Coral snake venoms from the Micrurus genus are a natural library of components with multiple targets, yet are poorly explored. In Brazil, 34 Micrurus species are currently described, and just a few have been investigated for their venom activities. Micrurus venoms are composed mainly of phospholipases A₂ and three-finger toxins, which are responsible for neuromuscular blockade—the main envenomation outcome in humans. Beyond these two major toxin families, minor components are also important for the global venom activity, including Kunitz-peptides, serine proteases, 5′ nucleotidases, among others. In the present study, we used the two-microelectrode voltage clamp technique to explore the crude venom activities of five different Micrurus species from the south and southeast of Brazil: M. altirostris, M. corallinus, M. frontalis, M. carvalhoi and M. decoratus. All five venoms induced full inhibition of the muscle-type α1β1δε nAChR with different levels of reversibility. We found M. altirostris and M. frontalis venoms acting as partial inhibitors of the neuronal-type α7 nAChR with an interesting subsequent potentiation after one washout. We discovered that M. altirostris and M. corallinus venoms modulate the α1β2 GABA_AR. Interestingly, the screening on K_V1.3 showed that all five Micrurus venoms act as inhibitors, being totally reversible after the washout. Since this activity seems to be conserved among different species, we hypothesized that the Micrurus venoms may rely on potassium channel inhibitory activity as an important feature of their envenomation strategy. Finally, tests on Na_V1.2 and Na_V1.4 showed that these channels do not seem to be targeted by Micrurus venoms. In summary, the venoms tested are multifunctional, each of them acting on at least two different types of targets.

• Arterial pressure
• Bioactive peptide mass spectrometry
• M2 muscarinic receptor
• Peptide folding
• Peptide sequencing, Drug screening Peptide synthesis
• Snake venom

Peptide toxins from venoms have undergone a long evolutionary process allowing host defense or prey capture and making them highly selective and potent for their target. This has resulted in the emergence of a large panel of toxins from a wide diversity of species, with varied structures and multiple associated biological functions. In this way, animal toxins constitute an inexhaustible reservoir of druggable molecules due to their interesting pharmacological properties. One of the most interesting classes of therapeutic targets is the G-protein coupled receptors (GPCRs). GPCRs represent the largest family of membrane receptors in mammals with approximately 800 different members. They are involved in almost all biological functions and are the target of almost 30% of drugs currently on the market. Given the interest of GPCRs in the therapeutic field, the study of toxins that can interact with and modulate their activity with the purpose of drug development is of particular importance. The present review focuses on toxins targeting GPCRs, including peptide-interacting receptors or aminergic receptors, with a particular focus on structural aspects and, when relevant, on potential medical applications. The toxins described here exhibit a great diversity in size, from 10 to 80 amino acids long, in disulfide bridges, from none to five, and belong to a large panel of structural scaffolds. Particular toxin structures developed here include inhibitory cystine knot (ICK), three-finger fold, and Kunitz-type toxins. We summarize current knowledge on the structural and functional diversity of toxins interacting with GPCRs, concerning first the agonist-mimicking toxins that act as endogenous agonists targeting the corresponding receptor, and second the toxins that differ structurally from natural agonists and which display agonist, antagonist, or allosteric properties.

• Kunitz peptide
• Snake venom
• V2R
• antagonist
• peptide sequencing
• peptide synthesis
• proteomics

• bradykinin-potentiating peptides
• cardioprotector
• cardiotoxin
• cardiovascular system
• natriuretic peptide
• snake venom

Studies on 3FTxs around the world are showing the amazing diversity in these proteins both in structure and function. In Brazil, we have not realized the broad variety of their amino acid sequences and probable diversified structures and targets. In this context, this work aims to conduct an in silico systematic study on available 3FTxs found in Micrurus species from Brazil. We elaborated a specific guideline for this toxin family. First, we grouped them according to their structural homologue predicted by HHPred server and further curated manually. For each group, we selected one sequence and constructed a representative structural model. By looking at conserved features and comparing with the information available in the literature for this toxin family, we managed to point to potential biological functions. In parallel, the phylogenetic relationship was estimated for our database by maximum likelihood analyses and a phylogenetic tree was constructed including the homologous 3FTx previously characterized. Our results highlighted an astonishing diversity inside this family of toxins, showing some groups with expected functional similarities to known 3FTxs, and pointing out others with potential novel roles and perhaps structures. Moreover, this classification guideline may be useful to aid future studies on these abundant toxins.

