) and phospholipases A2 (PLA2s). Twelve antigenic epitopes were identified via bioinformatics, leading to the design of a composite antigen peptide, EpiMPLA. It was engineered to be expressed via two expression systems, resulting in the recombinant immunogens, ProMPLA and p2AMPLA. Results: Immunization with ProMPLA and p2AMPLA produced robust antibody responses in mice, effectively inhibiting MPs and PLA2s. In vitro assays demonstrated that sera from immunized mice reduced the activity of these venom enzymes, minimized venom-induced hemorrhage and edema, and restored blood coagulation. At a venom dose of 2×LD50, all mice in the control group died, while survival rates were 90% for anti-ProMPLA and 70% for anti-p2AMPLA. Conclusion: The EpiMPLA epitope represents a promising candidate for generating neutralizing antibodies against D. acutusvenom, demonstrating its potential to address critical gaps in current antivenom therapy. These findings not only validate the feasibility of epitope-based antivenom development but also pave the way for further research to optimize this strategy.

Abstract Background: Mammary gland tumors are the most prevalent neoplasm in intact female dogs, and they are good natural models to study comparative oncology. Most canine mammary malignancies, as in women, are commonly refractory to conventional therapies and demand continuous new therapeutic approaches. Crotalus durissus terrificus, also called rattlesnake, has more than 60 different proteins in its venom with multiple pharmaceutical uses, such as antitumor, antiviral, and antimicrobial action. Crotoxin, a potent β-neurotoxin formed by the junction of two subunits, a basic subunit (CB-PLA2) and an acidic subunit (crotapotin), has already been reported to have anticancer properties in different types of cancers. Methods: In this work, we describe the cytotoxic potential of crotoxin and its subunits compared to doxorubicin (drug of choice) in two canine mammary carcinoma cell lines. Results: Crotoxin, CB-PLA2, crotalic venom, and doxorubicin decreased cell viability and the ability to migrate in a dose-dependent manner, and crotapotin did not present an antitumoral effect. For all compounds, the predominant cell death mechanism was apoptosis. In addition, crotoxin did not show toxicity in normal canine mammary gland cells. Conclusion: Therefore, this work showed that crotoxin and CB-PLA2 had cytotoxic activity, migration inhibition, and pro-apoptotic potential in canine mammary gland carcinoma cell lines, making their possible use in cancer research.

Abstract Background: In Colombia, several species of Buthidae scorpions belonging to the genera Centruroides and Tityus coexist, and their stings are considered life-threatening to humans because of their venom neurotoxins. Despite previous studies focusing on neurotoxins from these scorpion genera, little is known about the enzymes present in their venoms and their relationship with whole venom toxicity. Methods: Here, using proteomic and biochemical protocols the enzymatic activities of the venoms of three Colombian scorpion species, C. margaritatus, T. pachyurus, and T. n. sp. aff. metuendus, were compared to establish the presence and absence of enzymes such as phospholipases, hyaluronidases, and proteases that could be related to venom toxicity. Results: C. margaritatus was positive for hyaluronidases, T. n. sp. aff. metuendus for proteases, and T. pachyurus exhibited activity for all three mentioned enzymes. Conclusion: This information provides valuable insights into the specific enzyme diversity of each species’ venom and their potential role in venom toxicity, which could contribute to the development of better treatments and prevention strategies for scorpion envenomation.

