A Snake Venom Peptide and Its Derivatives Prevent Aß42 Aggregation and Eliminate Toxic Aß42 Aggregates In Vitro.

Over a century has passed since Alois Alzheimer first described Alzheimer's disease (AD), and since then, researchers have made significant strides in understanding its pathology. One key feature of AD is the presence of amyloid-ß (Aß) peptides, which form amyloid plaques, and therefore, it is a primary target for treatment studies. Naturally occurring peptides have garnered attention for their potential pharmacological benefits, particularly in the central nervous system. In this study, nine peptide derivatives of Crotamine, a polypeptide from Crotalus durissus terrificus Rattlesnake venom, as well as one d-enantiomer, were evaluated for their ability to modulate Aß42 aggregation through various assays such as ThT, QIAD, SPR, and sFIDA. All tested peptides were able to decrease Aß42 aggregation and eliminate Aß42 aggregates. Additionally, all of the peptides showed an affinity for Aß42. This study is the first to describe the potential of crotamine derivative peptides against Aß42 aggregation and to identify a promising d-peptide that could be used as an effective pharmacological tool against AD in the future.

Exploring snake venoms beyond the primary sequence: From proteoforms to protein-protein interactions.

Snakebite envenomation has been a long-standing global issue that is difficult to treat, largely owing to the flawed nature of current immunoglobulin-based antivenom therapy and the complexity of snake venoms as sophisticated mixtures of bioactive proteins and peptides. Comprehensive characterisation of venom compositions is essential to better understanding snake venom toxicity and inform effective and rationally designed antivenoms. Additionally, a greater understanding of snake venom composition will likely unearth novel biologically active proteins and peptides that have promising therapeutic or biotechnological applications. While a bottom-up proteomic workflow has been the main approach for cataloguing snake venom compositions at the toxin family level, it is unable to capture snake venom heterogeneity in the form of protein isoforms and higher-order protein interactions that are important in driving venom toxicity but remain underexplored. This review aims to highlight the importance of understanding snake venom heterogeneity beyond the primary sequence, in the form of post-translational modifications that give rise to different proteoforms and the myriad of higher-order protein complexes in snake venoms. We focus on current top-down proteomic workflows to identify snake venom proteoforms and further discuss alternative or novel separation, instrumentation, and data processing strategies that may improve proteoform identification. The current higher-order structural characterisation techniques implemented for snake venom proteins are also discussed; we emphasise the need for complementary and higher resolution structural bioanalytical techniques such as mass spectrometry-based approaches, X-ray crystallography and cryogenic electron microscopy, to elucidate poorly characterised tertiary and quaternary protein structures. We envisage that the expansion of the snake venom characterisation "toolbox" with top-down proteomics and high-resolution protein structure determination techniques will be pivotal in advancing structural understanding of snake venoms towards the development of improved therapeutic and biotechnology applications.

Exploring metalloproteins found in the secretion of venomous species: Biological role and therapeutical applications.

Several species during evolution suffered random mutations in response to various environmental factors, which resulted in the formation of venom in phylogenetically distant species. The composition of the venom of most species is poorly known. Snake venom is well characterized while most species have poorly known composition. In contrast, snake venoms are well characterized which proteins and peptides are the main active and most abundant constituents. 42 protein families have been identified, including metalloproteins known as metalloproteinases. These macromolecules are enzymes with zinc in their active site derived from the disintegrin A and metalloproteinase (ADAM) cellular family and are categorized into three classes (PI, PII and PIII) according to their domain organization. The snake venom metalloproteinases (SVMP) are cytotoxic, neurotoxic, myotoxic and/or hematotoxic with a crucial role in the defense and restraint of prey. In this scenario envenoming represents a danger to human health and has been considered a neglected disease worldwide, particularly in tropical and subtropical countries. Nevertheless, recently advances in "omics" technologies have demonstrated interesting biological activities of SVMPs such as antimicrobial, anticancer, against cardiovascular diseases and nervous system disorders. Metalloproteins have the therapeutic potential to be converted into drugs as other components of the venom have undergone this process (e.g., captopril, tirefiban and eptifibatide). So, this chapter is focused on the metalloproteins found in the secretions of venomous species, highlight some aspects such as structure, biological activity, pharmacological therapeutic potential and on.