• Micrurus venoms
• classification guideline
• sequence variability
• structure-function
• three-finger toxins

Latest advancement of omics technologies allows in-depth characterization of venom compositions. In the present work we present a proteomic study of two snake venoms of the genus Naja i.e., Naja naja (black cobra) and Naja oxiana (brown cobra) of Pakistani origin. The present study has shown that these snake venoms consist of a highly diversified proteome. Furthermore, the data also revealed variation among closely related species. High throughput mass spectrometric analysis of the venom proteome allowed to identify for the N. naja venom 34 protein families and for the N. oxiana 24 protein families. The comparative evaluation of the two venoms showed that N. naja consists of a more complex venom proteome than N. oxiana venom. Analysis also showed N-terminal acetylation (N-ace) of a few proteins in both venoms. To the best of our knowledge, this is the first study revealing this posttranslational modification in snake venom. N-ace can shed light on the mechanism of regulation of venom proteins inside the venom gland. Furthermore, our data showed the presence of other body proteins, e.g., ankyrin repeats, leucine repeats, zinc finger, cobra serum albumin, transferrin, insulin, deoxyribonuclease-2-alpha, and other regulatory proteins in these venoms. Interestingly, our data identified Ras-GTpase type of proteins, which indicate the presence of extracellular vesicles in the venom. The data can support the production of distinct and specific anti-venoms and also allow a better understanding of the envenomation and mechanism of distribution of toxins. Data are available via ProteomeXchange with identifier PXD018726.

• Ankyrin repeat
• N-terminal acetylation
• Naja naja
• Naja oxiana
• Ras-GTPase
• extracellular vesicles
• venom proteome

• Beta-cardiotoxin
• Cardiomyocyte
• Cytotoxicity
• King cobra
• Purification

• Biological activity
• Snake
• Structure
• Three-finger toxins
• Venom

Snake venoms are sources of molecules with proven and potential therapeutic applications. However, most activities assayed in venoms (or their components) are of hemorrhagic, hypotensive, edematogenic, neurotoxic or myotoxic natures. Thus, other relevant activities might remain unknown. Using functional genomics coupled to the connectivity map (C-map) approach, we undertook a wide range indirect search for biological activities within the venom of the South American pit viper Bothrops jararaca. For that effect, venom was incubated with human breast adenocarcinoma cell line (MCF7) followed by RNA extraction and gene expression analysis. A list of 90 differentially expressed genes was submitted to biosimilar drug discovery based on pattern recognition. Among the 100 highest-ranked positively correlated drugs, only the antihypertensive, antimicrobial (both antibiotic and antiparasitic), and antitumor classes had been previously reported for B. jararaca venom. The majority of drug classes identified were related to (1) antimicrobial activity; (2) treatment of neuropsychiatric illnesses (Parkinson’s disease, schizophrenia, depression, and epilepsy); (3) treatment of cardiovascular diseases, and (4) anti-inflammatory action. The C-map results also indicated that B. jararaca venom may have components that target G-protein-coupled receptors (muscarinic, serotonergic, histaminergic, dopaminergic, GABA, and adrenergic) and ion channels. Although validation experiments are still necessary, the C-map correlation to drugs with activities previously linked to snake venoms supports the efficacy of this strategy as a broad-spectrum approach for biological activity screening, and rekindles the snake venom-based search for new therapeutic agents.

