The scorpion toxin BeKm-1 blocks hERG cardiac potassium channels using an indispensable arginine residue.

BeKm-1 is a peptide toxin from scorpion venom that blocks the pore of the potassium channel hERG (Kv11.1) in the human heart. Although individual protein structures have been resolved, the structure of the complex between hERG and BeKm-1 is unknown. Here, we used molecular dynamics and ensemble docking, guided by previous double-mutant cycle analysis data, to obtain an in silico model of the hERG-BeKm-1 complex. Adding to the previous mutagenesis study of BeKm-1, our model uncovers the key role of residue Arg20, which forms three interactions (a salt bridge and hydrogen bonds) with the channel vestibule simultaneously. Replacement of this residue even by lysine weakens the interactions significantly. In accordance, the recombinantly produced BeKm-1R20K mutant exhibited dramatically decreased activity on hERG. Our model may be useful for future drug design attempts.

Divergence in toxin antigenicity and venom enzymes in Tityus melici, a medically important scorpion, despite transcriptomic and phylogenetic affinities with problematic Brazilian species.

The Brazilian scorpion Tityus melici, native to Minas Gerais and Bahia, is morphologically related to Tityus serrulatus, the most medically significant species in Brazil. Despite inhabiting scorpion-envenomation endemic regions, T. melici venom remains unexplored. This work evaluates T. melici venom composition and function using transcriptomics, enzymatic activities, and in vivo and in vitro immunological analyses. Next-Generation Sequencing unveiled 86 components putatively involved in venom toxicity: 39 toxins, 28 metalloproteases, seven disulfide isomerases, six hyaluronidases, three phospholipases and three amidating enzymes. T. serrulatus showed the highest number of toxin matches with 80-100 % sequence similarity. T. melici is of medical importance as it has a venom LD50 of 0.85 mg/kg in mice. We demonstrated venom phospholipase A2 activity, and elevated hyaluronidase and metalloprotease activities compared to T. serrulatus, paralleling our transcriptomic findings. Comparison of transcriptional levels for T. serrulatus and T. melici venom metalloenzymes suggests species-specific expression patterns in Tityus. Despite close phylogenetic association with T. serrulatus inferred from COI sequences and toxin similarities, partial neutralization of T. melici venom toxicity was achieved when using the anti-T. serrulatus antivenom, implying antigenic divergence among their toxins. We suggest that the Brazilian therapeutic scorpion antivenom could be improved to effectively neutralize T. melici venom.

Genome-wide identification, structural homology analysis, and evolutionary diversification of the phospholipase D gene family in the venom gland of three scorpion species.

BACKGROUND: Venom phospholipase D (PLDs), dermonecrotic toxins like, are the major molecules in the crude venom of scorpions, which are mainly responsible for lethality and dermonecrotic lesions during scorpion envenoming. The purpose of this study was fivefold: First, to identify transcripts coding for venom PLDs by transcriptomic analysis of the venom glands from Androctonus crassicauda, Hottentotta saulcyi, and Hemiscorpius lepturus; second, to classify them by sequence similarity to known PLDs and motif extraction method; third, to characterize scorpion PLDs; fourth to structural homology analysis with known dermonecrotic toxins; and fifth to investigate phylogenetic relationships of the PLD proteins. RESULTS: We found that the venom gland of scorpions encodes two PLD isoforms: PLD1 ScoTox-beta and PLD2 ScoTox-alpha I. Two highly conserved regions shared by all PLD1s beta are GAN and HPCDC (HX2PCDC), and the most important conserved regions shared by all PLD2s alpha are two copies of the HKDG (HxKx4Dx6G) motif. We found that PLD1 beta is a 31-43 kDa acidic protein containing signal sequences, and PLD2 alpha is a 128 kDa basic protein without known signal sequences. The gene structures of PLD1 beta and PLD2 alpha contain 6 and 21 exons, respectively. Significant structural homology and similarities were found between the modeled PLD1 ScoTox-beta and the crystal structure of dermonecrotic toxins from Loxosceles intermedia. CONCLUSIONS: This is the first report on identifying PLDs from A. crassicauda and H. saulcyi venom glands. Our work provides valuable insights into the diversity of scorpion PLD genes and could be helpful in future studies on recombinant antivenoms production.

Identification, Characterization, and Modeling of a Bioinsecticide Protein Isolated from Scorpion Venom gland: A Three-Finger Protein.

Background: The majority of insecticides target sodium channels. The increasing emergence of resistance to the current insecticides has persuaded researchers to search for alternative compounds. Scorpion venom gland as a reservoir of peptides or proteins, which selectively target insect sodium channels. These proteins would be an appropriate source for finding new suitable anti-insect components. Methods: Transcriptome of venom gland of scorpion Mesobuthus eupeus was obtained by RNA extraction and complementary DNA library synthesis. The obtained transcriptome was blasted against protein databases to find insect toxins against sodium channel based on the statistically significant similarity in sequence. Physicochemical properties of the identified protein were calculated using bioinformatics software. The three-dimensional structure of this protein was determined using homology modeling, and the final structure was assessed by molecular dynamics simulation. Results: The sodium channel blocker found in the transcriptome of M. eupeus venom gland was submitted to the GenBank under the name of meuNa10, a stable hydrophilic protein consisting of 69 amino acids, with the molecular weight of 7721.77 g/mol and pI of 8.7. The tertiary structure of meuNa10 revealed a conserved LCN-type cysteine-stabilized alpha/beta domain stabilized by eight cysteine residues. The meuNa10 is a member of the 3FP superfamily consisting of three finger-like beta strands. Conclusion: This study identified meuNa10 as a small insect sodium channel-interacting protein with some physicochemical properties, including stability and water-solubility, which make it a good candidate for further in vivo and in vitro experiments in order to develop a new bioinsecticide.

Production of recombinant scorpion antivenoms in E. coli: current state and perspectives.

Scorpion envenomation is a serious health problem in tropical and subtropical zones. The access to scorpion antivenom is sometimes limited in availability and specificity. The classical production process is cumbersome, from the hyper-immunization of the horses to the IgG digestion and purification of the F(ab)'2 antibody fragments. The production of recombinant antibody fragments in Escherichia coli is a popular trend due to the ability of this microbial host to produce correctly folded proteins. Small recombinant antibody fragments, such as single-chain variable fragments (scFv) and nanobodies (VHH), have been constructed to recognize and neutralize the neurotoxins responsible for the envenomation symptoms in humans. They are the focus of interest of the most recent studies and are proposed as potentially new generation of pharmaceuticals for their use in immunotherapy against scorpion stings of the Buthidae family. This literature review comprises the current status on the scorpion antivenom market and the analyses of cross-reactivity of commercial scorpion anti-serum against non-specific scorpion venoms. Recent studies on the production of new recombinant scFv and nanobodies will be presented, with a focus on the Androctonus and Centruroides scorpion species. Protein engineering-based technology could be the key to obtaining the next generation of therapeutics capable of neutralizing and cross-reacting against several types of scorpion venoms. KEY POINTS: • Commercial antivenoms consist of predominantly purified equine F(ab)'2fragments. • Nanobody-based antivenom can neutralize Androctonus venoms and have a low immunogenicity. • Affinity maturation and directed evolution are used to obtain potent scFv families against Centruroides scorpions.

A first molecular characterization of the scorpion telson microbiota of Hadrurus arizonensis and Smeringurus mesaensis.

Scorpions represent an ancient lineage of arachnids that have radiated across the globe and are incredibly resilient-since some thrive in harsh environments and can exist on minimal and intermittent feedings. Given the emerging importance of microbiomes to an organism's health, it is intriguing to suggest that the long-term success of the scorpion bauplan may be linked to the microbiome. Little is known about scorpion microbiomes, and what is known, concentrates on the gut. The microbiome is not limited to the gut, rather it can be found within tissues, fluids and on external surfaces. We tested whether the scorpion telson, the venom-producing organ, of two species, Smeringurus mesaensis and Hadrurus arizonensis, contain bacteria. We isolated telson DNA from each species, amplified bacterial 16S rRNA genes, and identified the collection of bacteria present within each scorpion species. Our results show for the first time that telsons of non-buthid scorpion species do indeed contain bacteria. Interestingly, each scorpion species has a phylogenetically unique telson microbiome including Mollicutes symbionts. This study may change how we view scorpion biology and their venoms.

An overview of Tityus cisandinus scorpion venom: Transcriptome and mass fingerprinting reveal conserved toxin homologs across the Amazon region and novel lipolytic components.

Tityus cisandinus, a neglected medically important scorpion in Ecuadorian and Peruvian Amazonia, belongs to a complex of species related to the eastern Amazon endemic Tityus obscurus, spanning a distribution of ca. 4000 km. Despite high morbidity and mortality rates, no effective scorpion antivenom is currently available in the Amazon region. Knowledge of the structural/functional relationships between T. cisandinus venom components and those from related Amazonian species is crucial for designing region-specific therapeutic antivenoms. In this work, we carried out the first venom gland transcriptomic study of an Amazonian scorpion outside Brazil, T. cisandinus. We also fingerprinted its total venom through MALDI-TOF MS, which supported our transcriptomic findings. We identified and calculated the expression level of 94 components: 60 toxins, 25 metalloproteases, five disulfide isomerases, three amidating enzymes, one hyaluronidase, and also uncovered transcripts encoding novel lipolytic beta subunits produced by New World buthid scorpions. This study demonstrates the high similarity between T. cisandinus and T. obscurus venoms, reinforcing the existence of a neglected complex of genetically and toxinologically related Amazonian scorpions of medical importance. Finally, we demonstrated the low recognition of currently available therapeutic sera against T. cisandinus and T. obscurus venoms, and concluded that these should be improved to protect against envenomation by Amazonian Tityus spp.

Differential venom gland gene expression analysis of juvenile and adult scorpions Androctonus crassicauda.

BACKGROUND: The Androctonus crassicauda, belonging to the genus Androctonus of the family Buthidae, is the most venomous scorpion in Middle East countries. However, the venom gland transcriptome profile of A. crassicauda scorpion has not yet been studied. In this study, we elucidated and compared the venom gland gene expression profiles of adult and juvenile male scorpion A. crassicauda using high-throughput transcriptome sequencing. This is the first report of transcriptional analysis of the venom glands of scorpions in different growth stages, with insights into the identification of the key genes during venom gland development. RESULTS: A total of 209,951 mRNA transcripts were identified from total RNA-seq data, of which 963 transcripts were differentially expressed (DE) in adult and juvenile scorpions (p < 0.01). Overall, we identified 558 up-regulated and 405 down-regulated transcripts in the adult compared to the juvenile scorpions, of which 397 and 269 unique unigenes were annotated, respectively. GO and KEGG enrichment analyses indicated that the metabolic, thermogenesis, cytoskeleton, estrogen signaling, GnRH signaling, growth hormone signaling, and melanogenesis pathways were affected by two different growth conditions and the results suggested that the DE genes related to those pathways are important genes associated with scorpion venom gland development, in which they may be important in future studies, including Chs, Elovl, MYH, RDX, ACTN, VCL, PIP5K, PP1C, FGFR, GNAS, EGFR, CREB, CoA, PLCB, CALM, CACNA, PKA and CAMK genes. CONCLUSIONS: These findings broadened our knowledge of the differences between adult and juvenile scorpion venom and opened new perspectives on the application of comparative transcriptome analysis to identify the special key genes.

Recombinantly expressed MeICT, a new toxin from Mesobuthus eupeus scorpion, inhibits glioma cell proliferation and downregulates Annexin A2 and FOXM1 genes.

Gliomas are highly invasive and lethal malignancy that do not respond to current therapeutic approaches. Novel therapeutic agents are required to target molecular mechanisms involved in glioma progression. MeICT is a new short-chain toxin isolated from Mesobuthus eupeus scorpion venom. This toxin contained 34 amino acid residues and belongs to chloride channels toxins. In this study, the coding sequence of MeICT was cloned into the pET32Rh vector and a high yield of soluble recombinant MeICT was expressed and purified. Recombinant MeICT-His significantly inhibited the proliferation and migration of glioma cells at low concentration. In vivo studies showed that MeICT was not toxic when administrated to mice at high doses. We also determined the effect of MeICT on the mRNA expression of MMP-2, Annexin A2 and FOXM-2 that are key molecules in the progression and invasion of glioma. Expression of Annexin A2 and FOXM1 mRNA was significantly down-regulated following treatment with MeICT. However, no significant decrease in the expression of MMP-2 gene was identified. In this study a short toxin with four disulfide bonds was successfully produced and its anti-cancer effects was detected. Our findings suggest that recombinant MeICT can be considered as a new potent agent for glioma targeting.

Phylogenomics of Scorpions Reveal Contemporaneous Diversification of Scorpion Mammalian Predators and Mammal-Active Sodium Channel Toxins.

Scorpions constitute a charismatic lineage of arthropods and comprise more than 2500 described species. Found throughout various tropical and temperate habitats, these predatory arachnids have a long evolutionary history, with a fossil record that began in the Silurian. While all scorpions are venomous, the asymmetrically diverse family Buthidae harbors nearly half the diversity of extant scorpions, and all but one of the 58 species that are medically significant to humans. However, the lack of a densely sampled scorpion phylogeny has hindered broader inferences of the diversification dynamics of scorpion toxins. To redress this gap, we assembled a phylogenomic data set of 100 scorpion venom gland transcriptomes and genomes, emphasizing the sampling of highly toxic buthid genera. To infer divergence times of venom gene families, we applied a phylogenomic node dating approach for the species tree in tandem with phylostratigraphic bracketing to estimate the minimum ages of mammal-specific toxins. Our analyses establish a robustly supported phylogeny of scorpions, particularly with regard to relationships between medically significant taxa. Analysis of venom gene families shows that mammal-active sodium channel toxins (NaTx) have independently evolved in five lineages within Buthidae. Temporal windows of mammal-targeting toxin origins are correlated with the basal diversification of major scorpion mammal predators such as shrews, bats, and rodents. These results suggest an evolutionary model of relatively recent diversification of buthid NaTx homologs in response to the diversification of scorpion predators. [Adaptation; arachnids; phylogenomic dating; phylostratigraphy; venom.].

Genomic Structure of Two Kv1.3 Channel Blockers from Scorpion Mesobuthus eupeus and Sea Anemone Stichodactyla haddoni and Construction of their Chimeric Peptide as a Novel Blocker.

Different toxins acting on Kv1.3 channel have been isolated from animal venom. MeuKTX toxin from Mesobuthus eupeus phillipsi scorpion and shtx-k toxin from Stichodactyla haddoni sea anemone have been identified as two effective Kv1.3 channel blockers. In this work, we characterized the genomic organization of both toxins. MeuKTX gene contains one intron and two exons, similar to the most scorpion toxins. There are a few reports of genomic structure of sea anemone toxins acting on Kv channels. The sequence encoding mature peptide of shtx-k was located in an exon separated by an intron from the coding exon of the propeptide and signal region. In order to make a peptide with more affinity for Kv1.3 channel and greater stability, the shtx-k/ MeuKTX chimeric peptide was designed and constructed using splicing by overlap extension-PCR (SOE-PCR) method. MeuKTX, shtx-k, and shtx-k/MeuKTX were cloned and the expression of the soluble proteins in E. coli was determined. Molecular docking studies indicated more inhibitory effect of shtx-k/MeuKTX on Kv1.3 channel compared to shtx-k and MeuKTX toxins. Key amino acids binding channel from both toxins, also involved in interaction of chimeric peptide with channel. Our results showed that the fusion peptide, shtx-k/MeuKTX could be an effective agent to target Kv1.3 channel.

Reduced Toxicity of Centruroides vittatus (Say, 1821) May Result from Lowered Sodium ß Toxin Gene Expression and Toxin Protein Production.

Body tissue and venom glands from an eastern population of the scorpion Centruroides vittatus (Say, 1821) were homogenized and molecular constituents removed to characterize putative sodium ß toxin gene diversity, RT-qPCR, transcriptomic, and proteomic variation. We cloned sodium ß toxins from genomic DNA, conducted RT-qPCR experiments with seven sodium ß toxin variants, performed venom gland tissue RNA-seq, and isolated venom proteins for mass spectrophotometry. We identified >70 putative novel sodium ß toxin genes, 111 toxin gene transcripts, 24 different toxin proteins, and quantified sodium ß toxin gene expression variation among individuals and between sexes. Our analyses contribute to the growing evidence that venom toxicity among scorpion taxa and their populations may be associated with toxin gene diversity, specific toxin transcripts variation, and subsequent protein production. Here, slight transcript variation among toxin gene variants may contribute to the major toxin protein variation in individual scorpion venom composition.

A non-lethal method for studying scorpion venom gland transcriptomes, with a review of potentially suitable taxa to which it can be applied.

Scorpion venoms are mixtures of proteins, peptides and small molecular compounds with high specificity for ion channels and are therefore considered to be promising candidates in the venoms-to-drugs pipeline. Transcriptomes are important tools for studying the composition and expression of scorpion venom. Unfortunately, studying the venom gland transcriptome traditionally requires sacrificing the animal and therefore is always a single snapshot in time. This paper describes a new way of generating a scorpion venom gland transcriptome without sacrificing the animal, thereby allowing the study of the transcriptome at various time points within a single individual. By comparing these venom-derived transcriptomes to the traditional whole-telson transcriptomes we show that the relative expression levels of the major toxin classes are similar. We further performed a multi-day extraction using our proposed method to show the possibility of doing a multiple time point transcriptome analysis. This allows for the study of patterns of toxin gene activation over time a single individual, and allows assessment of the effects of diet, season and other factors that are known or likely to influence intraindividual venom composition. We discuss the gland characteristics that may allow this method to be successful in scorpions and provide a review of other venomous taxa to which this method may potentially be successfully applied.

Expression and purification of recombinant alpha-toxin AnCra1 from the scorpion Androctonus crassicauda and its functional characterization on mammalian sodium channels.

BACKGROUND: Alpha-scorpion toxins with long-chain peptide and four disulfide bonds represent diverse pharmacological profiles for various subtypes of voltage-gated sodium channels. Obtaining the natural toxins are difficult and time-consuming process, which represents the major difficulty to interpreting analysis of their structural and functional properties. METHODS AND RESULTS: This study describes the toxin peptide and plasmid construct containing the gene coding for mammalian toxin AnCra1 from the scorpion Androctonus crassicauda venom. We have established genetic construction of fusion protein in pET32a + vector containing thioredoxin (Trx-tag), enterokinase cleavage site and 6xhistidine-tag for efficient expression in Escherichia coli strain RG2 (DE3). The soluble expressed peptide, then purified by Ni-NTA resin affinity chromatography and its purity was confirmed by reverse-phase HPLC and mass spectrometry (7433.54 Da.). The electrophysiological data showed that recombinant AnCra1 selectively inhibits the fast inactivation of hNav1.7 channel (EC50 = 136.7 ± 6.6 nM). CONCLUSIONS: Our findings demonstrate that the AnCra1 is structurally and functionally analogous to alpha excitatory toxins; furthermore, expression and purification of bioactive scorpion toxins in bacterial cells can be a practicable and efficient way to obtain a novel source of toxin peptides as tools to study the function and physiological responses of ion channels.

Mapping the interaction surface of scorpion ß-toxins with an insect sodium channel.

The interaction of insect-selective scorpion depressant ß-toxins (LqhIT2 and Lqh-dprIT3 from Leiurus quinquestriatus hebraeus) with the Blattella germanica sodium channel, BgNav1-1a, was investigated using site-directed mutagenesis, electrophysiological analyses, and structural modeling. Focusing on the pharmacologically defined binding site-4 of scorpion ß-toxins at the voltage-sensing domain II (VSD-II), we found that charge neutralization of D802 in VSD-II greatly enhanced the channel sensitivity to Lqh-dprIT3. This was consistent with the high sensitivity of the splice variant BgNav2-1, bearing G802, to Lqh-dprIT3, and low sensitivity of BgNav2-1 mutant, G802D, to the toxin. Further mutational and electrophysiological analyses revealed that the sensitivity of the WT = D802E < D802G < D802A < D802K channel mutants to Lqh-dprIT3 correlated with the depolarizing shifts of activation in toxin-free channels. However, the sensitivity of single mutants involving IIS4 basic residues (K4E = WT << R1E < R2E < R3E) or double mutants (D802K = K4E/D802K = R3E/D802K > R2E/D802K > R1E/D802K > WT) did not correlate with the activation shifts. Using the cryo-EM structure of the Periplaneta americana channel, NavPaS, as a template and the crystal structure of LqhIT2, we constructed structural models of LqhIT2 and Lqh-dprIT3-c in complex with BgNav1-1a. These models along with the mutational analysis suggest that depressant toxins approach the salt-bridge between R1 and D802 at VSD-II to form contacts with linkers IIS1-S2, IIS3-S4, IIIP5-P1 and IIIP2-S6. Elimination of this salt-bridge enables deeper penetration of the toxin into a VSD-II gorge to form new contacts with the channel, leading to increased channel sensitivity to Lqh-dprIT3.

Overexpression and purification of a toxic peptide LaIT2 from Japanese scorpion, Liocheles australasiae.

In Japan, there are two species of scorpions, Madara scorpion (Isometrus maculatus) and Yaeyama scorpion (Liocheles australasiae), and both of them are living in Yaeyama island. It has been shown that Liocheles australasiae has venom including ß-toxin acting on K+-channels (ß-KTx) (Juichi et al., 2018) [1]. Interestingly, LaIT2, one of the toxins found in the venom of Liocheles australasiae, displays the virulence for insects but almost not for mammals. Until now, molecular mechanism of the functional specificity of LaIT2 is unknown. To clear this issue, we tried to establish the overexpression system of LaIT2 in Rosetta-gami B (DE3) pLysS, which have trxB/gor mutations to induce the disulfide bond formation. In this study, we have succeeded to overexpress the recombinant LaIT2 (rLaIT2) as a thioredoxin (Trx)-tagged protein, and established the purification protocol with Ni2+-NTA column chromatography, enterokinase digestion, and HPLC. We succeeded to obtain approximately 0.5 mg of rLaIT2 from the E. coli cells cultured in 1 L of M9 culture medium. Intramolecular disulfide bonding pattern of rLaIT2 was identified by endopeptidase fragmentation and mass spectrometry. rLaIT2 showed insecticidal activity and antimicrobial activity, and these are almost identical to those of natural LaIT2. 1H-15N HSQC spectrum of 15N-labeled rLaIT2 indicated that the rLaIT2 has a stable conformation.

De novo transcriptomic and proteomic analysis and potential toxin screening of Mesobuthus martensii samples from four different provinces.

ETHNOPHARMACOLOGICAL RELEVANCE: As well-known medicinal materials in traditional Chinese medicine, scorpions, commonly called as Quanxie () in Chinese, have been widely used to treat several diseases such as rheumatoid arthritis, apoplexy, epilepsy and chronic pain for more than a thousand years. Not only in the ancient times, the scorpions have also been recorded nowadays in the Pharmacopoeia of the People's Republic of China since 1963. AIM OF STUDY: This study aims to explore the differences in composition of the venom of scorpions from different regions by using the method of transcriptomics and proteomics. MATERIALS AND METHODS: Whole de novo transcriptomes, proteomics and their bioinformatic analyses were performed on samples of the scorpion Mesobuthus martensii and their venoms from four different provinces with clear geographical boundaries, including Hebei, Henan, Shandong and Shanxi. RESULTS: The four captured samples had the same morphology, and the conserved CO-1 sequence matched that of M. martensii. A total of 141,003 of 174,653 transcripts were identified as unigenes, of which we successfully annotated 51,627 (36.61%), 21,970 (15.58%), 7,168 (5.08%), and 45,263 (32.10%) unigenes with the NR, GO, KEGG and SWISSPROT databases, respectively, while a total of 427 proteins were collected from the protein extracted from venoms. Both GO and KEGG annotations exhibited only slight differences among the four samples while the expression level of gene and protein was quite different. A total of 249 toxin-related unigenes were successfully screened, including 41 serine proteases and serine protease inhibitors, 39 potassium channel toxins, 38 phospholipases, 16 host defense peptides, 9 metalloproteases, and 50 other toxins. Although the toxin species were similar among the four samples, the gene expression of each toxin varied considerably, for example, the scorpion from HB province has the most abundant expression quality in sequences c48391_g1, c55239_g1 and c47749_g1 while the lowest expressions of c51178_g1, c62033_g3 and c63754_g2. CONCLUSION: The regional differences in the transcriptomes and proteomes of M. martensii are mainly from expression levels e.g. toxins rather than expression species, of which the method can be further extended to evaluate the qualities of traditional Chinese medicines obtained from different regions.

NMR three-dimensional structure of the cationic peptide Stigmurin from Tityus stigmurus scorpion venom: In vitro antioxidant and in vivo antibacterial and healing activity.

Infectious diseases and the rapid development of pathogens resistant to conventional drugs are a serious global public health problem, which motivates the search for new pharmacological agents. In this context, cationic peptides without disulfide bridges from different species of scorpion venom have been the target of scientific studies due to their multifunctional activities. Stigmurin is a linear peptide composed of 17 amino acid residues (Phe-Phe-Ser-Leu-Ile-Pro-Ser-Leu-Val-Gly-Gly-Leu-Ile-Ser-Ala-Phe-Lys-NH2), which is present in the venom gland of the scorpion Tityus stigmurus. Here we present investigations of the in vitro antioxidant action of Stigmurin together with the in vivo antibacterial and healing activity of this peptide in a wound infection model induced by Staphylococcus aureus. In addition, we have reports for the first time of the three-dimensional structure determined by NMR spectroscopy of a peptide without disulfide bridges present in scorpion venom from the Tityus genus. Stigmurin showed hydroxyl radical scavenging above 70 % at 10 µM and antibiotic action in the skin wound, reducing the number of viable microorganisms by 67.2 % on the 7 day after infection. Stigmurin (1 µg / µL) increased the retraction rate of the lesion, with wound area reduction of 43 % on the second day after skin injury, which indicates its ability to induce tissue repair. Stigmurin in trifluoroethanol:water exhibited a random conformation at the N-terminus region (Phe1 to Pro6), with a helical structure from Ser7 to Phe16. This structural information, allied with the multifunctional activity of Stigmurin, makes it an attractive candidate for the design of novel therapeutic agents.

A Venomics Approach Coupled to High-Throughput Toxin Production Strategies Identifies the First Venom-Derived Melanocortin Receptor Agonists.

Animal venoms are rich in hundreds of toxins with extraordinary biological activities. Their exploitation is difficult due to their complexity and the small quantities of venom available from most venomous species. We developed a Venomics approach combining transcriptomic and proteomic characterization of 191 species and identified 20,206 venom toxin sequences. Two complementary production strategies based on solid-phase synthesis and recombinant expression in Escherichia coli generated a physical bank of 3597 toxins. Screened on hMC4R, this bank gave an incredible hit rate of 8%. Here, we focus on two novel toxins: N-TRTX-Preg1a, exhibiting an inhibitory cystine knot (ICK) motif, and N-BUTX-Ptr1a, a short scorpion-CSαß structure. Neither N-TRTX-Preg1a nor N-BUTX-Ptr1a affects ion channels, the known targets of their toxin scaffolds, but binds to four melanocortin receptors with low micromolar affinities and activates the hMC1R/Gs pathway. Phylogenetically, these two toxins form new groups within their respective families and represent novel hMC1R agonists, structurally unrelated to the natural agonists.