Abstract The skin of anuran species is a protective barrier against predators and pathogens, showing also chemical defense by substances that represent a potential source for bioactive substances. This review describes the current chemical and biological knowledge from the skin secretions of Leptodactylidae species, one of the most diverse neotropical frog families. These skin secretions reveal a variety of substances such as amines (12), neuropeptides (16), and antimicrobial peptides (72). The amines include histamine and its methylated derivatives, tryptamine derivatives and quaternary amines. The peptides of Leptodactylidae species show molecular weight up to 3364 Da and ocellatins are the most reported. The peptides exhibit commonly glycine (G) or glycine-valine (GV) as C-terminal amino acids, and the most common N-terminal amino acids are glutamic acid (E), lysine (K), and valine (V). The substances from Leptodactylidae species have been evaluated against pathogenic microorganisms, particularly Escherichia coli and Staphylococcus aureus, and the most active peptides showed MIC of 1-15 µM. Furthermore, some compounds showed also pharmacological properties such as immunomodulation, treatment of degenerative diseases, anticancer, and antioxidant. Currently, only 9% of the species in this family have been properly studied, highlighting a large number of unstudied species such as an entire subfamily (Paratelmatobiinae). The ecological context, functions, and evolution of peptides and amines in this family are poorly understood and represent a large field for further exploration.

Abstract Background: Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease. There is no effective treatment for neurodegenerative diseases. Snake venoms are a cocktail of proteins and peptides with great therapeutic potential and might be useful in the treatment of neurodegenerative diseases. Crotapotin is the acid chain of crotoxin, the major component of Crotalus durissus collilineatus venom. PD is characterized by low levels of neurotrophins, and synaptic and axonal degeneration; therefore, neurotrophic compounds might delay the progression of PD. The neurotrophic potential of crotapotin has not been studied yet. Methods: We evaluated the neurotrophic potential of crotapotin in untreated PC12 cells, by assessing the induction of neurite outgrowth. The activation of the NGF signaling pathway was investigated through pharmacological inhibition of its main modulators. Additionally, its neuroprotective and neurorestorative effects were evaluated by assessing neurite outgrowth and cell viability in PC12 cells treated with the dopaminergic neurotoxin MPP+ (1-methyl-4-phenylpyridinium), known to induce Parkinsonism in humans and animal models. Results: Crotapotin induced neuritogenesis in PC12 cells through the NGF-signaling pathway, more specifically, by activating the NGF-selective receptor trkA, and the PI3K/Akt and the MAPK/ERK cascades, which are involved in neuronal survival and differentiation. In addition, crotapotin had no cytotoxic effect and protected PC12 cells against the inhibitory effects of MPP+ on cell viability and differentiation. Conclusion: These findings show, for the first time, that crotapotin has neurotrophic/neuroprotective/neurorestorative potential and might be beneficial in Parkinson's disease. Additional studies are necessary to evaluate the toxicity of crotapotin in other cell models.

Abstract Background: Medications currently used to treat pain are frequently associated with serious adverse effects and rapid development of tolerance. Thus, there is a need to develop more effective, and safer medicines for the population. Blocking NMDA receptors (NMDAR) has shown to be a promising target for the development of new drugs. That statement is due to NMDAR activation and glutamate release in the spinal cord which affects chronic pain modulation. Therefore, the aim of this study was to evaluate the possible spinal antinociceptive activity of PnTx4(5-5) toxin. The peptide is purified from the venom of the spider P. nigriventer and its affinity for NMDAR and sodium channels Nav1.2-1.6 has already been established. Methods: We compared its effect and safety with MK-801 (NMDAR antagonist) and evaluated its influence on glutamate and reactive oxygen species (ROS) levels in CSF. PnTx4(5-5) was administered intrathecally in the Formalin test and co-administered with NMDA in the Spontaneous pain test. After three minutes of observation, mice cerebrospinal fluid was collected to measure glutamate and ROS levels. Results: The spider peptide inhibited nociception as post-treatment in the inflammatory phase of the Formalin test. Furthermore, it inhibited spontaneous nociception induced by NMDA, being more potent and effective than MK-801 in both models tested. A glutamate rise level in the CSF of mice was significantly reduced by the toxin, but ROS increase was not affected. The animals’ motor skills were not affected by the tested doses of NMDAR inhibitors. Conclusion: In conclusion, the results suggest PnTx4(5-5) may mediate its antinociceptive effect in the spinal cord not only by inhibiting postsynaptic receptors but probably also by acting on autoreceptors. This effect does not affect the motricity of mice at the highest dose tested, which suggests that it has therapeutic potential and safety for use as a painkiller.