• Bothrops jararaca
• biosimilar drugs
• connectivity map
• drug discovery
• therapeutic potential

• Animals
• Bothrops
• Crotalid Venoms/pharmacology
• Crotalid Venoms/therapeutic use
• Drug Discovery
• Humans
• MCF-7 Cells
• Transcriptome/drug effects

Sperm contains a wealth of cell surface receptors and ion channels that are required for most of its basic functions such as motility and acrosome reaction. Conversely, animal venoms are enriched in bioactive compounds that primarily target those ion channels and cell surface receptors. We hypothesized, therefore, that animal venoms should be rich enough in sperm-modulating compounds for a drug discovery program. Our objective was to demonstrate this fact by using a sperm-based phenotypic screening to identify positive modulators from the venom of Walterinnesia aegyptia.

Herein, as proof of concept that venoms contain interesting compounds for sperm physiology, we fractionated Walterinnesia aegyptia snake venom by RP-HPLC and screened for bioactive fractions capable of accelerating mouse sperm motility (primary screening). Next, we purified each compound from the positive fraction by cation exchange and identified the bioactive peptide by secondary screening. The peptide sequence was established by Edman sequencing of the reduced/alkylated compound combined to LC-ESI-QTOF MS/MS analyses of reduced/alkylated fragment peptides following trypsin or V8 protease digestion.

Using this two-step purification protocol combined to cell phenotypic screening, we identified a new toxin of 7329.38 Da (actiflagelin) that activates sperm motility in vitro from OF1 male mice. Actiflagelin is 63 amino acids in length and contains five disulfide bridges along the proposed pattern of disulfide connectivity C₁-C₅, C₂-C₃, C₄-C₆, C₇-C₈ and C₉-C₁₀. Modeling of its structure suggests that it belongs to the family of three finger toxins with a noticeable homology with bucandin, a peptide from Bungarus candidus venom.

This report demonstrates the feasibility of identifying profertility compounds that may be of therapeutic potential for infertility cases where motility is an issue.

• Bioactive compounds
• De novo sequencing
• Edman degradation
• Fertility
• Snake venom
• Sperm motility
• Tandem mass spectrometry
• Venomics
• Walterinnesia aegyptia

• Kunitz peptide
• polycystic kidney disease
• snake toxin

Mamba venoms contain a multiplicity of three-finger fold aminergic toxins known to interact with various α-adrenergic, muscarinic and dopaminergic receptors with different pharmacological profiles. In order to generate novel functions on this structural scaffold and to avoid the daunting task of producing and screening an overwhelming number of variants generated by a classical protein engineering strategy, we accepted the challenge of resurrecting ancestral proteins, likely to have possessed functional properties. This innovative approach that exploits molecular evolution models to efficiently guide protein engineering, has allowed us to generate a small library of six ancestral toxin (AncTx) variants and associate their pharmacological profiles to key functional substitutions. Among these variants, we identified AncTx1 as the most α_1A-adrenoceptor selective peptide known to date and AncTx5 as the most potent inhibitor of the three α2 adrenoceptor subtypes. Three positions in the ρ-Da1a evolutionary pathway, positions 28, 38 and 43 have been identified as key modulators of the affinities for the α₁ and α_2C adrenoceptor subtypes. Here, we present a first attempt at rational engineering of the aminergic toxins, revealing an epistasis phenomenon.

• Black mamba venom
• Dendroaspis
• Eastern green mamba venom
• Elapidae
• Snake venomics
• Top-down proteomics

• Dendroaspis angusticeps
• Green mamba
• Proteomics
• Snake venom
• Toxicovenomics: antivenoms

• animal toxin
• screening approaches
• toxin engineering
• transient receptor potential channel
• voltage-gated ion channel

