Posters and presentation information
Thank you for considering to present your work as a poster at Venoms and Toxins 2023.
Digital poster preparation and submission
- Page size: Prepare your poster as you would normally do for printing. You can prepare your poster in sizes A1 or A0, as the page size is not important if only presenting digitally. However, print hardcopy posters in A1 portrait format. Larger posters and those in landscape format may not be displayed due to space constraints.
- Naming your poster files: Name your poster files as follows: <your surname>-Ven23-Poster.pdf. For example, for David Jones, name your file as Jones-Ven23-Poster.pdf. DO NOT name your poster files as, e.g., Oxford-poster, Venoms2023, Oxford-venoms-poster. Such files will be automatically rejected.
- Poster submission and deadlines: All poster presenters, whether attending virtually or in-person, are required to submit a digital version of their poster. Submit your final poster as PDF (<5MB) and via the link below no later than 14th August 2023 (we must have received your poster abstracts by 07th August). Late posters may not be included in the conference programme. Please DO NOT send your poster (or abstract) files by email. Please ensure you send us the very final version of your poster (as well as your poster abstract), as once published, it cannot be replaced.
Poster presentation
- The poster PDF files, whether the presenter is attending virtually or in-person, are required for presentation, and will be made available via the secure ‘Download VEN23 Documents’ page to the conference participants before the meeting. The participants will be able to ask questions via the Zoom chatbox during the conference. There is no specific time for presenting digital posters.
- Hardcopy posters: If attending in-person, you may bring along a printed copy of your poster (maximum A1 size) to be displayed during the conference. You may be assigned a specific day to display your poster.
- Any further information about the poster presentations will be available in the future.
Before uploading your poster, you must make sure that you follow ALL of the instructions above!
Accepted posters (unedited)
(Presenters in Bold)
If your abstract has been accepted for presentation but it does not appear in the list below, please let us know as soon as possible by email at VenomsOxford@gmail.com.
Monoclonal antibody discovery for snake venom toxins using yeast surface display
Camille J Abada, Mark Wilkinson, Nicholas R Casewell, Stuart Ainsworth, Stefanie K Menzies
Centre for Snakebite Research & Interventions, Department of Tropical Disease Biology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool UK
Snakebite envenoming is a global health crisis, affecting thousands of individuals each year. Currently, antivenom is the recommended treatment however, there is a need for next generation antivenoms with higher therapeutic potential, sufficient safety, and cost-effectiveness. Traditional monoclonal antibody (mAb) discovery relies on animal immunization, which is time consuming and an expensive process. Here, we present early-stage data showing the application of yeast surface display (YSD) technology to discover new mAbs against snake venom toxins. This approach utilizes an existing nonimmune human library in which each yeast cell typically displays up to 1 x 105 copies of a unique single-chain antibody fragment (scFv) fused to a surface protein. ScFv are isolated through rounds of magnetic-activated cell sorting (MACS), a high throughput screening method, followed by rounds of fluorescence-activated cell sorting (FACS). YSD is an advantageous platform utilizing eukaryotic protein-directed evolution, it is highly versatile in protein selection, relatively cheap and screening strategies are based on quantitative FACS. Moreover, scFv candidates can undergo iterative maturation processes through hypermutation and further affinity-based sorting to enrich biochemical properties. We chose alpha-bungarotoxin (α-Bgtx) as a proof-of-concept target and successfully isolated diverse antibody clones for which maturation and subsequent neutralizing assays are ongoing. The preliminary success with α-Bgtx selection prompted us to extend our pipeline to target snake venom metalloproteinases (SVMP) proteins. The ongoing selection process is expected to identify potent antibodies with potential therapeutic and diagnostic applications for snakebite.
Maternal effects on animal venoms? An exploratory study using the green lacewing, Chrysoperla carnea (Neuroptera: Chrysopidae)
Gustavo A Agudelo-Cantero1, Thomas F Hansen1, Jeroen Kool2, Christophe Pelabon3, Eivind AB Undheim1
1Centre for Ecological and Evolutionary Synthesis, Department of Biosciences, University of Oslo, PO Box 1066 Blindern, 0316 Oslo, Norway
2AIMMS, Division of BioAnalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, Faculty of Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands
3Center for Biodiversity Dynamics, Department of Biology, Norwegian University of Science and Technology (NTNU), 7491 Trondheim, Norway
Despite much research has been conducted on the evolution of animal venoms, our understanding about the factors that generate variation on venom phenotypes remains limited and concentrated to a few lineages. In this context, the extent to which the environment experienced by parents affects offspring venom phenotypes has been rarely, if ever, explored. To fill this gap, we are exploring, without an a priori hypothesis, whether the venom of the green lacewing Chrysoperla carnea (Neuroptera: Chrysopidae) is subject to maternal effects. The green lacewing is a good model species for this purpose because only larvae are venomous and adult females are very prolific (up to 1200 eggs during lifespan), making possible to compare the venom of larvae with known dam origin (half-sibs) while manipulating the maternal environment. We collected 42 adult lacewing females in Læsø (Denmark) in May 2023 in order to obtain 3rd-instar larvae for milking their venom. Some females started laying eggs in their individual containers since collection, so we kept field-laid eggs 3-6 days until arriving to the laboratory in Oslo (Norway). In the laboratory, we took up to 5 field-laid eggs/female and put them individually in petri dishes with moth eggs, and transferred females to new containers to lay more eggs. Two days after, we collected up to 5 lab-laid eggs/female as before. In total, we milked the venom of 108 larvae obtained from 19 dams (55 from field-laid eggs and 53 from lab-laid eggs), so that each dam had at least one larvae in a given group. Venom samples are being analyzed by a combination of high-throughput venomics techniques (e.g., RP-HPLC-nanofractionation, nanoLC–MS/MS). Our results will clarify potential causal links between venom phenotypes of offspring to the maternal genotype/phenotype, a relevant aspect to understand the ecological and evolutionary processes that shape animal venoms.
Venom variation analytics with LC-MS: from 5 months to 5 hours
Luis L Alonso1, Jory van Thiel1, Julien Slagboom1, Saer Samanipour2, Jeroen Kool1
1AIMMS Division of BioAnalytical Chemistry, Vrije Universiteit Amsterdam, The Netherlands
2Van’t Hoff Institute of Molecular Sciences, Universiteit van Amsterdam, The Netherlands
Snakebite is considered one of the most lethal Neglected Tropical Diseases. Snake venoms are known for their complex nature and venom composition can be investigated with liquid chromatography mass spectrometry (LC-MS). The LC-MS data processing after analysis of the toxins in venoms however is a tedious and long process. Besides venom complexity regarding composition at the interspecies level, individual venoms at the intraspecies level also can vary heavily in terms of composition, which depends on many different factors. While venom variation between species has been well-studied, the extent of intraspecific variation is starting to gain significant interest. The main bottleneck in studying such a complex biomatrix at the chemical level (i.e., at the toxin accurate mass level) includes the manual toxin deconvolution and identification from the measured LC-MS data. This study provides a new methodology that allows us to rapidly analyze and process the measured data of numerous venom samples in an automated fashion. We verify this principle through LC-MS analysis of venoms of two genera of snakes, Pseudonaja and Oxyuranus, which include some of the most venomous snakes in the world. This was done both manually –which took 5 months–, and automatically –which took 5 hours–. The automated data extraction and deconvolution of the LC-MS raw files was performed directly after analysis. Next, in-house written scripts were used to process and sort all data. Finally, the results between the automatically and the manually extracted and sorted data were compared, and we found that the same results were obtained. This workflow provides opportunities to perform fast in-depth venom variation and venom composition research. It opens new possibilities for studying animal venoms as evolutionary model systems and study venom variation in the aid of developing better antivenoms.
Dermonecrosis caused by spitting cobra snakebite is the result of toxin potentiation and is prevented by the repurposed drug varespladib
Keirah E Bartlett1†, Steven R Hall1,2†, Sean A Rasmussen3, Edouard Crittenden1, Charlotte A Dawson1, Laura-Oana Albulescu1,2, Robert A Harrison1,2, Anthony J Saviola4, Cassandra M Modahl1, Mark C Wilkinson1, José María Gutiérrez5, Nicholas R Casewell1,2
1Centre for Snakebite Research & Interventions, Department of Tropical Disease Biology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, UK
2Centre for Drugs & Diagnostics, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, UK
3Department of Pathology and Laboratory Medicine, Queen Elizabeth II Health Sciences Centre and Dalhousie University, 7th Floor of MacKenzie Building, 5788 University Avenue, Halifax, Nova Scotia, Canada
4Department of USA Biochemistry and Molecular Genetics, 12801 East 17th Avenue, University of Colorado Denver, Aurora, USA
5Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica
Snakebite envenoming is a neglected tropical disease that causes substantial mortality and morbidity globally. The venom of African spitting cobras often causes permanent injury via tissue-destructive dermonecrosis at the bite site, which is ineffectively treated by current antivenoms. To address this therapeutic gap, we identified the aetiological venom toxins responsible for causing local dermonecrosis. While cytotoxic three-finger toxins were primarily responsible for causing spitting cobra cytotoxicity in cultured keratinocytes, their potentiation by phospholipases A2 toxins was essential to cause dermonecrosis in vivo. This evidence of probable toxin synergism suggests that a single toxin-family inhibiting drug could prevent local envenoming. We show that local injection with the repurposed phospholipase A2-inhibiting drug varespladib significantly prevents local tissue damage caused by several spitting cobra venoms in murine models of envenoming. Our findings therefore provide a new therapeutic strategy to more effectively prevent life-changing morbidity caused by snakebite in rural Africa.
Therapeutic applications of snake venom toxins: a comprehensive review – in-silico analysis and future directions
Anas Bedraoui1, Montamas Suntravat2,3, Zakaria Alouani1, Salim El Mejjad1, Najib Ramzi1, Salwa Enezari1, Naoual Oukkache5, Elda, E Sanchez2,3, Jacob A Galan4, Rachid El Fatimy1 and Tariq Daouda1
1Institute of Biological Sciences (ISSB-P), UM6P- Faculty of Medical Sciences (FMS), Mohammed VI Polytechnic University (UM6P), Ben Guerir, Morocco
2National Natural Toxins Research Center (NNTRC), Texas A&M University-Kingsville, Kingsville, TX, USA
3Department of Chemistry, Texas A&M University-Kingsville, Kingsville, TX, USA
4Department of Human Genetics, University of Texas Rio Grande Valley, Brownsville, TX, USA
5Laboratoire des Venins et Toxines, Département de Recherche, Institut Pasteur du Maroc, 1, Place Louis Pasteur, Casablanca 20360, Morocco
Toxins derived from snake venom are a promising source of bioactive molecules for medical applications. They contain a plethora of compounds that can be leveraged to treat Cardiovascular Disease (CVD), diabetes, stroke, cancer, and pain. For instance, Bradykinin Potentiating Peptide (BPP), extracted from Bothrops jararaca snake venom, has been instrumental in the development of Captopril, a pivotal drug in the treatment of CVD and diabetes. Other bioactive compounds like Three-Finger Toxins (3FTx) have also shown significant promise in treating CVD and pain, while Snake Venom Metalloproteinases (SVMPs) and Phospholipase A2s (PLA2s) have largely contributed to advancements in cancer treatments. Further, Disintegrins have proven to be beneficial for stroke therapy. However, the full extent to which snake venom toxins can be used for therapeutic purposes remains to be defined. Here, we present approved snake venom-based drugs for treating CVD, diabetes, stroke, and pain. Then, we present a statistical overview of the distribution of toxins within 130 snake species from the Crotalidae, Viperidae, and Elapidae families. Using machine learning dimensionality reduction methods t-SNE, PCA and UMAP, we generate 2D cluster visualizations of the 130 snake species based on their respective families and associated Lethal Dose 50% (LD50) levels. Finally, we discuss future perspectives on how Artificial Intelligence (AI) models could potentially lead to the discovery of new toxin-derived drugs.
Bloody insights: using organ-on-chip technology to study haemorrhagic activities of snake venoms on endothelial tubules
Mátyás A Bittenbinder1,2, Flavio Bonanini3, Dorota Kurek3, Freek J Vonk1,2, Jeroen Kool2
1Naturalis Biodiversity Center, 2333 CR Leiden, The Netherlands
2AIMMS Division of BioAnalytical Chemistry, Vrije Universiteit Amsterdam, The Netherlands
3Mimetas, Leiden, The Netherlands
Snakebite envenomation is a major public health issue which causes severe morbidity and mortality affecting millions of people annually. Studying the haemorrhagic effects in vitro has been challenging as the effects observed in vivo (i.e., rapid onset of haemorrhagic activities) is not observed during in vitro studies. To date, the study of tissue-damaging effects of snake venom toxins has been largely based on two-dimensional cell culture models. These do not have the tubular morphology of vasculature found in vivo and lack important environmental cues from the cellular microenvironment, such as interaction with the extracellular matrix (ECM) and exposure to flow. To bridge this ‘discrepancy’ in the tissue-damaging effects observed in vitro and in vivo models, we have implemented organ-on-a-chip technology to investigate the effects of four different snake venoms on a microfluidic blood vessel model. We were able to multiplex several readouts to comprehensively assess the tissue-damaging activities of a panel of snake venoms on a human blood vessel model grown in an organ-on-chip platform. We adapted image-based protocols using a fluorescence microscope platereader to assess endothelial barrier function for monitoring the vascular leakage kinetically after venom exposure. Simultaneously, we assessed morphology and viability allowing us to differentiate between different modes of toxicity of the different venoms, such as blood vessel disruption without significant cell killing or extensive cell death without apparent structural disruption. Using this approach, we were able to differentiate between at least two distinct mechanisms by which the microvasculature is being affected. The first mechanism being delamination of the endothelial cell monolayer from its surroundings, the second mechanism involving disruption of the endothelial cell membrane.
Molecular identification of the disintegrin protein in the venom of four snake species of the genus Bothrops from Peru
Tania A Broncano1, Daniel A Torrejón1, Fanny Lazo1, Edith Rodríguez1, Armando Yarlequé1,2, Dan E Vivas-Ruiz1,2
1Laboratorio de Biología Molecular, Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos (Perú)
2Network for Snake Venom Research and Drug Discovery, http://snake-research.com Disintegrins (DIS) are produced by proteolysis by snake venom metalloproteinases type II (SVMP-II). These proteins bind to integrins generally through the RGD motif, altering cell adhesion and migration, apoptosis, platelet aggregation, and angiogenesis. Due to these interesting properties, the objective of this work was to molecularly identify the DIS in the venom of Bothrops atrox, B. pictus, B. barnetti and B. brazili snakes of greatest clinical importance in Peru, and with this achieve their cloning. It began with the isolation of RNA from lyophilized venom, its conversion to cDNA, and amplification. The amplified products were analyzed by electrophoresis and purified for their insertion into the pET101/D-TOPO cloning vector. The transformation was performed in chemically competent E. coli OneShot strain TOP10 cells by heat shock. The gene cassette of the transformed colonies was confirmed by colony PCR and subsequent sequencing. Sequences were identified by homology using BLASTn and the Translate tool – ExPASy program was used to obtain the amino acid sequence. A multiple alignment was performed using Clustal Omega. The prediction of the physicochemical properties was carried out with the Protparam program and of the secondary structure with the PRABI server. As results, the DIS of the four analyzed species were identified, which showed high homology with those reported in the database. Interestingly, the B. pictus DIS has a three amino acid insert close to the N-terminal region. Regarding the secondary structure, there are few conformational variants, predominantly random folding (~90%). It is concluded that the four species analyzed express the disintegrin protein in their venom and that they show homology in both primary and secondary structure with others reported in the database. This is the first report of disintegrins in the venom of Peruvian snakes. Financial support: VRIP-UNMSM (Grant: PINTERDIS B2110007i) and PROCIENCIA (Grant: 079-2021-PROCIENCIA).
Intraspecific variability in the venom of Bothrops pictus “jergón de la costa”
Juan J Cabrejos1, Dan E Vivas Ruiz1,Daniel A Torrejon1, Jorge A Roque1, Alex D Proleon1, Angie Regalado1, Raul Rivas1, Jordano Espinoza1, Paola Rosas1, Fanny Lazo1, Javier Cárdenas2, Armando Yarlequé1
1Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos, Lima, Perú
2Facultad de Enfermería, Universidad Nacional del Callao, Callao, Perú
The variability of venom in snakes is an intrinsic faculty that is conditioned by environmental and ecological factors. Bothrops pictus is an endemic Peruvian viper of which very little is known about its venom and the implications of its variability. In the present work we focus on studying the variation of the venom of 9 B.pictus specimens from different localities and venom coloration (yellow and white), using reducing and non-reducing SDS PAGE plus three enzymatic assays: phospholipase A2, serine proteases, and hyaluronidases. According to the electrophoretic profiles, it was evidenced that the Bothrops pictus specimens presented variability in the lower molecular weight protein bands. Regarding the enzymatic activities in PLA2, SVSP and HYA, the yellow venoms presented (0.018 -0.025 AU), (13.3 – 15.3 AU) and (7.9 – 14.7 UDI) respectively, while the white venoms (0.005 – 0.019 AU ), (4.0 – 12.3 AU) and (4.1 – 38.3) respectively. In general, it is evident that the most variable activity was hyaluronidase and that the yellow venom specimens presented greater activity than the white ones. The remarkable variation of the venom is concluded and emphasis is placed on continuing to investigate the activities of the different toxins, the implications of the antivenom and the factors that can condition this type of variation, one of the most important being phylogenetic, a criterion that has not yet been determined been elucidated in this species.
Evaluation of Varespladib action against the myotoxic effects of Micrurus spixii venom
Nathalia M Cantuaria1, Marisa MT da Rocha2, Caroline S Silva2, Stephen Hyslop3, Rafael S Floriano4, Matthew Lewin5, José M Gutierrez6, Yoko O Franco1
1University of Sorocaba (UNISO), Sorocaba, SP, Brazil
2Laboratory of Herpetology, Butantan Institute, São Paulo, SP, Brazil
3Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Campinas, SP, Brazil
4University of West São Paulo, Presidente Prudente, SP, Brazil
5Research and Development, Ophirex, Inc., Corte Madera, CA 94925, USA
6Clodomiro Picado Institute, Faculty of Microbiology, University of Costa Rica, San José, Costa Rica
Faced with the global public health issue involving snakebites and in line with the World Health Organization’s proposal to reduce 50% the harmful consequences of poisoning until 2030, Varespladib could be an alternative proposal. Varespladib, a phospholipase inhibitor, is effective against the toxic activities of different snake venoms rich in phospholipases. The myotoxic action present in some venoms can be related to phospholipases. The Micrurus spixii (M. spixii) venom presents phospholipase as one of the main toxins, and studies with M. spixii and Varespladib do not exist in the literature. Besides, the venom is less studied than other species of Micrurus. A traditional biomarker of myotoxicity, creatine kinase (CK NAC UV, Bioclin), was selected for this study with male Swiss mice approved by animal ethics (1807290922). Animals were divided in 5 groups (n=5) to receive intramuscularly in the right hind paw: (1) 50 µL of saline; (2) 10 µg of venom dissolved in 50 µL of saline; (3) 10 µg of venom + 2X effective dose (ED) of anti-elapid serum; (4) 10 µg venom + 250 µM Varespladib and (5) 10 µg of venom + 2X ED serum + 250 µM Varespladib. The animals were euthanized 3 h after each treatment for blood collection. The CK (U/L) values were (1): 119.35 ± 48.76; (2): 1953.89 ± 600.43; (3): 1384.35 ± 126.78; (4): 628.41 ± 193.64 and (5): 218.32 ± 180.95. We can conclude that the venom of the M. spixii species has myotoxic activity, causing a significant increase in CK levels. The antivenom did protect against the systemic effects, but did not against myotoxicity. Varespladib was more effective than antivenom in inhibiting myotoxicity. The association of Varespladib and antivenom showed a synergistic effect to inhibit the myotoxicity-induced by the venom.
Mapping the post-synaptic neurotoxic profile of mamba venoms by functional and ligand binding assays
Iara A Cardoso1, Mark C Wilkinson1, Christiane Berger-Schaffitzel2, Rohit N Patel1, Nicholas R Casewell1, Stefanie K Menzies1
1Centre for Snakebite Research and Interventions, Liverpool School of Tropical Medicine, Liverpool, UK
2BrisSynBio Bristol Synthetic Biology Research Centre, University of Bristol, Bristol, UK
Snakes from the genus Dendroaspis (popularly known as mambas) are among the most medically important snakes in Africa. Mamba envenoming is one of the most severe cases of snakebite, causing progressive descending paralysis and respiratory failure, which may become lethal. Their venom contains several neurotoxins, including toxins of the three-finger toxin (3FTx) family, which possess diverse biological functions, including the blockage of muscular nicotinic acetylcholine receptors (nAChRs) located on the post-synaptic membranes of neuromuscular junctions (these 3FTxs are known as long or short chain α-neurotoxins). The work presented here aims to characterise the post-synaptic neurotoxic effects of venoms from 5 mamba species from sub-Saharan Africa: D. polylepis, D. angusticeps, D. j. jamesoni, D. j. kaimosae, and D. viridis. The venoms were first fractionated through gel filtration and/or cation exchange chromatography (CX) and subsequent fractions were applied on SDS-PAGE. Fractions from CX were used in a cell-based assay of nAChR function using immortalised TE671 cells expressing muscle-type nAChR, and the results demonstrated the presence of α-neurotoxins in several venom fractions in all 5 species. Also, CX fractions from D. polylepis and D. angusticeps were analysed by Surface Plasmon Resonance, providing their profile of binding to a recombinant AChBP (a protein with binding properties similar to the α7-type nicotinic receptors). The fractions that showed activity in cell assays were submitted to reversed phase chromatography to isolate individual toxins and will be further identified by mass spectrometry. The neutralising efficacy of SAIMR Polyvalent antivenom was also determined for mamba venoms in the cell-based neurotoxicity assay. These results will provide useful information about mamba venom composition and highlight the post-synaptic antagonist potential of their α-neurotoxins.
IL-1R/IL-1β signaling involvement in CD68 induced intestinal inflammation and mucine depletion after Aah venom exposure
Nehla Chabane, Fatima Laraba-Djebari, Djelila Hammoudi-Triki
USTHB, Faculty of Biological Sciences, Laboratory of Cellular and Molecular Biology, Algiers 16111 Bab‑Ezzouar, Algeria
Scorpion venom result in the dysregulation of immune response caracterized by inflammatory cells accumulation and oxidative stress. Immune cells residing in the lamina propria participate in the initiation of inflammation. In fact, activated macrophages during colitis produced several of oxygen-derived free radicals and secrete various proinflammatory cytokines such as TNF-α, IL-1β, and IL-6. IL-1β is released after NLRP3 inflammasome activation, it contribute to maintain intestinal epithelial barrier. The mucus on the surface of the intestinal mucosa participates plays an important role in protecting the epithelium. Our study aims to investigate the mechanisms of canonical pathway mediated by IL-1 receptor in Aah venom induced colitis. To evaluate the impact of inhibition of IL-1R on immune response induced by Aah venom, we have used an inhibiteur of IL-1 receptor in envenomed mice. ROS generation was assay with quanting nitrite oxide and H202 levels in colon supernantant as well as MDA level. For histological analysis the degree of histological damage of the colonic mucosa was evaluated after haematoxylin and eosin staining. Immunohistochemestry targeting CD68 and blue alcian staining for mucosecretion evaluation were carry on colonic sections. Our data demonstrated that the Aah venom induced increase CD-68 accumulation and overproduction of ROS in colon tissue after Aah venom exposure. Moroever, histopathological analysis revealed colonic damage associate with mucine depletion. However, blocking IL-1R reduced macrophage infiltrating resulting in the decrease of nitrooxidative stress, prevent histopathological alteration and mucine depletion were also improved. Our study demonstrated that IL-1R/IL-1β can modulte the intestinal inflammation induced by Aah venom, this data could help to developp biotherapy thargeting signaling pathway in the management of inflammation of the gut.
Discovery and development of repurposed small molecules to neutralise snake venom toxins
Rachel H Clare1, Laura-Oana Albulescu1, Adam Westhorpe1, Rohit N. Patel1, Nada Mosallam2, Daniel Chong-Jun-Weng2, Ramachandran Gunasekar2, Charlotte A Dawson1, Steven Hall1, Evy Bore2, Christopher Woodley2, Nivya James2, Edouard Crittenden1, Emma Stars1, Amy E Mariott1, Jeroen Kool3, Neil G Berry2, Paul M O’Neill2, Nicholas R Casewell1
1Centre for Snakebite Research and Interventions, Liverpool School of Tropical Medicine, UK
2Department of Chemistry, University of Liverpool, UK
3Division of BioAnalytical Chemistry, Vrije Universiteit Amsterdam, The Netherlands
Snakebite results in >100 000 deaths annually. Despite antivenom being the only currently approved therapeutic for the treatment of snakebite, these therapies have several limitations, i) requirement for administration in a healthcare setting due to intravenous delivery and management of severe adverse reactions, ii) species-restrictive efficacy as well as poor dose efficacy due to variation in the venoms used for immunisations, iii) access is compounded by the inconsistency in availability, largely attributed to the requirements for refrigeration, and the high purchasing cost. Therefore there is considerable interest in next generation therapies. A handful of toxin-inhibiting drugs have demonstrated highly promising preclinical efficacy against snakebite, and the potential to administer such drugs orally in snakebite-affected communities offers an exciting new treatment strategy. Of the many venom components found across medically important venomous snakes, the three finger toxins (3FTx), phospholipases A2 (PLA2) and snake venom metalloproteinases (SVMP) are considered amongst the most important therapeutic targets due to their contribution to severe pathology. However, the chemical space explored to date for snakebite drugs is highly limited (varespladib against PLA2 and two SVMP candidates, DMPS and marimastat). To address this limited drug portfolio, we present the progress of three parallel toxin specific drug discovery programs, initiated by high-throughput screening of large diverse repurposed drug libraries. Our aim is to broaden the chemical space of hit molecules against SVMP, PLA2 and 3FTx toxins. Across our three projects we have tested >18,000 drugs in different assays, against different venoms. From this we have designed ~60 novel analogs across 6 chemical series with three lead series against SVMP and PLA2 toxins progressing into pre-clinical testing.
Excitatory action of peptides from Bothrops bilineatus smaragdinus (Viperidae: Crotalinae) venom on the motor cholinergic transmission in vitro
Fernanda YGM Couceiro1, Isabele N Oliveira1, Francis L Pacagnelli1, Bruno Lomonte2, Rafael J Borges3, Daniel C Pimenta4, Kristian A Torres-Bonilla5, Stephen Hyslop5, Rafael S Floriano1
1Laboratory of Toxinology and Cardiovascular Research, University of Western São Paulo (UNOESTE), Presidente Prudente, SP, Brazil
2Clodomiro Picado Institute, University of Costa Rica (UCR), San José, Costa Rica
3The Center of Medicinal Chemistry, Center for Molecular Biology and Genetic Engineering, State University of Campinas (UNICAMP), Campinas, SP, Brazil
4Laboratory of Biochemistry and Biophysics, Butantan Institute (IB), São Paulo, SP, Brazil.
5Department of Translational Medicine, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Campinas, SP, Brazil
Bothrops bilineatus smaragdinus (Amazonian viper) venom is characterized by inducing an intense neuromuscular facilitation followed by irreversible blockade in mouse nerve phrenic-diaphragm (PND) preparation. In this study, we identified two peptides potentially involved in the uncommon B. b. smaragdinus venom-induced neuromuscular facilitation and examined their influence on the nicotinic (cholinergic) transmission using electrophysiological approaches in mouse PND preparation. Venom was fractionated by gel filtration using a TSKgelÒ G2000SWXL column coupled to a HPLC Shimadzu SCL-10AVP system, with fractions being afterwards subjected to residual neuromuscular activity through an Ugo Basile S.R.L. 4400 myographic system. Peptides were purified by HPLC-RP using a JupiterÒ C18 column and their intact mass determined by mass spectrometry. Evoked (EPPs) and miniature (MEPPs) end-plate potentials were determined by conventional electrophysiology techniques. Gel filtration revealed eight fractions (Peaks: P1–P8) from venom, with fraction P8 (15 mg/ml) presenting intense increase in the frequency of MEPPs [MEPPs/min: from 8.7±2.9 (basal) to 75.7±7.1 (t30 min), p<0.05, n=4] and amplitude of EPPs [EPP/mV: from 7.5±2.3 (basal) to 28.6±9.2 (t30 min), p<0.05, n=4]. HPLC-RP analysis of the fraction P8 determined the presence of two peptides with 652,4849 Da (P8-1) and 531,4108 Da (P8-2) of intact mass. Peptide P8-1 [8.3 mg/ml (55% of P8 mass)] reproduced partially the P8-induced acetylcholine excitatory action [MEPPs/min: from 6.3±2.3 (basal) to 40.2±13 (t30 min), p<0.05, n=4], being more effective when combined with P8-2 [6.8 mg/ml (45% of P8 mass)] to produce increase frequency of MEPPs [MEPPs/min: from 10.2±3.4 (basal) to 58±13.7 (t30 min), p<0.05, n=4]. In conclusion, P8-1 appears to be primarily involved with the neuromuscular excitatory effect previously seen with B. b. smaragdinus venom in vitro, most likely stimulating acetylcholine release from motor neuron terminals.
Severe systemic features following Lion’s Mane Jellyfish envenomation
Nathaniel I Keymer1, Michael Eddleston2, Simon L Hill3, James M Coulson1
1National Poisons Information Service, Cardiff, UK
2National Poisons Information Service, Edinburgh, UK
3National Poisons Information Service, Newcastle, UK
A 60-year-old female patient was discussed with our service following a lion’s mane jellyfish (Cyanea capillata) envenomation in Scotland. Positive identification was made by the patient. She had been stung across her left anterior upper arm, shoulder and chest. Following a hot shower at home, she presented to A&E at three hours post exposure. Her clinical course was marked by severe pain bilaterally in her hands and feet which was refractory to morphine (the patient remarked the pain was worse than childbirth), along with an episode of hypertension followed by bradycardia. Her highest blood pressure was 193 mmHg systolic and her lowest heart rate was 35 bpm; these had stabilised to around 70-80 bpm and 150 mmHg respectively by six hours post exposure with supportive care, IV fluids and chlorphenamine. She also experienced nausea and “writhing movements”. Her symptoms had predominantly resolved by nine hours post exposure with the exception of erythematous rash from the tentacles. A similar Irukandji-like reaction has been reported previously in a Scottish C. capillata envenomation; however, the patient reported above was bradycardic rather than tachycardic. Irukandji syndrome is suggested to be the result of a catecholamine storm, and the (assumedly reflex) bradycardia present in this patient is therefore surprising. A directly neurotoxic component has recently been identified in C. capillata venom. The possibility that this or another as-yet-unidentified venom may act centrally, along with the difficulties with pain control identified in this patient, suggest that an alternative approach to pain management may be required. Clinicians should consider other modes of analgesia early in patients with severe pain unresponsive to opioids or systemic features following C. capillata envenomation.
Trends in Lion’s Mane Jellyfish envenomation reported to the National Poisons Information Service
Nathaniel I Keymer1, Sally M Bradberry2, Laurence A Gray1, Euan A Sandilands3, Ruben HK Thanacoody4, James M Coulson1
1National Poisons Information Service, Cardiff, UK
2National Poisons Information Service, Birmingham, UK
3National Poisons Information Service, Edinburgh, UK
4National Poisons Information Service, Newcastle, UK
Eleven cases of jellyfish envenomation tentatively or confidently identified as caused by the lion’s mane jellyfish (Cyanea capillata) have been reported to our service since 2008 (55% female, age range 5-60 years). Most occurred in Scotland, with one case each in Galway, Ireland and Cramlington, England. C. capillata have been observed throughout the UK, but most commonly in Scotland. Most occurred in August and September, with one case in June and two in July. All patients developed local effects from the sting (erythema 6/11, oedema 4/11, paraesthesia 4/11, pruritis 3/11). Pain (including myalgia and arthralgia) was reported in 8/11 patients which was described as severe in 6 patients; body parts distant from the site of envenomation experienced pain in 4 patients. Morphine was reported to have little-to-no effect on the severity of the pain in 2/10 patients. Further signs of systemic envenomation were present in 3/11 patients and included sweating (1/11), cramps (1/11), nausea (2/11), shaking/writhing movements (2/11), raised creatinine kinase (1/11), raised liver function tests (1/11), hypertension (2/11) and bradycardia (1/11). In 2/11 patients where the clinical course was known, systemic symptoms including pain resolved within 9 and 12 hours. 4/11 patients were stung on the arm or hand, 3/11 on the face or eye, 3/11 across the torso and the sting site was unknown in 3/11 (1 patient was stung on both the arm and torso, another was stung on the hand and then rubbed their eye). All 3 patients stung across the torso developed severe pain or systemic symptoms. 1/2 patients stung only on the arm or hand developed systemic symptoms. No patient stung on the face developed systemic symptoms, though swelling threatening the airway and significant pain were noted. Significant lion’s mane envenomation is marked with severe, whole-body pain refractory to morphine. Systemic features and severe pain are more common following exposure across a larger area of skin.
Delayed toxicity following jellyfish stings reported to the National Poisons Information Service (NPIS)
Stephen SD Jones1, Sally M Bradberry2, Laurence A Gray1, Euan A Sandilands3, Ruben HK Thanacoody4, James M Coulson1
1National Poisons Information Service, Cardiff, UK
2National Poisons Information Service, Birmingham, UK
3National Poisons Information Service, Edinburgh, UK
4National Poisons Information Service, Newcastle, UK
Jellyfish stings to humans are relatively common and often result in mild, short-lived features. Delayed toxicity may occur. From 1st January 2008 to 31st May 2023 the NPIS received 48 enquiries concerning patients who presented to healthcare providers more than 24 hours after a jellyfish sting (52% of all jellyfish enquiries). The median time since exposure was 7 days (range 1 to 35 days). Twenty-eight patients were female (58%) and most patients (n=40, 83%) were adult. Six patients were less than 16 years old. Poisons Severity Score (PSS) at the time of enquiry was minor (PSS = 1) in 39 cases, moderate (PSS=2) in 5 cases and none (PSS=0) in 3 cases. No cases of severe toxicity (PSS=3) were reported. Patients were most commonly stung on the arms (n=11, 23%) and legs (n=10, 21%). In most cases (n=28, 58%) it was not known if the patient had received first aid at the time of exposure but 11 patients were taking antihistamines and 5 were prescribed antibiotics. A further 5 patients (10%) had treated the initial sting with vinegar. The median age of patients with moderate features (PSS=2) was 30 years (IQR=38.5). Four patients with PSS=2 were female. Of the five cases with PSS =2 the jellyfish was tentatively identified as a Mauve stinger (Pelagia noctiluca) (n=3) and Portugese Man o’ war (Physalia physalis) (n=1). Four patients were stung on the arm or wrists. Two patients reported being stung several times during the exposure. Common features among moderately poisoned patients included pain and inflammation at the site. One patient developed areas of anaesthesia affecting the skin distal to the sting. Another patient developed a low grade fever with rigors, diaphoresis, leukopenia and thrombocytopenia and presented five weeks after the initial exposure. The persisting features were attributed to prolonged antigenic effect of embedded foreign proteins. Patients may continue to experience local and systemic features of toxicity several weeks after a jellyfish sting.
Histopathological analysis and in situ localization of the venom from the most dangerous scorpion species in North Africa: Androctonus mauritanicus, Androctonus australis hector and Buthus occitanus
Bouchra Darkaoui1,2, Mohammed Aksim3, Ayoub Lafnoune1,2, Soukaina Khourcha1, Ayoub Aarab4, Rachida Cadi2 and Naoual Oukkache1
1Laboratory of Venoms and Toxins, Pasteur Institute of Morocco, 1 Place Louis Pasteur, Casablanca 20250, Morocco
2Laboratory of Molecular Genetics, Physiopathology and Biotechnology, Faculty of Sciences Ain Chock, Hassan II University of Casablanca, Morocco
3Laboratory of Anatomic Pathology, the Regional Hospital Centre Hassan II, Agadir, Morocco
4Laboratory of Pathological Anatomy Marrakech, Agadir, Morocco
In North Africa, scorpion sting poses a significant and urgent public health concern, particularly for children, with high morbidity and mortality rates. The main culprits are scorpion species from the Buthidae family, notably Androctonus mauritanicus (Am), Androctonus australis hector (Aah), and Buthus occitanus (Bo). These encounters lead to a cascade of harmful effects, starting with localized tissue damage and progressing to systemic manifestations. The venom of these scorpions is highly neurotoxic and myotoxic, worsening the severity of envenomation. This study aims to evaluate histopathological changes and identify venom components’ specific locations in organs namely the brain and the heart. Toxicity testing using the LD50 test on Swiss mice weighing 18-22 g was conducted to assess the venom’s toxic activity. Sublethal doses of venom were then injected into mice for histopathological analysis and in situ localization. After four hours, the mice were sacrificed, and the collected organs were examined. Immunohistochemical staining was performed using antibodies against the Am, Aah, and Bo venoms. Our findings demonstrated that the Am venom exhibited the highest toxicity, followed by the Aah venom, and then the Bo venom. The study revealed severe tissue damage, including cellular loss in the brain, myofiber degeneration in the heart. Notably, the Am venom displayed the most toxic effects across all organs studied, followed by the Aah venom and then the Bo venom. Additionally, our results highlighted the important cytoplasmic and membranous staining in the heart compared to the brain tissue for the three venoms studied with 60% in the heart and 40% in the brain for the Am venom, 80% in the heart and 50% in the brain for the Aah venom, 60% in the heart and 40% in the brain for the Bo venom.
Cases of ibogaine toxicity reported to the UK National Poisons Information Service (UK NPIS) over a 10-year period
Ella P Edwards1, Callum W Welfoot1, Sally M Bradberry2, Laurence A Gray1, Euan A Sandilands3, Ruben H K Thanacoody4, and James M Coulson1
1National Poisons Information Service, Cardiff, United Kingdom
2National Poisons Information Service, Birmingham, United Kingdom
3National Poisons Information Service, Newcastle, United Kingdom
4National Poisons Information Service, Edinburgh, United Kingdom
Ibogaine is a psychoactive alkaloid derived from the root bark of the West African shrub Tabernanthe iboga. It is not licensed in the UK but is used by individuals to treat drug and alcohol addiction. A retrospective analysis of telephone enquiries involving ibogaine between 1 January 2012 and 31 December 2022 to the UK NPIS was performed. Eleven enquiries relating to ibogaine were received in this period. Of these, four related to the same patient therefore 7 cases in total. Five (71%) patients were male and 2 (29%) were female. The majority (57%) were in the age category 31-40. All enquiries were from hospitals and related to exposure during recreational misuse. Three sources of ibogaine were reported: bought online, from a drug dealer and from a shaman. The indication was unspecified in 3 cases but in two cases ibogaine was being used to self-medicate for heroin addiction and in two cases it was to treat insomnia. The dose, where reported, ranged from 5 g to 34 g. Of the 44 symptoms reported, 21 (48%) were neuropsychological including hallucinations (14%), anxiety (10%), speech disorder (10%), stupor (10%) and coma (10%). Cardiovascular symptoms made up 10 (23%) of the total symptoms, including bradycardia (30%), QT prolongation (20%), cardiac arrest (20%) and torsades de pointes (20%). Gastrointestinal symptoms accounted for five (14%) of the symptoms with vomiting the most common. In 1 (14%) case the MAXPSS was minor, in 3 (43%) it was moderate and in 3 (43%) it was severe. Ibogaine enquiries account for a small number of total enquiries to the UK NPIS. However, the number of moderate and severe cases is significantly greater for ibogaine compared to the total number of enquiries for this period (chi-square 96.6, p <0.01). Despite data limitations, this report suggests individuals using ibogaine in variable doses for recreational purposes are at risk of developing significant toxicity. Further studies of dose-response relationship are required.
Molecular and bioinformatic identification of a new serine protease transcript from Bothrops barnetti venom, pit viper snake from Perú
Jordano Espinoza1, Dan E Vivas-Ruiz1,2, Daniel Torrejón1, Félix A Urra2,3, Alex Proleón1, Frey Romero4, Fanny Lazo1, Edith Rodríguez1,2, Armando Yarlequé1,2
1Laboratorio de Biología Molecular, Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos (Perú)
2Network for Snake Venom Research and Drug Discovery, http://snake-research.com
3Clinical and Molecular Pharmacology Program, Institute of Biomedical Sciences (ICBM), Faculty of Medicine, University of Chile, Santiago – Chile
4Facultad de Ciencias Biológicas, Universidad Nacional San Agustín, Arequipa, Perú
Snake venom is a complex mixture of peptides and proteins that act synergistically during the bite poisoning process of these snakes. Within this pool of proteins, we have the serine proteases that are known for their functional diversity and their action on specific components of the hemostatic system. It is known that serine proteases may be present in viperid venom that act as: coagulation factor activators, plasminogen activators, fibrino(geno)lytic enzymes, coagulating enzymes, kininogenases, among others. In 2013, our research group identified and characterized barnetobin, a thrombin-like enzyme (SVTLE) from Bothrops barnetti snake venom. Enzyme that presented unique structural and functional characteristics. For this reason, we proceeded to continue with more studies through the construction of a cDNA library, reaching the molecular identification of a new serine protease transcript from the venom of this snake. The methodology was based on the extraction of total RNA from the lyophilized venom of a specimen of the snake B. barnetti, the conversion to complementary cDNA and the amplification by means of polymerase chain action (PCR) using previously designed primers. Then, the purification of the amplified DNA carried out, the construction of cDNA library in commercial strains of Escherichia coli for their cultivation in Luria Bertani medium. Finally, the molecular and bioinformatic characteristics of the new serine protease, under the name of SVSP-Bb2, were determined after sequencing and subsequent deduction in silico of the protein codified. The results were the identification of a new sequence transcript for 702 bp long which deduced sequence had an ORF for a protein sequence of 233 aa. The bioinformatics predictions of the deduced protein (SVSP-Bb2) showed a similar sequence (90.56%) and phylogenetically more related to the protease A from Bothrops jararaca snake venom than the barnetobin (67.3% similarity).
Jatropha gossypiifolia L. (Euphorbiaceae) aqueous extract reduces hemostatic disorders induced by Bothrops erythromelas snake venom in mice via inhibition of fibrinogenolytic and procoagulant proteases
Juliana Felix-Silva1,2, Jacinthia B Xavier-Santos2, Julia G R Passos2, Jacyra A S Gomes2, Denise V Tambourgi3, Ivanise M M Rebbechi1, Matheus F Fernandes-Pedrosa2
1Clinical Haematology Laboratory, Department of Clinical and Toxicological Analysis, Faculty of Pharmacy, Federal University of Rio Grande do Norte, Natal, RN, Brazil
2Laboratory of Pharmaceutical Technology and Biotechnology (TecBioFar), Faculty of Pharmacy, Federal University of Rio Grande do Norte, Natal, RN, Brazil
3Immunochemistry Laboratory, Butantan Institute, São Paulo, SP, Brazil
Jatropha gossypiifolia L. (Euphorbiaceae) is a medicinal plant used in folk medicine for snakebite treatment, especially in Northeastern Brazil where antivenom availability is limited. Bothrops erythromelas is the primary snake species causing these incidents in this region. Given the significance of systemic toxicity in Bothrops envenomation, we evaluated the inhibitory efficacy of J. gossypiifolia leaf aqueous extract (AE) on hemostatic effects induced by B. erythromelas venom (BeV). In mice, AE (100-200 mg/kg, i.p.) significantly reduced thrombocytopenia (72.5% inhibition, p<0.01) and defibrinogenemia (50.3% inhibition, p<0.001) caused by BeV (10 µg, i.p.), indicating protection against BeV-induced hemostatic effects. Due their relevance in venom-induced consumption coagulopathy (VICC), common in Bothrops snakes, the in vitro inhibitory action of AE against fibrinogenolytic and procoagulant activities of BeV was also assessed. Using bovine fibrinogen as substrate and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) with densitometric analysis, we observed that pre-incubating BeV with AE reduced Aα and Bβ chains degradation by approximately 64% and 75%, respectively. BeV’s Minimum Coagulant Concentration (MCC, smallest amount coagulating plasma in 60 s) was 0.5 μg, and AE pre-incubation significantly prolonged the clotting time of 1 MCC-P to 195.5 s, demonstrating AE’s inhibitory potential against procoagulant activity of BeV. These in vitro results indicate that the in vivo protective effects of AE against hemostatic disorders induced by BeV may be related with an inhibition of snake venom proteases involved in VICC. Thus, is concluded that J. gossypiifolia contains compounds reducing B. erythromelas-induced hemostatic disorders, indicating the potentiality of this vegetal species as a source of new bioactive molecules against Bothrops venom.
The Fast and The Furriest: Investigating the rate of selection on mammalian toxins
Leah Lucy Joscelyne Fitzpatrick1,2, Vincent Nijman1,2, Rodrigo Ligabue-Braun and K.Anne-Isola Nekaris1,2
1Nocturnal Primate Research Group, Department of Social Sciences, Oxford Brookes University, Oxford OX3 0BP, UK
2Centre for Functional Genomics, Department of Health and Life Sciences, Oxford Brookes University, Oxford OX3 0BP, UK
3Department of Pharmacosciences, Federal University of Health Sciences of Porto Alegre (UFCSPA), Avenida Sarmento Leite 245, Porto Alegre 90050-130, Brazil
The evolution of venom and the selection pressures that act on toxins have been increasingly researched within toxinology in the last two decades, in part due to the exceptionally high rates of diversifying selection observed in animal toxins. In 2015, Sungar and Moran proposed the ‘two-speed’ model of toxin evolution linking evolutionary age of a group to the rates of selection acting on toxins but due to a lack of data, mammals were not included as less than 30 species of venomous mammal have been recorded, represented by elusive species which produce small amounts of venom. Due to advances in genomics and transcriptomics, the availability of toxin sequences from venomous mammals has been increasing. Using branch- and site-specific selection models, we present the rates of both episodic and pervasive selection acting upon venomous mammal toxins as a group for the first time. We identified seven toxin groups present within venomous mammals, representing Chiroptera, Eulipotyphla and Monotremata: KLK1, Plasminogen Activator, Desmallipins, PACAP, CRiSP, Kunitz Domain One and Kunitz Domain Two. All but one group (KLK1) was identified by our results to be evolving under both episodic and pervasive diversifying selection with four toxin groups having sites that were implicated in the fitness of the animal by TreeSAAP (Selection on Amino Acid Properties). Our results suggest that venomous mammal ecology, behaviour or genomic evolution are the main drivers of selection, although evolutionary age may still be a factor. Our conclusion from these results indicates that mammalian toxins are following the two-speed model of selection, evolving predominately under diversifying selection, fitting in with other younger venomous taxa like snakes and cone snails—with high amounts of accumulating mutations, leading to more novel adaptions in their toxins.
Beyond the bloodstream: Unravelling the impact of i.v. or i.m. administered antivenom on venom neutralization in the lymphatic system
Erika Gamulin1, Maja Lang Balija1, Sanja Mateljak Lukačević1, Dražen Vnuk2, Ana Smajlović2, Beata Halassy1, Tihana Kurtović1
1Centre for Research and Knowledge Transfer in Biotechnology, University of Zagreb, Zagreb, Croatia
2Clinic for Surgery, Orthopaedics and Ophthalmology, Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia
Envenoming induced by snakebites constitutes a significant public health burden. Commonly, venoms are introduced into the subcutaneous or muscle tissue and carried to the interstitial space for distribution. It is widely recognized that those of vipers are primarily absorbed through the lymphatic system before being transported into the systemic circulation. This lymphatic absorption influences distribution and systemic availability of the venom constituents and serves as a “gateway” for their entrance into the circulation, allowing them to reach target organs. Even though a standardized protocol does not exist, parenterally administered antivenom remains the mainstay in the snakebite therapy. Widely held belief that i.v. administration of antivenoms is more effective than i.m. application is based on the studies on venom/antivenom pharmacokinetics in the bloodstream. Lately, it has been demonstrated that i.v. antivenom-mediated neutralization not only in the systemic circulation, but also in the lymphatic system might be highly important for clinical outcome. The role of i.m. antivenoms in the elimination of lymph-absorbed venom might be even greater, but it has not been studied yet. Our aim was to define the route of antivenom administration with the more pronounced impact on bioavailability of the s.c. injected venom and decrement of its quantities in the lymphatic compartment. Sheep, as large animal model, was employed. The dosage of venom corresponded to the typical amount yielded in milking of a mature snake and chosen to be representative of a typical envenomation. Specific antivenom was given either via i.m. bolus or i.v. infusion. Lymph samples were collected by a continuous drainage technique from the ductus thoracicus. Venom and antivenom concentrations were measured by using a respective in-house ELISA assay and their cumulative absorption was determined. The results on venom/antivenom distribution in the lymphatic system will be presented.
Mass-Spectrometry-Based Proteome Profiling of scorpion venoms: From species diversity to venom variability
Ines HILAL1,2, Soukaina KHOURCHA1,2, Amal SAFI2, Abdelaziz HMYENE2, Reto STÖCKLIN3 and Naoual OUKKACHE1
1Laboratory of Venoms and Toxins, Pasteur Institute of Morocco, Casablanca, Morocco
2Laboratory of Biochemistry, Environment and Food Technology, Faculty of Sciences and Techniques of Mohammedia, Morocco.
3Atheris Laboratories, Case Postale 314, CH-1233 Bernex, Geneva, Switzerland.
From its lethal sting to its medicinal potential, scorpion venom is a paradoxical elixir of nature, this venom contains a variety of molecules, including enzymes, molecules involved in allergic reactions, and, most notably, neurotoxins that exert a potent pharmacological activity on the ion channels of excitable cells’ membrane. In this study, we conducted an exhaustive analysis of the venoms derived from 26 Moroccan scorpions belonging to the Androctonus, Buthus, Hottentotta, and Scorpio genus. Using electrospray mass spectrometry coupled with high-performance liquid chromatography (HPLC), we aimed to provide detailed information regarding the composition and molecular nature of these venoms. We initially fractionated the venoms using HPLC, and the fractions were subsequently analyzed via mass spectrometry to establish peptide maps and fingerprints for each venom. We obtained a wide range of molecular weights, varying between 236 and 622, which were predominantly found between 2000 and 4000 Da and corresponded mainly to short toxins that blocked the K+ channels. Larger molecular weights exceeding 4000 Da were identified as long toxins modulating the Na+ channels. Moreover, we discovered other peptides below 2000 Da that were previously unknown. Our observations also indicated that the venom of the Androctonus, Buthus, and Hottentotta genera contained higher quantities of masses corresponding to both long and short toxins, while the venom of Scorpio maurus displayed a relatively lower toxin content. This study provides valuable insight into the complex composition of scorpion venoms and underscores the significance of understanding their composition in the development of novel therapeutic approaches.
Ontogenetic changes in adenosine abundance in Bitis arietans venom
Taline D Kazandjian, Mark Wilkinson, Charlotte A Dawson, Nicholas R Casewell
Centre for Snakebite Research and Interventions, The Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, UK
The puff adder (Bitis arietans) is a medically important snake with a wide geographical range, spanning across sub-Saharan Africa and parts of the Arabian Peninsula. Snake venoms are generally viewed as a proteinaceous mixture but can also contain small molecules such as adenosine, a hypotensive nucleoside previously detected in B. arietans venom. Although prior studies have shown ontogenetic changes in venom toxin composition can occur, changes in small molecule content are not well documented. Utilising high-performance liquid chromatography (HPLC), this study compared the adenosine abundance detected in the venoms of six hatchling B. arietans (3 male, 3 female; eSwatini heritage), with samples collected from 4 time points (4, 10, 16 and 22 months). The resulting quantified abundance data was compared between time points, and against venom pooled from adult B. arietans from the same geographical origin, to investigate whether adenosine content varies over time and to begin elucidating the ecological role of adenosine in B. arietans venom. No significant differences in adenosine abundance were detected between the 4 time points (ANOVA, F = 1.30, df = 3, p = 0.39) or between the sexes (unpaired t-test, t = 1.01, df = 22, p = 0.32) of the hatchlings, however young B. arietans consistently had nearly three times the relative abundance of adenosine than adult venom (15.2-16.7% per mg venom, compared to the adult mean of 5.3%). The higher relative adenosine content in juvenile B. arietans venom may reflect a compensatory effect for the reduced volume of venom injected during predatory bites, though this needs to be explored in future functional studies.
Quercetin-PLGA nanoparticles alleviate inflammation, oxidative stress and kidney tissue injury induced by Cerastes cerastes venom
Kahina Kiouas, Fatima Laraba-Djebari and Habiba Oussedik-Oumehdi
USTHB, Faculty of Biological Sciences, Laboratory of Cellular and Molecular Biology, BP 32, El-Alia, Bab Ezzouar, 16111, Algiers, Algeria
Viper venoms contain a diverse array of biologically active compounds that give rise to a wide range of clinical effects, spanning from local tissue damage to severe systemic complications. These include pain, edema, hemostatic disturbances, hemorrhage, necrosis and vital organ failure. Inflammation and oxidative stress are widely recognized to be closely associated with the onset of local and systemic toxicity caused by viper venoms, emphasizing the potential advantages of antioxidant therapy in the management of this burden. The aim of this study was to evaluate therapeutic potential of quercetin-PLGA nanoparticles (QT-NPs) on the systemic effects of C. cerastes venom. QT was encapsulated into PLGA nanoparticles by the nanoprecipitation method. The developed QT-NPs underwent physicochemical characterization and were then evaluated in the ‘challenge then treat’ model of envenoming. The efficiency of QT-NPs was assessed by histopathological and immunohistochemical analysis of kidney biopsies and by evaluation of inflammation and redox status markers. Result indicated that formulated NPs exhibited a spherical shape, an average hydrodynamic size of 122.91 ± 7.06 nm and a negative charge as measured by dynamic light scattering. Data revealed that the administration of QT or QT-NPs effectively reduced the oxidative stress and inflammation. Indeed, a significant reduction in NO and MDA levels was observed. Serum TNF-a levels and MPO activity were also significantly reduced. Further, histopathological analysis of kidney biopsies evidenced a reduction of hemorrhage, edema, and inflammatory cell infiltration, mainly macrophages and T lymphocytes as revealed by IHC analysis. Interestingly, data showed that QT-NPs were significantly more effective than free QT, especially at 24 hours after envenomation. These findings highlighted the therapeutic potential of QT-NPs on venom-induced toxicity, and opens up the avenue for their use in the management of snakebite envenoming.
National snakebite envenoming strategy for prevention and control in Pakistan: An urgent need for integration of the neglected tropical disease within a global strategy to achieve SDG 2030
Amina Najam1, Saboor-ul- Hassan1, Syeda Shazia Adeel2, Aamer Ikram3
1Vaccine and Biological Products Centre, National Institute of Health, Islamabad, Pakistan
2Quality Assurance Department, National Institute of Health, Islamabad, Pakistan
3National Institute of Health, Islamabad, Pakistan
Snakebite envenoming (SBE) is a life-threatening neglected tropical disease (NTD), resulting in enormous suffering, disability and deaths. In Pakistan, the estimated burden of SBE is around 30,000 bite cases and thousand deaths annually, leading to a substantial social and economic backlash in rural areas. The extreme climate and topography of the country makes it an ideal place for harboring a variety of venomous snake species. The snakebite incidence is higher during summers, especially in the monsoon season in the entire region. This problem is exacerbated by the lack of adequate monitoring and surveillance systems at the national level. Engaging and empowering local communities through well-designed initiatives is critical to educate the public about the complications associated with it. Clinical management is a big challenge due to factors including shortage of critical care medical facilities, long distances to reach proper treatment centers, cultural beliefs, confidence of common people on traditional healers and efficacy issues of imported antivenoms. Thus, there is an imminent need for setting up a comprehensive plan of action for better prevention and control in Pakistan by adopting WHO strategy 2019 for SBE for 50% reduction of morbidity and mortality. The recent launch of National Snakebite Envenoming (Prevention & Control) Project (NSBEP&C) with the support of Hamish Ogston Foundation, UK, offers an excellent opportunity to tackle this serious public health issue in Pakistan. The scope of this project is defined by four basic pillars: i) estimating current snakebite incidence in various regions of Pakistan and integrating the data with the centralized national surveillance system at NIH; ii) to assure access to safe and effective treatment and build a stable and sustainable market for locally manufactured antivenoms; iii) launching snakebite awareness and education programs for high risk population; iv) national policy and guidelines development for better clinical management practices at local healthcare setups. This initiative is being developed to achieve long-term impact for better prevention and control of SBE in Pakistan till 2030 with the support of local authorities. With international scientific collaborations and regional coordination, the project will be expanded throughout the country in future for improvement in antivenom production, quality control and availability to the neediest patients.
Analysis and identification of Some Putative Novel Peptides purified from Iranian endemic Echis carinatus sochureki snake venom by MALDI-TOF Mass Spectrometry
Nafiseh Nasri Nasrabadi1, Nasser Mohammadpour Dounighi2, Hossein Vatanpour 3
1Pharmaceutical Sciences Research Centre, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran
2Department of Venomous Animals and Anti-venom, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization, Karaj, Iran
3Department of Toxicology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran
This study was aimed to assess novel pro-coagulant, anti-coagulant and anti-platelet proteins, named EC1.5 (a), EC5.1 (b) and EC4 (a) from Iranian Echis Carinatus (IEC) venom. These peptides were purified by multi-step chromatography methods. Hematological properties were measured using activated clotting tests, platelet aggregation studies, and hemorrhage assessment. Subsequently, these proteins were identified through both their intact molecular mass and peptide mass fingerprint (PMF) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Multiple sequence alignments were performed by ClustalW, Bioedit software. Molegro Data Modeller (MDM) 3.0 software was used to predict the putative tertiary structure of proteins. EC1.5 (a), a single-band protein with a molecular mass of 66 and 55 kDa, was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a reduced and non-reduced state, respectively. Based on the Mascot results, we considered that EC1.5 (a) is a metalloproteinase of group ΙΙ which exhibited potent pro-coagulant activity. It is predicted that the EC1.5 (a) with hemorrhagic activity, potentially is a metalloproteinase/disintegrin region that constitutes the disintegrin-like domains. Our findings demonstrate that the disintegrin domain of EC1.5 (a) lacks platelet aggregation inhibitory activity. On the contrary, this factor shows the property of a platelet aggregation inducer. Also, the EC5.1 (b) was observed as a single-band protein with a molecular mass of 7.5 kDa. EC5.1 (b) showed both anti-coagulant and anti-platelet properties. Additionally, the structure of the EC5.1 (b) fraction is expected to be similar to that of phospholipase A2, while EC4 (a) structure is potentially very similar to that of Echistatin with 5 kDa molecular mass.
Rhaebo gutattus (Anura: Bufonidae) skin secretion venomics
Daniel C Pimenta1, Gabriela M M Vianna1, Laís C Mendes1, Cleópatra A da S Caldeira2, Maria A Juliano3 and Emídio Beraldo-Neto1
1Laboratório de Bioquímica, Instituto Butantan, São Paulo, SP, 05503-900, Brazil
2Centro de Estudos em Biomoléculas Aplicadas à Saúde, FioCruz, Porto Velho, RO, 76812-245, Brazil 3Departamento de Biofísica, EPM-UNIFESP, São Paulo, SP, 04044-020, Brazil Animal venoms and secretions are among the most rich and diverse biological fluids in Nature. These complex mixtures of compounds have been selected through evolutionary pressures, dependent on predators, microorganisms, and environmental factors. Classically, animal toxins related to predation, including prey immobilization and digestion are enzymes, proteins, and peptides, while shorter peptides, alkaloids, and steroids act in innate immunity as antibiotic molecules. However, these molecules may also serve as pharmaceutical prototypes for medicinal chemistry studies. R. guttatus, due to its peculiar mode of active defence, were investigated here regarding the composition of its skin secretion. We have performed mass spectrometry analyses, including proteomics, LC-MS and de novo peptide sequencing. Moreover, in solution shotgun proteomics was performed, aiming to analyse the protein secreted into the skin. Besides alkaloids and steroids, we were able to identify 25 peptides, ranging from ~1500-8500 Da in the LC-MS analyses. De novo peptide sequencing BLAST analyses matched antimicrobial peptides from other amphibians, such as dermaseptin, phylloseptin and ocellatin. Proteomics analyses revealed 829 proteins in the skin secretion solution, most of them constitutive (actin, tubulin, filamin, annexin) as well as some metabolic enzymes (proteasome, peroxidase, catalase). Although proteins in the skin have already been described for other Bufonides, this would be the first report of bioactive peptides in the skin secretion of these animals. In conclusion, this study highlights the importance of understanding the complex composition of R. guttatus skin secretion, particularly the involvement of peptides in the defence mechanisms. Further research in this area holds great potential for enhancing the knowledge of on the Toxinology of the Amphibian skin secretion and, therefore, enable the discovery of novel therapeutic agents.
Purification of recombinant PLA2 from B. pictus (rBpMtx) expressed in Pichia pastoris and his functional comparison with native PLA2 (BpMtx)
Alex Proleón1, Daniel Torrejón1, Dan Vivas-Ruiz1,a, Fanny Lazo1, Angie Regalado1, Felix A. Urra2,a, Armando Yarlequé1,a
1Laboratorio de Biología Molecular, Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos, Av. Venezuela Cdra 34 S/N, Ciudad Universitaria, Lima 01, Peru
2Laboratorio Laboratorio de Plasticidad Metabólica y Bioenergética, Programa de Farmacología Molecular y Clínica, Facultad de Medicina, Universidad de Chile, Santiago, Chile
The study of snake venom toxins is important due to the toxicological and pharmacological activities that they show; however, this has multiple limitations such as laborious chromatographic processes and the total dependence of snake venom. An alternative solution is the recombinant expression. The aim of this study was comparing the functional characteristics of BpMtx, a Lys49-PLA2s which makes up 2.8 % of Bothrops pictus snake venom (an endemic and an endangered Peruvian snake) whit its recombinant version (rBpMtx), expressed in Pichia pastoris strain KM71 cells. rBpMtx was purified from culture medium BMMY of induced P. pastoris through one cation exchange chromatographic step (CM Sephadex C-25) and the purification was evaluated through SDS-PAGE and Western blot. This recombinant protein was recognized with serum anti-Myotoxin (rabbit serum) and showed a molecular weight of 21 kDa. Comparison was performed with PLA2 enzymatic activity and myotoxic activity. PLA2 enzymatic activity comparison was made with chromogenic substrate NOBA (4-nitro-3-octanoyloxy-benzoic-acid) and myotoxic activity comparison was made with Creatine kinase kit (assay). rBpMtx did not showed PLA2 enzymatic activity while BpMtx showed a significant enzymatic activity (0.216±0.035 U/mg). On the other hand, rBpMtx showed myotoxic activity (3335.029±303.682 U/L with 25 µg of protein) which is not significantly different from the value of BpMtx. Lys49-PLA2s are unable to hydrolyse phospholipids, the enzymatic activity of native protein is due to low levels of PLA2 catalytically active. Moreover, Lys49-PLA2s are characterized by triggering myotoxicity. The expression recombinant of this protein can be the first step to not depend on an endangered species. Furthermore, analysis of the toxic and pharmacological activities will allow to elucidate the structure-function relationship of these proteins of biotechnological interest. Financial support: Fondo Nacional de Desarrollo Científico, Tecnológico y de Innovación Tecnológica (FONDECYT) mediante el Contrato N° 079-2021- FONDECYT y el Vicerrectorado de Investigación y Posgrado de la UNMSM de acuerdo con el Proyecto N° B17101271.
Analysis of the Micrurus accident profile in the state of Goiás, central-west Brazil between 2002 and 2019
Júlia S Ramos1, Thayná C S Macedo1, Raphael L Alcântara2 and Nelson J Silva Jr1,2
1Undergraduate Program in Medicine, School of Medical and Life Sciences, Pontifical Catholic University of Goiás, Goiânia, GO, Brazil
2Graduate Program in Environmental Sciences and Health, School of Medical and Life Sciences, Pontifical Catholic University of Goiás, Goiânia, GO, Brazil
Accidents with venomous animals are a public health problem in Brazil owing to the wide extension of the national territory and the great diversity of fauna between the different regions of the country. Between 2002 and 2019, 27,547 accidents involving arthropods and snakes were reported in the state of Goiás, central Brazil. There were 1,032 cases with snakes (3.74%), 2,623 with spiders (9.52%), 17,500 with scorpions (63.53%) and 6,392 (23.21%) with other invertebrates. The most involved genera were Bothrops (66.8%), Crotalus (14.4%) and Micrurus (1.84%), totaling 19 cases. Accidents with Micrurus (coralsnakes) have a lower incidence owing to the semifossorial habits of these snakes, which corroborates the profile of the accidents found, with 57.8% related to rural areas and 73.6% affecting male subjects, with a predominance of the age group between 31 and 50 years. The accidents were classified as mild (42.1%), moderate (5.2%) and severe (42.1%), with the bite site equally distributed between upper and lower limbs (47.3%) and 5,2% without information. The use of antielapid serum was performed in 11 cases (57.8%), with the prevalence of the use of 10 ampoules (31.5%) followed by 5 ampoules (15.7%). There were no records of medication use. The main local manifestations reported were pain (73.6%), paresthesia (31.5%) and edema (26.3%), while the most reported systemic manifestations were neuroparalytic (31.5%), vagal (10.5%) and myolytic (5.2%). There were no records of complications or sequelae. In view of this, the idea is reinforced that there should be a classification of accidents with coralsnakes and that the application of the serum is carried out according to the patient’s clinical condition and its manifestations, following the recommendations of keeping the subjetc under observation even without the presence of symptoms.
Biological characterization of BpPLA2-I, an acidic phospholipase A2 from the Bothrops pictus venom
Angie Regalado1, Alex Proleón1, Félix Urra2, Fanny Lazo1, Nadia Vera1, Dan Vivas-Ruiz1,2, Armando Yarlequé1,2
1Laboratorio de Biología Molecular, Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos (Perú)
2Network for Snake Venom Research and Drug Discovery, http://snake-research.com
The cases of ophidism in the central coast of Peru are mainly caused by the Bothrops pictus snake, an endemic Peruvian species, whose venom is composed of various toxins including phospholipase A2 (PLA2) responsible for various pathophysiological effects on envenoming. In the present work, the aim was to isolate and characterize an acidic PLA2 from the venom of B. pictus called BpPLA2-I. B. pictus venom was fractionated using CM Sephadex C-25 cation exchange chromatography followed by size exclusion chromatography on MPLC (ENrich™Q SEC 70) equipment. The purified BpPLA2-I represented 1.03% of the venom. The purity was confirmed by SDS-PAGE (15%) revealing a band of 13 kDa at reducing conditions and by MALDI-TOF obtaining a peak corresponding to 13.86 kDa. BpPLA2-I showed enzymatic activity of 1.54 U*μmol/min*mL*mg on the NOBA chromogenic substrate; likewise, it produced edema (MED = 10.127±3.632 μg); however, it did not generate any myotoxic effect. On the other hand, ELISA assays found a high reactivity of commercial antibotropic serum (INS) against BpPLA2-I and B. pictus with a titers of 32 000 and 64 000, respectively. This recognition was corroborated by Western Blot. In conclusion, this is the first report of an acidic PLA2 from Peruvian Bothrops pictus venom called BpPLA2-I. Financial support: VRIP-UNMSM (Grant: PINTERDIS B2110007i and PCONFIGI N° B22100151).
Identification of a new isoform of serine protease from the venom of the peruvian snake Bothrops pictus (Desert Lancehead)
Raul Rivas-Amado1,Dan E. Vivas-Ruiz1, Fanny E. Lazo Manrique1 Edith F. Rodríguez Quispe1 Javier J. Cárdenas Tenorio2 Walter H. Silva Suarez3 Armando Yarlequé Chocas1
1Laboratorio de Biología Molecular de la Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos, Lima, Perú
2Laboratorio de Bioquímica, Facultad de Ciencias de la Salud, Universidad Nacional del Callao, Callao, Perú
3Servicio Nacional Forestal y de Fauna Silvestre (SERFOR) – MINAGRI
Bothrops pictus (Viperidae), is an endemic snake of Peru that inhabits the valleys of the central coast. A serine protease called Pictobin has previously been characterized from its venom, the pharmacological use of which is currently under study by its migrastatic and mitochondriotopic effects. Recently, our research group has found a new serinoprotease whose preliminary characterization is the objective of this work. The purification began with a first cation exchange chromatographic step CM sephadex C-50, followed by a Sephadex G-100 molecular filtration chromatography and a final step on Sephadex G-50, using, throughout the procedure, acetate buffer. 0.05M ammonium pH 5.0. The enzyme was purified 51.95 times with a yield of 20.8 %, the analysis by PAGE – SDS showed a single band of 52 kDa under reducing conditions and 49.5 kDa under non-reducing conditions, so it is inferred that the enzyme is single-chain with intrachain disulfide bonds. The enzyme has a low coagulant activity with respect to crude venom and Pictobin. Incubation with fibrinogen produced insoluble aggregates suggesting that the enzyme only releases a portion of the fibrinogen chain. The new serine protease hydrolyses the substrate BApNa and the commercial chromogenic synthetic tripeptides: S-2266 (substrate for glandular kallikrein), S-2160 (substrate for thrombin) and S-2222 (substrate for factor Xa) where the tripeptide D-Phe -Val-Arg-pNa (S-2160) was considered the best substrate. Finally, enzymatic inhibition with PMSF confirms the presence of serine as the main residue of the active site. It is concluded that a new serinoprotease isoform has been identified in the venom of the B. pictus snake, being the basis for other molecular, biological or pharmacological studies. Financial support: VRIP-UNMSM (Grants: B2110007i, B21100241), PROCIENCIA (Grant: 079-2021-FONDECYT).
A synthetic derivative of Lawsone as a new inhibitor of the venom of three clinically relevant snake species in Peru
Jorge Alejandro Roque, Felix Urra2,3, Jordano Espinoza1, Edith Rodríguez1, Fanny Lazo1, Ramiro Araya-Maturana3,4, Armando Yarlequé1,3, Dan Vivas-Ruiz1,3
1 Animal Toxins and Antivenoms Research Group (TOXIVEN) – Molecular Biology Laboratory, Faculty of Biological Sciences, National University of San Marcos, Lima, Peru
2 Metabolic Plasticity and Bioenergetics Laboratory, Molecular and Clinical Pharmacology Program, Faculty of Medicine, University of Chile, Santiago, Chile
3Network for Snake Venom Research and Drug Discovery, Santiago, Chile
4 Instituto de Química de Recursos Naturales, MIBI: Interdisciplinary Group on Mitochondrial Targeting and Bioenergetics, Universidad de Talca, Talca, Chile
Ophidism is caused by components of venom that are injected through the bite of a venomous snake. Currently, the only validated treatment is the administration of animal-derived antivenoms. However, their use has limitations, and efforts are being made to find alternative therapeutic approaches, including small molecules with inhibitory effects on enzymatic activities of snake venom toxins. This study evaluates the inhibitory capacity of a synthetic molecule derived from Lawsone (2-hydroxy-1,4-naphthoquinone) on the diffusive, proteolytic, and phospholipase activities of the venoms from the species Bothrops atrox, B. pictus, and B. barnetti, which are the major causes of snakebite envenoming in Peru. In the in vitro kinetic inhibitory assays, inhibition parameters and neutralization percentages were obtained for each of the venom activities. For the proteolytic activity, BApNA and azocasein were used as substrates, and an increase of approximately 20% in Km was observed for both cases, with constant Vmax. For the diffusive activity, hyaluronic acid was used as the substrate, and an increase of approximately 25% in Vmax was observed with constant Km. Lastly, using monodisperse 4N3OBA substrate, the phospholipase activity showed an increase of approximately 30% in Km with constant Vmax. These results suggest the nature of the molecule as a competitive inhibitor in most cases. In addition, the inhibition assays showed a neutralization of over 50% for proteolytic, diffusive, and phospholipase activities when using 10 mg of the synthetic molecule against 25 mg of venom, compared to the 800 mg of protein used in the commercial antivenom. Therefore, our results suggest that Lawsone can be a promising chemical scaffold for the design of new snake toxin inhibitors, with relevance in the blocking effect of small molecules on the venom of the Peruvian Bothrops species. Financial support: VRIP-UNMSM (Grant: PINTERDIS B2110007i and PCONFIGI N° B22100151), ACT210097 (ANID-Chile).
Structural analysis of snake venom thrombin-like enzymes (SVTLEs) from Bothrops atrox venom, more than just isoforms
Anthony Salcedo-Prudencio1, Diego Leonardo2, Daniel Torrejon1, Wolfram Seifert-Dávila3, Javier Cárdenas4, Fanny Lazo1, Armando Yarlequé1, Dan Vivas-Ruiz1
1Research Group on Toxins of Animal Origin and their Antivenoms (TOXIVEN)- Molecular Biology Laboratory, Faculty of Biological Sciences, Universidad Nacional Mayor de San Marcos, Lima, Peru
2Physics Institute of São Carlos, University of São Paulo, São Carlos – São Paulo, Brazil
3Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
4Biochemistry Laboratory, Faculty of Health Sciences, National University of Callao, Callao, Peru
Snake venom Thrombin-Like Enzymes (SVTLEs) are serine proteases generally glycosylated that constitute the third group of enzymes in Viperid venom. SVTLEs cleave fibrinogen causing coagulopathy and hypofibrinogenemia in victims. Given the medical relevance of the Bothrops, especially Bothrops atrox, its toxins have been a constant subject of study. However, little is currently known about the three-dimensional structure of the SVTLE isoforms and their implication in snakebite envenomation. Consequently, the aim of this work is the structural characterization of B. atrox SVTLEs in order to understand the degree of variation among its isoforms and its implication in snakebite envenomation. We examine 27 structures (22 from B. atrox) which we obtained using X-ray crystallography, deduced from sequence and available in the PDB. A phylogenetic analysis was carried out contemplating all their sequences and we observed that 2 of the Peruvian isoforms, Atroxobin and SVTLE-13, were very similar to the isoforms with the highest expression according to the bibliography. The glycosylation sites of all Peruvian isoforms as well as other SVTLEs were predicted and it was observed that, both in the prediction and in the experimental structure, SVTLE-13 has its glycosylation in an Asn very close to the catalytic triad (Ser195, His57, Asp102), which is absent in Atroxobin. On the other hand, it was also observed that SVTLE-13 has a displacement in the orientation of His57 of the triad, which does not occur in Atroxobin, but does in other SVTLEs. All these findings suggest a high structural variation, both intraspecific in the isoforms of the B. atrox SVTLEs and interspecific with respect to other SVTLEs, which would constitute for the first-time preliminary evidence of the existence of families of SVTLEs, as well as has been suggested for other groups of toxins such as metalloproteases and phospholipases. Financial support: Office of the Vice President for Research and Graduate Studies UNMSM (PCONFIGI B23100451). National Program for Scientific Research and Advanced Studies (Contract No. 079-2021-FONDECYT).
Targeting Snake Venom Metalloproteinase Toxin with Indian Medicinal Plants: An In-silico Docking study and to Develop Emergency Loading Dose of Oral Disintegrating Granules for Ophitoxaemia
Navanita Sivaramakumar1, Neeru Dugar2, Veera Venkata Satyanarayana Reddy Karri3, Prakash Yoganandam G4, Vasanth Raj Palanimuthu1
1Department of Pharmaceutical Biotechnology, JSS College of Pharmacy, JSS Academy of Higher Education & Research, Ooty, Nilgiris, Tamil Nadu, India
2Department of Pharmaceutical Chemistry, JSS College of Pharmacy, JSS Academy of Higher Education & Research, Ooty, Nilgiris, Tamil Nadu, India
3Department of Pharmaceutics, JSS College of Pharmacy, JSS Academy of Higher Education & Research, Ooty, Nilgiris, Tamil Nadu, India
4Department of Pharmacognosy, College of Pharmacy, Mother Theresa Post Graduate and Research Institute of Health Sciences, A Government of Puducherry Institution, Puducherry, India
Snake bites pose a major public health concern, particularly in India’s tropical and subtropical regions. India’s diverse flora boasts numerous medicinal plants with established therapeutic properties. This study delves into utilizing these plants to create rapid-absorption oral disintegrating granules, complementing conventional anti-snake venom therapies. Such an integrated approach could enhance treatment efficacy, especially where medical resources are limited. This research focuses on indigenous Indian medicinal plants role in developing emergency oral disintegrating granules. Initially, in-silico docking, pharmacokinetic and toxicity studies have been done targeting snake venom metalloproteinase (SVMP) toxin. SVMP a key snake venom component causing local tissue damage and systemic effects, is scrutinized. Docking simulations employing Schrödinger software and the relevant SVMP toxin structure (Protein Data Bank ID 2E3X) reveal potential SVMP-toxin interactions with medicinal plant compounds. Furthermore, ADME and toxicity studies assess potential drug candidate’s pharmacokinetics and safety. The study highlights Ternstroside F, with a maximum docking score of -12.7251, alongside other phytoconstituents scoring between -4.3613 and -12.7251 across ten medicinal plants. This underscores India’s medicinal plants’ importance in snake bite management, promoting herbal-derived supplement dosage forms as emergency treatments alongside anti-snake venom therapies. In silico techniques, including molecular docking, ADME, and toxicity studies, offer an efficient approach to identify promising drug candidates. The oral disintegrating granules serve as a loading dosage for snakebite until the patient reaches the hospital for anti-snake venom therapy, where the time it takes for individuals living in remote regions to get to the hospital is critical.
High-Throughput venom glycoproteomics
Amalia Kontochristou, Jeroen Kool, Melissa Bärenfänger, Julien Slagboom
AIMMS Division of BioAnalytical Chemistry, Vrije Universiteit Amsterdam, The Netherlands
Snakebite envenoming is considered a neglected tropical disease responsible for more than 100,000 deaths annually. Snakes use their venom to immobilise, incapacitate and kill their prey. Snake venoms are significantly complex and diverse proteinaceous mixtures that provokes a wide range of pathologies, including haemotoxicity. Snake venom toxins contain a great number of post-translational modifications (PTMs). Among them, N-glycosylation is one of the most common. N-glycosylation of proteins is known to be related to protein folding and function; however, there is little understanding of how N-glycosylation impacts the venom proteome. This study aimed to develop a high-throughput venom glycoproteomics methodology for screening snake venom toxins for N-glycosylation, characterising the effect of N-glycosylation on snake venom haemotoxicity and analysing N-glycans released from glycosylated snake venom toxins. For this purpose, the venoms of three snakes from the Viperidae family (Cerastes cerastes, Crotalus atrox and Echis ocellatus) were first separated using reverse-phase liquid chromatography and analysed with mass spectrometry in parallel to nanofractionation onto 384 well plates. The snake venom toxins were identified using bottom-up proteomics and the profiling of the coagulopathic activity of snake venoms was achieved by using a plasma coagulation bioassay. Our findings confirmed the presence of N-glycans on snake venom toxins and revealed that N-glycosylation affected the activity of a number of haemotoxic snake venom toxins. In two of the three analysed venoms, a decrease in haemotoxic activity was observed after releasing the N-glycans from snake venom toxins. Additionally, the structures of N-glycans separated from individual venom toxins were proposed. The analytical approach developed can be used for screening snake venom toxins for N-glycosylation and thereby enable a better understanding of the effect of glycosylation on venom haemotoxicity.
Accidents with scorpions in Central Brazil: a temporal analysis for the State of Goiás
Sarah C Talone1, Winglidy A Carvalho1, Raphael L Alcântara2 and Nelson J Silva Jr1,2
1Undergraduate Program in Biomedicine, School of Medical and Life Sciences, Pontifical Catholic University of Goiás, Goiânia, GO, Brazil
2Graduate Program in Environmental Sciences and Health, School of Medical and Life Sciences, Pontifical Catholic University of Goiás, Goiânia, GO, Brazil
Between 2002 and 2019, 27,547 cases of accidents with venomous animals were reported in the State of Goiás, of which: 1,047 with vertebrates (3.8%), 22,145 with invertebrates (80.4%), 4,098 with others (14.9%) and 257 with a non reported animal (0.9%). Among vertebrates, 1,032 cases with snakes (98.5%) and 15 with freshwater stingrays (1.43%) were recorded. Among snakes: 690 cases were caused by Bothrops (pitvipers), 149 by Crotalus (rattlesnakes), and 19 by Micrurus (coralsnakes). The cases with invertebrates (N=22,145) were represented by 17,500 – scorpions (79%); 2,623 – spiders (11.8%); 626 – centipedes (2.9%); and 1,396 – insects (6. 3%). Males were the most affected gender, with 51.1% of cases and females with 48.4%. The accidents by age group prevailed between 20 and 49 years (48.7%) justified by the longer exposure time in places where these animals are found. The most affected anatomical region was hands/feet (68.2%). Local symptoms prevailed as pain (18.6%) and pain+numbness+paresthesia+edema+hyperemia (19.8%). Systemic symptoms involved headache, blurred vision, neuroparalytic, myolytic and hemolytic action, and vagal symptoms (4.7%), but most were asymptomatic (95.3%). Cases were classified as mild (77.5%), moderate (14.3%), severe (3.3%) and unclassified (4.9%). The cases evolved to cure (72%), cure with sequelae (0.06%), deaths (0.14%) and not informed (27.75%). Anti-scorpion serum, anti-arachnid serum and other combinations were used. The distribution of accidents is influenced by the disorganized urbanization process. Deaths are mainly associated with the pediatric age group. The seasonality of the accidents has little evident characteristics in relation to precipitation and annual temperature in the State of Goiás. It is necessary a continuous training of health professionals regarding the amount of serum to be used and the completion and forwarding of notification forms.
Functional expression in Pichia pastoris of Barnettobin, a thrombin-like enzyme from Bothrops barnetti venom
Daniel Torrejón1, Dan E Vivas-Ruiz1,2, Alex Proleón1, Jordano Espinoza1, Félix A Urra2,3, Carlos Chávez-Olortegui4, Fanny Lazo1, Edith Rodríguez1,2, Armando Yarlequé1,2
1Laboratorio de Biología Molecular, Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos (Perú)
2Network for Snake Venom Research and Drug Discovery, http://snake-research.com
3Clinical and Molecular Pharmacology Program, Institute of Biomedical Sciences (ICBM), Faculty of Medicine, University of Chile, Santiago – Chile
4Departamento de Bioquímica-Inmunología, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, Minas Gerais, Brazil
Snake venoms from the Bothrops genus are renowned for their potent pharmacological activities, attributed to a rich repertoire of bioactive components. Among these, serine proteases stand out as key effectors that contribute to the complex pathophysiological effects of envenomation. These proteases play pivotal roles in the disruption of vital physiological processes by cleaving proteins involved in hemostasis, fibrinolysis, and inflammation. For instance, Barnettobin, a thrombin-like enzyme from B. barnetti, is important due to its contribution to the pathophysiology of envenomation, its potential therapeutic applications as an antithrombotic agent. Due to the challenges in obtaining such protein, heterologous expression becomes a viable strategy. This work focused on obtaining the recombinant version of Barnettobin, named as rBarnettobin, in Pichia pastoris. The study began with the optimization and synthesis of the coding sequence of Barnettobin, followed by its cloning into the pPICZαC vector in Escherichia coli TOP10 cells. Sequencing confirmed the proper integration of the optimized Barnettobin coding insert. Following propagation in E. coli TOP10 cells, the recombinant vector rBarnetobin-pPICZαC were transformed into Pichia pastoris strain KM71H, respectively, with a transformation efficiency of 1.5 x 105 CFU/µg of linearized plasmid. Expression and purification of the target proteins were successful as confirmed by SDS-PAGE and Western Blot analyses, revealing two bands for rBarnetobin, suggesting one corresponds to the glycosylated variant (53 kDa), while the other to the non-glycosylated version (52 kDa). The tests carried out indicated the functional conservation of rBarnetobin exhibited amidolytic activity of 0.90 U/mg and 1 µg induced fibrinogen coagulation within 197 seconds. Funding: Fondo Nacional de Desarrollo Científico, Tecnológico y de Innovación Tecnológica (FONDECYT) mediante el Contrato N° 079-2021- FONDECYT y el Vicerrectorado de Investigación y Posgrado de la UNMSM de acuerdo con el Proyecto N° B17101271.
Pictolysin-III is an SVMP-III from Bothrops pictus snake venom that reduces mitochondrial bioenergetics in cancer cells
Félix A. Urra1,2, Christopher Almarza1,2, Francisco Guajardo1,2, Paola Rosas2,3, Fanny Lazo2,3, Luciana Oliveira2,4, Armando Yarlequé2,3, Dan Vivas-Ruiz2,3
1Metabolic plasticity and bioenergetics Lab, Program of Molecular and Clinical Pharmacology, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile, Santiago CL
2Network for Snake Venom Research and Drug Discovery
3Laboratory of Molecular Biology, Faculty of Biological Sciences, Universidad Nacional Mayor de San Marcos, Lima, Perú. 4Laboratory of Biochemistry of Proteins from Animal Venoms, Research, and Development Center, Ezequiel Dias Foundation, Belo Horizonte 30510-010, Brazil. Snake venom metalloproteinases (SVMPs) are recognized for acting on extracellular matrix (ECM)-cell interaction, disrupting the adhesion and migration of non-cancer and cancer cell lines. Although recent studies have shown that several snake toxin classes can interfere with mitochondrial metabolism in cancer cells, the effect of SVMPs on bioenergetics remains extensively unknown. Since ECM-cell interaction determines the cellular metabolism, we evaluate the effect of a novel SVMP type-III, called Pictolysin-III (Pic-III), isolated from Bothrops pictus venom on mitochondrial bioenergetics in triple-negative breast cancer (MDA-MB-231) and human breast fibroblast (RMF-621). Using XFe96 technology, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were determined for evaluating mitochondrial respiration and glycolysis. Moreover, NAD(P)H, mitochondrial ROS, and ATP changes were determined using fluorescence and luminescence-based methods. Our results show that Pic-III triggers a decrease in basal and FCCP-stimulated mitochondrial respiration and reduced glycolysis and glycolytic capacity. Consistent with these results, Pic-III decreased the intracellular ATP levels and increased NAD(P)H and mitochondrial ROS under non-cytotoxic conditions. To our knowledge, Pic-III is the first SVMP type III reported with action on mitochondrial bioenergetics and may offer novel opportunities for promising lead compounds that inhibit ECM–cancer-cell interactions. Financial support: Contrato N° 079-FONDECYT-2021 (FONDECYT-Peru), UNMSM- VRIP (B22100151), ACT210097 (ANID-Chile), UM-03/22 (UChile), EQM220164 (ANID-Chile), FONDECYT 11201322 (ANID-Chile).
The snake venom metalloproteinase Pictolysin-III produces cytokine secretion and sensitization to anticancer BH3 mimetic drugs
Dan Vivas-Ruiz1,2, Christopher Almarza2,3, Francisco Guajardo2,3, Víctor Andrades2,3, Jessica Astorga2,3, Daniel Oropesa2,3, Alex Proleon1,2, Daniel Torrejón1,2, Jorge Toledo4, Luciana Oliveira2,5, Armando Yarlequé1,2, Félix A. Urra2,3
1Laboratory of Molecular Biology, Faculty of Biological Sciences, Universidad Nacional Mayor de San Marcos, Lima, Perú
2Network for Snake Venom Research and Drug Discovery, Santiago, Chile
3Metabolic plasticity and bioenergetics Lab, Program of Molecular and Clinical Pharmacology, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile, Santiago CL 4Advanced Scientific Equipment Network (REDECA), Faculty of Medicine, Universidad de Chile, Santiago 8380453, Chile 5Laboratory of Biochemistry of Proteins from Animal Venoms, Research and Development Center, Ezequiel Dias Foundation, Belo Horizonte 30510-010, Brazil. The effects of snake venom metalloproteinases (SVMPs) on adhesion and migration in cancer cell lines as well as the induction of pro-inflammatory cytokine secretion in immune cells have been extensively studied however, the mechanisms involved remain poorly understood. In this work, we evaluate the effect of a novel type-III SVMP, called Pictolysin-III (Pic-III), isolated from Bothrops pictus venom on cell morphology using immunofluorescence and time-lapse records, viability by MTT reduction, and secretion of cytokines by CBA in Caco-2 and MDA-MB-231 cells and human fibroblast RMF621. Pic-III blocked the formation of cellular projections, producing disruptions in the actin network and reducing the cell spreading. These morphological changes were characterized by an aggregation of actin, increased circularity, and loss of cell polarization in a concentration-dependent manner in Caco-2 cells. Moreover, Pic-III generated changes in the cytokine secretion profile of RMF-621 cells, increasing the IL-8, IL-1β, and TNF levels with no changes in IL-10 secretion. Pic-III induced the production of IL-8 and IL-1β, decreasing IL-10 in Caco-2 cells. Since our data suggest that Pic-III induces disruption of mitochondrial respiration, we speculate that this toxin may sensitize the cytotoxic effect of ABT-199, a BCL2-selective inhibitor with cytotoxic action in several cancer cells, including triple-negative breast cancer cells. Notably, the combination Pic-III + ABT-199 reduced the viability to 0.15 ± 0.05 folds of the control, suggesting that Pic-III-sensitizes to BH3 mimetic ABT-199 (Venetoclax) in MDA-MB-231 cells. Taken together, our results suggest that Pic-III may offer novel opportunities for promising lead compounds that modify the tumoral environment and response to cytotoxic drugs. Financial support: Contrato N° 079-FONDECYT-2021 (PROCIENCIA-Peru), ACT210097 (ANID-Chile), UM-03/22 (UChile), EQM220164 (ANID-Chile), FONDECYT 11201322 (ANID-Chile).
Cytotoxicity profiling of toxins in snake venoms enabled by nanofractionation analytics and high throughput venomics
Haifeng Xu1,2, Matyas A Bittenbinder1, Julien Slagboom1, Nicholas R Casewel3 Paul Jennings2, Jeroen Kool1
1AIMMS Division of BioAnalytical Chemistry, Vrije Universiteit Amsterdam, The Netherlands
2AIMMS Division of Molecular and Computational Toxicology, Vrije Universiteit Amsterdam, The Netherlands
3Centre for Snakebite Research and Interventions, LSTM, Liverpool, UK Glasgow
UK Snake venom is well-known for causing severe heamotoxicity, neurotoxicity, and cytotoxicity upon envenoming. We evaluated four in vitro mammalian cell lines (RPTEC, HepaRG, HUVEC, and Hacat) against elapid venoms and their nanofractionated toxins for their cytotoxic properties. Fluorescence assays with Hoechst 33342 and propidium iodide, and the resazurin reduction assay were used to assess cell permeability and viability, respectively. Three crude cobra venoms (Naja mossambica, Naja haje, and Naja naja) showed dose-response cytotoxicity for all the cell lines by causing disruption of cell permeability and cell viability. Bungarus multicinctus venom only showed slight selective cytotoxicity in the HepaRG cell line. Subsequently, nanofractionation analytics coupled in parallel to mass spectrometry was applied for separation and high-resolution fractionation of toxins with accurate masses. Fractionated toxins were subjected to cellular assays for assessing the cytotoxicity profile of all individual toxins for four cell lines, which are representative of renal proximal tubule (RPTEC), liver (HepaRG), human umbilical vein (HUVEC), and epidermis (Hacat) tissues. Selective cytotoxicity was observed for some toxins from N mossambica. Next, a separate well plate with nanofractionated toxins was used for High Throughput venomics to chemically identify all fractionated toxins at the toxin ID level. PLA2s and 3FTXs were the prominent toxins found in N mossambica and N naja snakes that contributed to cytotoxicity. Through this, we developed and validated a cytotoxicity-assessing analytical platform that can be used to rapidly study cytotoxic venoms to pinpoint and identify their cytotoxins and get a clearer view of their cytotoxic mechanism of action, and selectivity towards different cell types.