Molecular Memory Micromotors for Fast Snake Venom Toxin Dynamic Detection.

The analysis and detection of snake venom toxins are a matter of great importance in clinical diagnosis for fast treatment and the discovery of new pharmaceutical products. Current detection methods have high associated costs and require the use of sophisticated bioreceptors, which in some cases are difficult to obtain. Herein, we report the synthesis of template-based molecularly imprinted micromotors for dynamic detection of α-bungarotoxin as a model toxin present in the venom of many-banded krait (Bungarus multicinctus). The specific recognition sites are built-in in the micromotors by incubation of the membrane template with the target toxin, followed by a controlled electrodeposition of a poly(3,4-ethylenedioxythiophene)/poly(sodium 4-styrenesulfonate) polymeric layer, a magnetic Ni layer to promote magnetic guidance and facilitate washing steps, and a Pt layer for autonomous propulsion in the presence of hydrogen peroxide. The enhanced fluid mixing and autonomous propulsion increase the likelihood of interactions with the target analyte as compared with static counterparts, retaining the tetramethylrhodamine-labeled α-bungarotoxin on the micromotor surface with extremely fast dynamic sensor response (after just 20 s navigation) in only 3 µL of water, urine, or serum samples. The sensitivity achieved meets the clinically relevant concentration postsnakebite (from 0.1 to 100 µg/mL), illustrating the feasibility of the approach for practical applications. The selectivity of the protocol is very high, as illustrated by the absence of fluorescence in the micromotor surface in the presence of α-cobratoxin as a representative toxin with a size and structure similar to those of α-bungarotoxin. Recoveries higher than 95% are obtained in the analysis of urine- and serum-fortified samples. The new strategy holds considerable promise for fast, inexpensive, and even onsite detection of several toxins using multiple molecularly imprinted micromotors with tailored recognition abilities.

Streamlining the Analysis of Proteins from Snake Venom.

Investigating snake venom is necessary for developing new treatments for envenoming and harnessing the therapeutic potential that lies within venom toxins. Despite considerable efforts in previous research, several technical challenges remain for characterizing the individual components within such complex mixtures. Here, we present native and top-down mass spectrometry (MS) workflows that enable the analysis of individual venom proteins within complex mixtures and showcase the utility of these methodologies on King cobra (Ophiophagus hannah) venom. First, we coupled ion mobility spectrometry for separation and electron capture dissociation for charge reduction to resolve highly convoluted mass spectra containing multiple proteins with masses ranging from 55 to 127 kDa. Next, we performed a top-down glycomic analysis of a 25.5 kDa toxin, showing that this protein contains a fucosylated complex glycan. Finally, temperature-controlled nanoelectrospray mass spectrometry facilitated the top-down sequence analysis of a ß-cardiotoxin, which cannot be fragmented by collisional energy due to its disulfide bond pattern. The work presented here demonstrates the applicability of new and promising MS methods for snake venom analysis.

Emerging anticancer potential and mechanisms of snake venom toxins: A review.

Animal-derived venom, like snake venom, has been proven to be valuable natural resources for the drug development. Previously, snake venom was mainly investigated in its pharmacological activities in regulating coagulation, vasodilation, and cardiovascular function, and several marketed cardiovascular drugs were successfully developed from snake venom. In recent years, snake venom fractions have been demonstrated with anticancer properties of inducing apoptotic and autophagic cell death, restraining proliferation, suppressing angiogenesis, inhibiting cell adhesion and migration, improving immunity, and so on. A number of active anticancer enzymes and peptides have been identified from snake venom toxins, such as L-amino acid oxidases (LAAOs), phospholipase A2 (PLA2), metalloproteinases (MPs), three-finger toxins (3FTxs), serine proteinases (SPs), disintegrins, C-type lectin-like proteins (CTLPs), cell-penetrating peptides, cysteine-rich secretory proteins (CRISPs). In this review, we focus on summarizing these snake venom-derived anticancer components on their anticancer activities and underlying mechanisms. We will also discuss their potential to be developed as anticancer drugs in the future.

High-Voltage Toxin'Roll: Electrostatic Charge Repulsion as a Dynamic Venom Resistance Trait in Pythonid Snakes.

The evolutionary interplay between predator and prey has significantly shaped the development of snake venom, a critical adaptation for subduing prey. This arms race has spurred the diversification of the components of venom and the corresponding emergence of resistance mechanisms in the prey and predators of venomous snakes. Our study investigates the molecular basis of venom resistance in pythons, focusing on electrostatic charge repulsion as a defense against α-neurotoxins binding to the alpha-1 subunit of the postsynaptic nicotinic acetylcholine receptor. Through phylogenetic and bioactivity analyses of orthosteric site sequences from various python species, we explore the prevalence and evolution of amino acid substitutions that confer resistance by electrostatic repulsion, which initially evolved in response to predatory pressure by Naja (cobra) species (which occurs across Africa and Asia). The small African species Python regius retains the two resistance-conferring lysines (positions 189 and 191) of the ancestral Python genus, conferring resistance to sympatric Naja venoms. This differed from the giant African species Python sebae, which has secondarily lost one of these lysines, potentially due to its rapid growth out of the prey size range of sympatric Naja species. In contrast, the two Asian species Python brongersmai (small) and Python bivittatus (giant) share an identical orthosteric site, which exhibits the highest degree of resistance, attributed to three lysine residues in the orthosteric sites. One of these lysines (at orthosteric position 195) evolved in the last common ancestor of these two species, which may reflect an adaptive response to increased predation pressures from the sympatric α-neurotoxic snake-eating genus Ophiophagus (King Cobras) in Asia. All these terrestrial Python species, however, were less neurotoxin-susceptible than pythons in other genera which have evolved under different predatory pressure as: the Asian species Malayopython reticulatus which is arboreal as neonates and juveniles before rapidly reaching sizes as terrestrial adults too large for sympatric Ophiophagus species to consider as prey; and the terrestrial Australian species Aspidites melanocephalus which occupies a niche, devoid of selection pressure from α-neurotoxic predatory snakes. Our findings underline the importance of positive selection in the evolution of venom resistance and suggest a complex evolutionary history involving both conserved traits and secondary evolution. This study enhances our understanding of the molecular adaptations that enable pythons to survive in environments laden with venomous threats and offers insights into the ongoing co-evolution between venomous snakes and their prey.

Analysis of the Snake Venom Peptidome.

Snake venom peptidomes are known to be a large source of molecules with different pharmacological properties. The complexity and variability of snake venoms, the presence of proteinases, and the lack of complete species-specific genome sequences make snake venom peptidome profiling a challenging task that requires especial technical strategies for sample processing and mass spectrometric analysis. Here, we describe a method for assessing the content of snake venom peptides and highlight the importance of sampling procedures, as they substantially influence the peptidomic complexity of snake venoms.

Immunological Cross-Reactivity and Preclinical Assessment of a Colombian Anticoral Antivenom against the Venoms of Three Micrurus Species.

Snakebite accident treatment requires the administration of antivenoms that provide efficacy and effectiveness against several snake venoms of the same genus or family. The low number of immunogenic components in venom mixtures that allow the production of antivenoms consequently gives them partial neutralization and a suboptimal pharmacological response. This study evaluates the immunorecognition and neutralizing efficacy of the polyvalent anticoral antivenom from the Instituto Nacional de Salud (INS) of Colombia against the heterologous endemic venoms of Micrurus medemi, and M. sangilensis, and M. helleri by assessing immunoreactivity through affinity chromatography, ELISA, Western blot, and neutralization capability. Immunorecognition towards the venoms of M. medemi and M. sangilensis showed values of 62% and 68% of the protein composition according to the immunoaffinity matrix, respectively. The analysis by Western blot depicted the highest recognition patterns for M. medemi, followed by M. sangilensis, and finally by M. helleri. These findings suggest that the venom compositions are closely related and exhibit similar recognition by the antivenom. According to enzyme immunoassays, M. helleri requires a higher amount of antivenom to achieve recognition than the others. Besides reinforcing the evaluation of INS antivenom capability, this work recommends the use of M. helleri in the production of Colombian antisera.

Distinct regulatory networks control toxin gene expression in elapid and viperid snakes.

BACKGROUND: Venom systems are ideal models to study genetic regulatory mechanisms that underpin evolutionary novelty. Snake venom glands are thought to share a common origin, but there are major distinctions between venom toxins from the medically significant snake families Elapidae and Viperidae, and toxin gene regulatory investigations in elapid snakes have been limited. Here, we used high-throughput RNA-sequencing to profile gene expression and microRNAs between active (milked) and resting (unmilked) venom glands in an elapid (Eastern Brown Snake, Pseudonaja textilis), in addition to comparative genomics, to identify cis- and trans-acting regulation of venom production in an elapid in comparison to viperids (Crotalus viridis and C. tigris). RESULTS: Although there is conservation in high-level mechanistic pathways regulating venom production (unfolded protein response, Notch signaling and cholesterol homeostasis), there are differences in the regulation of histone methylation enzymes, transcription factors, and microRNAs in venom glands from these two snake families. Histone methyltransferases and transcription factor (TF) specificity protein 1 (Sp1) were highly upregulated in the milked elapid venom gland in comparison to the viperids, whereas nuclear factor I (NFI) TFs were upregulated after viperid venom milking. Sp1 and NFI cis-regulatory elements were common to toxin gene promoter regions, but many unique elements were also present between elapid and viperid toxins. The presence of Sp1 binding sites across multiple elapid toxin gene promoter regions that have been experimentally determined to regulate expression, in addition to upregulation of Sp1 after venom milking, suggests this transcription factor is involved in elapid toxin expression. microRNA profiles were distinctive between milked and unmilked venom glands for both snake families, and microRNAs were predicted to target a diversity of toxin transcripts in the elapid P. textilis venom gland, but only snake venom metalloproteinase transcripts in the viperid C. viridis venom gland. These results suggest differences in toxin gene posttranscriptional regulation between the elapid P. textilis and viperid C. viridis. CONCLUSIONS: Our comparative transcriptomic and genomic analyses between toxin genes and isoforms in elapid and viperid snakes suggests independent toxin regulation between these two snake families, demonstrating multiple different regulatory mechanisms underpin a venomous phenotype.

A novel metalloproteinase-derived cryptide from Bothrops cotiara venom inhibits angiotensin-converting enzyme activity.

Snake venoms are primarily composed of proteins and peptides, which selectively interact with specific molecular targets, disrupting prey homeostasis. Identifying toxins and the mechanisms involved in envenoming can lead to the discovery of new drugs based on natural peptide scaffolds. In this study, we used mass spectrometry-based peptidomics to sequence 197 peptides in the venom of Bothrops cotiara, including a novel 7-residue peptide derived from a snake venom metalloproteinase. This peptide, named Bc-7a, features a pyroglutamic acid at the N-terminal and a PFR motif at the C-terminal, homologous to bradykinin. Using FRET (fluorescence resonance energy transfer) substrate assays, we demonstrated that Bc-7a strongly inhibits the two domains of angiotensin converting enzyme (Ki < 1 µM). Our findings contribute to the repertoire of biologically active peptides from snake venoms capable of inhibiting angiotensin-converting enzyme (ACE), beyond current known structural motifs and precursors. In summary, we report a novel snake venom peptide with ACE inhibitory activity, suggesting its potential contribution to the hypotensive effect observed in envenomation.

Snake venom toxins: Potential anticancer therapeutics.

Snake venom contains a cocktail of compounds dominated by proteins and peptides, which make up the toxin. The toxin components of snake venom attack several targets in the human body including the neuromuscular system, kidney and blood coagulation system and cause pathologies. As such, the venom toxins can be managed and used for the treatment of these diseases. In this regard, Captopril used in the treatment of cardiovascular diseases was the first animal venom toxin-based drug approved by the US Food and Drug Administration and the European Medicines Agency. Cancers cause morbidity and mortality worldwide. Due to side effects associated with the current cancer treatments including chemotherapy, radiotherapy, immunotherapy, hormonal therapy and surgery, there is a need to improve the efficacy of current treatments and/or develop novel drugs from natural sources including animal toxin-based drugs. There is a long history of earlier and ongoing studies implicating snake venom toxins as potential anticancer therapies. Here, we review the role of crude snake venoms and toxins including phospholipase A2, L-amino acid oxidase, C-type lectin and disintegrin as potential anticancer agents tested in cancer cell lines and animal tumour models in comparison to normal cell lines. Some of the anti-tumour activities of snake venom toxins include induction of cytotoxicity, apoptosis, cell cycle arrest and inhibition of metastasis, angiogenesis and tumour growth. We thus propose the advancement of multidisciplinary approaches to more pre-clinical and clinical studies for enhanced bioavailability and targeted delivery of snake venom toxin-based anticancer drugs.

Characterisation of the forest cobra (Naja melanoleuca) venom using a multifaceted mass spectrometric-based approach.

Snake venoms consist of highly biologically active proteins and peptides that are responsible for the lethal physiological effects of snakebite envenomation. In order to guide the development of targeted antivenom strategies, comprehensive understanding of venom compositions and in-depth characterisation of various proteoforms, often not captured by traditional bottom-up proteomic workflows, is necessary. Here, we employ an integrated 'omics' and intact mass spectrometry (MS)-based approach to profile the heterogeneity within the venom of the forest cobra (Naja melanoleuca), adopting different analytical strategies to accommodate for the dynamic molecular mass range of venom proteins present. The venom proteome of N. melanoleuca was catalogued using a venom gland transcriptome-guided bottom-up proteomics approach, revealing a venom consisting of six toxin superfamilies. The subtle diversity present in the venom components was further explored using reversed phase-ultra performance liquid chromatography (RP-UPLC) coupled to intact MS. This approach showed a significant increase in the number of venom proteoforms within various toxin families that were not captured in previous studies. Furthermore, we probed at the higher-order structures of the larger venom proteins using a combination of native MS and mass photometry and revealed significant structural heterogeneity along with extensive post-translational modifications in the form of glycosylation in these larger toxins. Here, we show the diverse structural heterogeneity of snake venom proteins in the venom of N. melanoleuca using an integrated workflow that incorporates analytical strategies that profile snake venom at the proteoform level, complementing traditional venom characterisation approaches.

A comparative study of protein structure prediction tools for challenging targets: Snake venom toxins.

Protein structure determination is a critical aspect of biological research, enabling us to understand protein function and potential applications. Recent advances in deep learning and artificial intelligence have led to the development of several protein structure prediction tools, such as AlphaFold2 and ColabFold. However, their performance has primarily been evaluated on well-characterised proteins and their ability to predict sturtctures of proteins lacking experimental structures, such as many snake venom toxins, has been less scrutinised. In this study, we evaluated three modelling tools on their prediction of over 1000 snake venom toxin structures for which no experimental structures exist. Our findings show that AlphaFold2 (AF2) performed the best across all assessed parameters. We also observed that ColabFold (CF) only scored slightly worse than AF2, while being computationally less intensive. All tools struggled with regions of intrinsic disorder, such as loops and propeptide regions, and performed well in predicting the structure of functional domains. Overall, our study highlights the importance of exercising caution when working with proteins with no experimental structures available, particularly those that are large and contain flexible regions. Nonetheless, leveraging computational structure prediction tools can provide valuable insights into the modelling of protein interactions with different targets and reveal potential binding sites, active sites, and conformational changes, as well as into the design of potential molecular binders for reagent, diagnostic, or therapeutic purposes.

Snake and arthropod venoms: Search for inflammatory activity in human cells involved in joint diseases.

Most anti-inflammatory drugs currently adopted to treat chronic inflammatory joint diseases can alleviate symptoms but they do not lead to remission. Therefore, new and more efficient drugs are needed to block the course of joint inflammatory diseases. Animal venoms, rich in bioactive compounds, can contribute as valuable tools in this field of research. In this study, we first demonstrate the direct action of venoms on cells that constitute the articular joints. We established a platform consisting of cell-based assays to evaluate the release of cytokines (IL-6, IL-8, TNFα, IL-1ß, and IL-10) by human chondrocytes, synoviocytes and THP1 macrophages, as well as the release of neuropeptides (substance-P and ß-endorphin) by differentiated sensory neuron-like cells, 24 h after stimulation of cells with 21 animal venoms from snake and arthropod species, sourced from different taxonomic families and geographic origins. Results demonstrated that at non-cytotoxic concentrations, the venoms activate at varying degrees the secretion of inflammatory mediators involved in the pathology of articular diseases, such as IL-6, IL-8, and TNF-α by chondrocytes, synoviocytes, and macrophages and of substance P by neuron-like cells. Venoms of the Viperidae snake family were more inflammatory than those of the Elapidae family, while venoms of Arthropods were less inflammatory than snake venoms. Notably, some venoms also induced the release of the anti-inflammatory IL-10 by macrophages. However, the scorpion Buthus occitanus venom induced the release of IL-10 without increasing the release of inflammatory cytokines by macrophages. Since the cell types used in the experiments are crucial elements in joint inflammatory processes, the results of this work may guide future research on the activation of receptors and inflammatory signaling pathways by selected venoms in these particular cells, aiming at discovering new targets for therapeutic intervention.

Bothrops snake venom L-amino acid oxidases impair biofilm formation of clinically relevant bacteria.

The present work addressed the abilities of two L-amino acid oxidases isolated from Bothrops moojeni (BmooLAAO-I) and Bothrops jararacussu (BjussuLAAO-II) snake venoms to control the growth and prevent the biofilm formation of clinically relevant bacterial pathogens. Upon S. aureus (ATCC BAA44) and S. aureus (clinical isolates), BmooLAAO-I (MIC = 0.12 and 0.24 µg/mL, respectively) and BjussuLAAO-II (MIC = 0.15 µg/mL) showed a potent bacteriostatic effect. Against E. coli (ATCC BAA198) and E. coli (clinical isolates), BmooLAAO-I (MIC = 15.6 and 62.5 µg/mL, respectively) and BjussuLAAO-II (MIC = 4.88 and 9.76 µg/mL, respectively) presented a lower extent effect. Also, BmooLAAO-I (MICB50 = 0.195 µg/mL) and BjussuLAAO-II (MICB50 = 0.39 µg/mL) inhibited the biofilm formation of S. aureus (clinical isolates) in 88% and 89%, respectively, and in 89% and 53% of E. coli (clinical isolates). Moreover, scanning electron microscopy confirmed that the toxins affected bacterial morphology by increasing the roughness of the cell surface and inhibited the biofilm formation. Furthermore, analysis of the tridimensional structures of the toxins showed that the surface-charge distribution presents a remarkable positive region close to the glycosylation motif, which is more pronounced in BmooLAAO-I than BjussuLAAO-II. This region may assist the interaction with bacterial and biofilm surfaces. Collectively, our findings propose that venom-derived antibiofilm agents are promising biotechnological tools which could provide novel strategies for biofilm-associated infections.

Composition characterization of various viperidae snake venoms using MS-based proteomics N-glycoproteomics and N-glycomics.

Viperidae snake species is widely abundant and responsible for most envenomation cases in Turkey. The structural and compositional profiles of snake venom have been investigated to study the venom component variation across different species and to profile the venom biological activity variation against prey. In this context, we used proteomics, glycoproteomics and glycomics strategies to characterize the protein, glycoproteins and glycan structural and compositional profiles of various snake venoms in the Viperidae family. Moreover, we compared these profiles using the downstream bioinformatics and machine learning classification modules. The overall mass spectrometry profiles identified 144 different proteins, 36 glycoproteins and 78 distinct N-glycan structures varying in composition across the five venoms. A high amount of the characterized proteins belongs to the glycosylated protein family Trypsin-like serine protease (Tryp_SPc), Disintegrin (DISIN), and ADAM Cysteine-Rich (ACR). Most identified N-glycans have a complex chain carrying galactosylated N-glycans abundantly. The glycan composition data obtained from glycoproteomics aligns consistently with the findings from glycomics. The clustering and principal component analyses (PCA) illustrated the composition-based similarities and differences between each snake venom species' proteome, glycoproteome and glycan profiles. Specifically, the N-glycan profiles of M. xanthina (Mx) and V. a. ammodytes (Vaa) venoms were identical and difficult to differentiate; in contrast, their proteome profiles were distinct. Interestingly, the variety of the proteins across the species highlighted the impact of glycosylation on the diversity of the glycosylated protein families. This proposed high throughput approach provides accurate and comprehensive profiles of the composition and function of various Viperidae snake venoms.

Snake Venom Components as Therapeutic Drugs in Ischemic Heart Disease.

Ischemic heart disease (IHD), especially myocardial infarction (MI), is a leading cause of death worldwide. Although coronary reperfusion is the most straightforward treatment for limiting the MI size, it has nevertheless been shown to exacerbate ischemic myocardial injury. Therefore, identifying and developing therapeutic strategies to treat IHD is a major medical challenge. Snake venoms contain biologically active proteins and peptides that are of major interest for pharmacological applications in the cardiovascular system (CVS). This has led to their use for the development and design of new drugs, such as the first-in-class angiotensin-converting enzyme inhibitor captopril, developed from a peptide present in Bothrops jararaca snake venom. This review discusses the potential usefulness of snake venom toxins for developing effective treatments against IHD and related diseases such as hypertension and atherosclerosis. It describes their biological effects at the molecular scale, their mechanisms of action according to their different pharmacological properties, as well as their subsequent molecular pathways and therapeutic targets. The molecules reported here have either been approved for human medical use and are currently available on the drug market or are still in the clinical or preclinical developmental stages. The information summarized here may be useful in providing insights into the development of future snake venom-derived drugs.

Mouse skin peptidomic analysis of the hemorrhage induced by a snake venom metalloprotease.

Hemorrhage induced by snake venom metalloproteases (SVMPs) results from proteolysis, capillary disruption, and blood extravasation. HF3, a potent SVMP of Bothrops jararaca, induces hemorrhage at pmol doses in the mouse skin. To gain insight into the hemorrhagic process, the main goal of this study was to analyze changes in the skin peptidome generated by injection of HF3, using approaches of mass spectrometry-based untargeted peptidomics. The results revealed that the sets of peptides found in the control and HF3-treated skin samples were distinct and derived from the cleavage of different proteins. Peptide bond cleavage site identification in the HF3-treated skin showed compatibility with trypsin-like serine proteases and cathepsins, suggesting the activation of host proteinases. Acetylated peptides, which originated from the cleavage at positions in the N-terminal region of proteins in both samples, were identified for the first time in the mouse skin peptidome. The number of peptides acetylated at the residue after the first Met residue, mostly Ser and Ala, was higher than that of peptides acetylated at the initial Met. Proteins cleaved in the hemorrhagic skin participate in cholesterol metabolism, PPAR signaling, and in the complement and coagulation cascades, indicating the impairment of these biological processes. The peptidomic analysis also indicated the emergence of peptides with potential biological activities, including pheromone, cell penetrating, quorum sensing, defense, and cell-cell communication in the mouse skin. Interestingly, peptides generated in the hemorrhagic skin promoted the inhibition of collagen-induced platelet aggregation and could act synergistically in the local tissue damage induced by HF3.

Next-Generation Sequencing for Venomics: Application of Multi-Enzymatic Limited Digestion for Inventorying the Snake Venom Arsenal.

To improve the characterization of snake venom protein profiles, we report the application of a new generation of proteomic methodology to deeply characterize complex protein mixtures. The new approach, combining a synergic multi-enzymatic and a time-limited digestion (MELD), is a versatile and straightforward protocol previously developed by our group. The higher number of overlapping peptides generated during MELD increases the quality of downstream peptide sequencing and of protein identification. In this context, this work aims at applying the MELD strategy to a venomics purpose for the first time, and especially for the characterization of snake venoms. We used four venoms as the test models for this proof of concept: two Elapidae (Dendroaspis polylepis and Naja naja) and two Viperidae (Bitis arietans and Echis ocellatus). Each venom was reduced and alkylated before being submitted to two different protocols: the classical bottom-up proteomics strategy including a digestion step with trypsin only, or MELD, which combines the activities of trypsin, Glu-C and chymotrypsin with a limited digestion approach. The resulting samples were then injected on an M-Class chromatographic system, and hyphenated to a Q-Exactive Mass Spectrometer. Toxins and protein identification were performed by Peaks Studio X+. The results show that MELD considerably improves the number of sequenced (de novo) peptides and identified peptides from protein databases, leading to the unambiguous identification of a greater number of toxins and proteins. For each venom, MELD was successful, not only in terms of the identification of the major toxins (increasing of sequence coverage), but also concerning the less abundant cellular components (identification of new groups of proteins). In light of these results, MELD represents a credible methodology to be applied as the next generation of proteomics approaches dedicated to venomic analysis. It may open new perspectives for the sequencing and inventorying of the venom arsenal and should expand global knowledge about venom composition.