ρ-Da1a is a three-finger fold toxin from green mamba venom that is highly selective for the α_1A-adrenoceptor. This toxin has atypical pharmacological properties, including incomplete inhibition of ³H-prazosin or ¹²⁵I-HEAT binding and insurmountable antagonist action. We aimed to clarify its mode of action at the α_1A-adrenoceptor. The affinity (pKi 9.26) and selectivity of ρ-Da1a for the α_1A-adrenoceptor were confirmed by comparing binding to human adrenoceptors expressed in eukaryotic cells. Equilibrium and kinetic binding experiments were used to demonstrate that ρ-Da1a, prazosin and HEAT compete at the α_1A-adrenoceptor. ρ-Da1a did not affect the dissociation kinetics of ³H-prazosin or ¹²⁵I-HEAT, and the IC₅₀ of ρ-Da1a, determined by competition experiments, increased linearly with the concentration of radioligands used, while the residual binding by ρ-Da1a remained stable. The effect of ρ-Da1a on agonist-stimulated Ca²⁺ release was insurmountable in the presence of phenethylamine- or imidazoline-type agonists. Ten mutations in the orthosteric binding pocket of the α_1A-adrenoceptor were evaluated for alterations in ρ-Da1a affinity. The D106^3.32A and the S188^5.42A/S192^5.46A receptor mutations reduced toxin affinity moderately (6 and 7.6 times, respectively), while the F86^2.64A, F288^6.51A and F312^7.39A mutations diminished it dramatically by 18- to 93-fold. In addition, residue F86^2.64 was identified as a key interaction point for ¹²⁵I-HEAT, as the variant F86^2.64A induced a 23-fold reduction in HEAT affinity. Unlike the M1 muscarinic acetylcholine receptor toxin MT7, ρ-Da1a interacts with the human α_1A-adrenoceptor orthosteric pocket and shares receptor interaction points with antagonist (F86^2.64, F288^6.51 and F312^7.39) and agonist (F288^6.51 and F312^7.39) ligands. Its selectivity for the α_1A-adrenoceptor may result, at least partly, from its interaction with the residue F86^2.64, which appears to be important also for HEAT binding.

• Adrenergic alpha-1 Receptor Agonists/chemistry
• Adrenergic alpha-1 Receptor Agonists/pharmacology
• Adrenergic alpha-1 Receptor Antagonists/chemistry
• Adrenergic alpha-1 Receptor Antagonists/pharmacology
• Animals
• Binding Sites
• Binding, Competitive
• CHO Cells
• Cricetulus
• Elapid Venoms/chemistry
• Elapid Venoms/pharmacology
• Elapidae/metabolism
• Humans
• Kinetics
• Ligands
• Models, Molecular
• Mutation
• Prazosin/chemistry
• Prazosin/pharmacology
• Protein Binding
• Radioligand Assay
• Receptors, Adrenergic, alpha-1/chemistry
• Receptors, Adrenergic, alpha-1/genetics
• Receptors, Adrenergic, alpha-1/metabolism
• Tetralones/chemistry
• Tetralones/pharmacology

• 5' Untranslated Regions
• Amino Acid Sequence
• Animals
• Base Sequence
• Computational Biology
• Conserved Sequence
• Cysteine/chemistry
• Evolution, Molecular
• Insect Proteins/chemistry
• Insect Proteins/genetics
• Introns
• Molecular Sequence Data
• Peptides/chemistry
• Peptides/genetics
• Phylogeny
• Polymorphism, Genetic
• Scorpions/genetics
• Sequence Homology, Amino Acid

Excessive hepatocyte apoptosis is a common event in acute and chronic liver diseases leading to loss of functional liver tissue. Approaches to prevent apoptosis have therefore high potential for the treatment of liver disease. G-protein coupled receptors (GPCR) play crucial roles in cell fate (proliferation, cell death) and act through heterotrimeric G-proteins. G_αiPCRs have been shown to regulate lipoapoptosis in hepatocytes, but their role in inflammation- or bile acid-induced apoptosis is unknown. Here, we analyzed the effect of inhibiting G_αiPCR function, using pertussis toxin (PT), on bile acid- and cytokine-induced apoptosis in hepatocytes. Primary rat hepatocytes, HepG2-rNtcp cells (human hepatocellular carcinoma cells) or H-4-II-E cells (rat hepatoma cells) were exposed to glycochenodeoxycholic acid (GCDCA) or tumor necrosis factor-α (TNFα)/actinomycin D (ActD). PT (50–200 nmol/L) was added 30 minutes prior to the apoptotic stimulus. Apoptosis (caspase-3 activity, acridine orange staining) and necrosis (sytox green staining) were assessed. PT significantly reduced GCDCA- and TNFα/ActD-induced apoptosis in rat hepatocytes (−60%, p<0.05) in a dose-dependent manner (with no shift to necrosis), but not in HepG2-rNtcp cells or rat H-4-II-E cells. The protective effect of pertussis toxin was independent of the activation of selected cell survival signal transduction pathways, including ERK, p38 MAPK, PI3K and PKC pathways, as specific protein kinase inhibitors did not reverse the protective effects of pertussis toxin in GCDCA-exposed hepatocytes. Conclusion: Pertussis toxin, an inhibitor of G_αiPCRs, protects hepatocytes, but not hepatocellular carcinoma cells, against bile acid- and cytokine-induced apoptosis and has therapeutic potential as primary hepatoprotective drug, as well as adjuvant in anti-cancer therapy.

• Animals
• Apoptosis/drug effects
• Bile Acids and Salts/pharmacology
• Caspase 3/metabolism
• Cytokines/pharmacology
• Enzyme Activation/drug effects
• Glycochenodeoxycholic Acid/pharmacology
• Hep G2 Cells
• Hepatocytes/drug effects
• Hepatocytes/metabolism
• Humans
• Male
• Mitogen-Activated Protein Kinase 1/metabolism
• Mitogen-Activated Protein Kinase 3/metabolism
• Pertussis Toxin/pharmacology
• Phosphatidylinositol 3-Kinases/metabolism
• Protein Kinase C/metabolism
• Rats
• Rats, Wistar
• Receptors, G-Protein-Coupled/antagonists & inhibitors
• Time Factors
• p38 Mitogen-Activated Protein Kinases/metabolism

By screening extracts of venom from the Asian scorpion Buthus martensii Karsch (BmK) for their abilities to activate opioid receptors, we have identified BmK-YA, an amidated peptide containing an enkephalin-like sequence. BmK-YA is encoded by a precursor that displays a signal sequence and contains four copies of BmK-YA sequences and four of His⁴-BmK-YA, all flanked by single amino acid residues. BmK-YA and His⁴-BmK-YA are amidated and thus fulfill the characteristics expected of bioactive peptides. BmK-YA can activate mammalian opioid receptors with selectivity for the δ subtype while His⁴-BmK-YA is inactive at opioid receptors. The discovery of BmK-YA suggests that scorpion venom may represent a novel source of bioactive molecules targeting G protein-coupled receptors (GPCRs) and reveal additional insights on the evolution of the opioid precursors.

• Amino Acid Sequence
• Analgesics, Opioid/isolation & purification
• Analgesics, Opioid/pharmacology
• Animals
• Arthropod Proteins/genetics
• Arthropod Proteins/isolation & purification
• Arthropod Proteins/pharmacology
• Calcium Signaling/drug effects
• Cloning, Molecular
• Enkephalins/genetics
• Enkephalins/isolation & purification
• Enkephalins/pharmacology
• HEK293 Cells
• Humans
• Molecular Sequence Data
• Protein Precursors/genetics
• Protein Precursors/isolation & purification
• Protein Precursors/pharmacology
• Receptors, Opioid/agonists
• Receptors, Opioid/metabolism
• Scorpion Venoms/genetics
• Scorpion Venoms/isolation & purification
• Scorpion Venoms/pharmacology
• Scorpions
• Sequence Analysis, Protein

• R01 DK063001 NIDDK NIH HHS
• MH60231 NIMH NIH HHS
• DA024746 NIDA NIH HHS
• R01 MH060231 NIMH NIH HHS
• R01 DA024746 NIDA NIH HHS

• Amino Acid Sequence
• Animals
• Chromatography, Ion Exchange
• Elapid Venoms/chemistry
• Elapidae
• Glycosylation
• Molecular Sequence Data
• Peptides/chemistry
• Sequence Analysis, Protein/methods
• Spectrometry, Mass, Electrospray Ionization
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods