The systematic annotation of novel peptides found in the venom of scorpions needs revision. The commonly used two-letter acronym with the initials of the genus and the species is not discriminative and induces confusion. A new universal five-letter abbreviated code is here proposed. With this code, every species can be unambiguously identified. The code contains the initial capital letter of the genus, followed by four letters from the species. This code discriminates the large majority of the species. For the few others from the same genus and with coinciding initial letters of the species name, a change in the fifth letter ensures uniqueness. For scorpions belonging to different genera with identical initial letters and the same exact species name, a five-letter identifier can be generated by using two letters from the genus (in uppercase) and three letters from the species (in lowercase). Following this proposal, the peptides belonging to all scorpion species can be properly annotated.

Hundreds of toxins, particularly from scorpions of lesser medical significance, remain unknown, especially those from species endemic to specific ecosystems, such as Tityus fasciolatus. Their discovery could contribute to the development of new drugs for channelopathies and other diseases. Tf5 is a new peptide that has been identified from the venom of Tityus fasciolatus, a scorpion species endemic to the Brazilian Cerrado ecosystem. A full-length cDNA sequence of the Tf5 gene was obtained through a previously constructed transcriptomic library, where an ORF (Open Reading Frame) sequence with a length of 180 was found, including the 37 aa mature KTx domain, which has six Cys residues. Tf5 was purified from the crude venom, resulting in a peptide with a molecular mass of 3983.95 Da. Its K+ channel blocker activity was evaluated on Kv1.1, Kv1.2, Kv1.3, and Kv1.4 subtypes. Of these Kv channels, the peptide demonstrated an ability to block Kv1.2 and Kv1.3 with an IC50 of 15.53 nM and 116.41 nM, respectively. Additionally, Tf5 shares a high degree of sequence identity with toxins from the α-KTx4 subfamily, which led to it being classified as α-KTx4.9. This is the first Kv channel blocker described from the T. fasciolatus scorpion.

Antibiotic-resistant bacteria pose a significant global health threat, particularly pathogens resistant to last-resort antibiotics, such as those listed as priority pathogens by the World Health Organization. Addressing this challenge requires the development of novel antimicrobial agents. Previously, we identified a blue 1,4-benzoquinone isolated from the venom of the Mexican scorpion Diplocentrus melici as a potent antimicrobial compound effective against Staphylococcus aureus and Mycobacterium tuberculosis. Moreover, we devised a cost-effective synthetic route for its production. In this study, we demonstrate that the blue benzoquinone exhibits antibacterial activity against additional pathogens, including the priority pathogen Acinetobacter baumannii. Notably, the compound effectively killed clinical strains of A. baumannii resistant to multiple antibiotics, including carbapenem and colistin. Furthermore, A. baumannii did not develop resistance to the benzoquinone even after multiple growth cycles under sub-inhibitory concentrations, unlike the tested antibiotics. These findings underscore the potential of this blue benzoquinone as a lead compound for the development of a new class of antibiotics targeting multidrug-resistant bacteria.

The venom from the Mexican scorpion Centruroides mascota was separated by chromatographic procedures and three new toxins for mammals (mice used as model) were found and characterized. They were named Cmasc1 to 3, and the full amino acid of Cmasc1 with a molecular mass of 7577.8 Da was determined. Similarly, the amino acid sequence of, Cmasc2 was determined. It has the molecular weight of 7693.8 Da Cmasc3 had molecular mass of 7542.5 Da, but the full amino acid sequence was not determined, however the first 62 amino acid residues are identical to Cmasc2. They are all packed by four disulfide bridges. Electrophysiological experiments using patch-clamp heterologous expressing Nav1.6 human channels showed that they are bona fide toxins of the beta type scorpion toxins (β-ScTx). A phylogenetic tree analysis showed that they are similar to other toxins from the genus Centruroides, as somehow expected. We are currently evaluating some single-chain antibodies potentially capable of protecting humans against intoxication by these toxins.

Centruroides baergi is a scorpion species distributed in the biogeographical province of the Balsas Basin in Mexico. Health officials have reported acute envenomation in human populations living on the western side of this scorpion's range, but none in the eastern region. This disparity in toxicity suggested that there may be two distinct species. We used two different approaches, including venom analysis and morphometric specimens from both regions, to test our hypothesis. We performed chromatographic, electrophoretic, and mass spectrometry analysis to identify the known β-toxins involved in the intoxication. The most remarkable finding was the absence of Cb3 β-toxin in the eastern population. Consequently, the LD50 of the eastern population was lower than that of the western population. We analyzed linear and ratio body measurements with parametric and nonparametric statistics to test species limits. These analyses indicated that all putative populations of C. baergi are significantly similar, suggesting that they may represent a single species. Unexpectedly, the population of scorpions in the center of the study area, Suchixtlahuaca, previously identified as C. baergi, showed significant morphological and venom composition differences. We provided empirical evidence of an abrupt change of highly toxic peptides around the 98th meridian that limits populations of Centruroides baergi to the east of Mexico.

Five peptides were isolated from the venom of the Mexican scorpion Centruroides bonito by chromatographic procedures (molecular weight sieving, ion exchange columns, and HPLC) and were denoted Cbo1 to Cbo5. The first four peptides contain 66 amino acid residues and the last one contains 65 amino acids, stabilized by four disulfide bonds, with a molecular weight spanning from about 7.5 to 7.8 kDa. Four of them are toxic to mice, and their function on human Na+ channels expressed in HEK and CHO cells was verified. One of them (Cbo5) did not show any physiological effects. The ones toxic to mice showed that they are modifiers of the gating mechanism of the channels and belong to the beta type scorpion toxin (β-ScTx), affecting mainly the Nav1.6 channels. A phylogenetic tree analysis of their sequences confirmed the high degree of amino acid similarities with other known bona fide β-ScTx. The envenomation caused by this venom in mice is treated by using commercially horse antivenom available in Mexico. The potential neutralization of the toxic components was evaluated by means of surface plasmon resonance using four antibody fragments (10FG2, HV, LR, and 11F) which have been developed by our group. These antitoxins are antibody fragments of single-chain antibody type, expressed in E. coli and capable of recognizing Cbo1 to Cbo4 toxins to various degrees.

Scorpion species diversity in Colima was investigated with a multigene approach. Fieldwork produced 34 lots of scorpions that were analyzed with 12S rDNA, 16S rDNA, COI, and 28S rDNA genetic markers. Our results confirmed prior phylogenetic results recovering the monophyly of the families Buthidae and Vaejovidae, some species groups, and genera. We recorded 11 described species of scorpions and found 3 putatively undescribed species of Centruroides, 1 of Mesomexovis, and 1 of Vaejovis. Furthermore, we obtained evidence that Centruroides elegans, C. infamatus, and C. limpidus do not occur in Colima, contrary to prior reports. Seven genetically different and medically relevant species of Centruroides for Colima are recorded for the first time. We used the InDRE database (Instituto de Diagnóstico y Referencia Epidemiológicos), which contains georeferenced points of scorpions, to estimate the distribution of the scorpion species found in our fieldwork. Finally, we discuss from a biogeographical, ecological, and medical point of view the presence and origin of the 14 scorpion species found in Colima.

Alternative recombinant sources of antivenoms have been successfully generated. The application of such strategies requires the characterization of the venoms for the development of specific neutralizing molecules against the toxic components. Five toxic peptides to mammals from the Mexican scorpion Centruroides villegasi were isolated by chromatographic procedures by means of gel filtration on Sephadex G-50, followed by ion-exchange columns on carboxy-methyl-cellulose (CMC) resins and finally purified by high-performance chromatography (HPLC) columns. Their primary structures were determined by Edman degradation. They contain 66 amino acids and are maintained well packed by four disulfide bridges, with molecular mass from 7511.3 to 7750.1 Da. They are all relatively toxic and deadly to mice and show high sequence identity with known peptides that are specific modifiers of the gating mechanisms of Na+ ion channels of type beta-toxin (β-ScTx). They were named Cv1 to Cv5 and used to test their recognition by single-chain variable fragments (scFv) of antibodies, using surface plasmon resonance. Three different scFvs generated in our laboratory (10FG2, HV, LR) were tested for recognizing the various new peptides described here, paving the way for the development of a novel type of scorpion antivenom.

Scorpine is an antimicrobial and antimalarial peptide isolated from Pandinus imperator scorpion venom. As there are few functional and structural studies reported on scorpine-like peptides, we investigated the recombinant truncated N- and C-terminal domains as well as complete scorpine using biological assays and determined the N- and C-terminal structures using solution nuclear magnetic resonance. The study was conducted using recombinant N- and C-terminal peptides and complete scorpine expressed in Escherichia coli. The results showed that N-scorpine presented a random coil structure in water and adopted α-helical folding in the presence of 50% trifluoroethanol (TFE). C-scorpine contains three disulfide bonds with two structural domains: an unstructured N-terminal domain in water that can form a typical secondary alpha-helix structure in 50% TFE and a C-terminal domain with the CS-αβ motif. Our findings demonstrate cytolytic activity associated with C-scorpine, N-scorpine, and scorpine, as well as channel blocking activity associated with the C-scorpine domain.

Scorpions are a group of arthropods that strike fear in many people due to their severe medical symptoms, even death, caused by their venomous stings. Even so, not all scorpion species contain harmful venoms against humans but still have valuable bioactive molecules, which could be used in developing new pharmaceutical leads for treating important diseases. This work conducted a comprehensive analysis of the venom from the scorpion Thorellius intrepidus. The venom of T. intrepidus was separated by size exclusion chromatography, and four main fractions were obtained. Fraction IV (FIV) contained small molecules representing over 90% of the total absorbance at 280 nm. Analysis of fraction FIV by RP-HPLC indicated the presence of three main molecules (FIV.1, FIV.2, and FIV.3) with similar UV absorbance spectra profiles. The molecular masses of FIV.1, FIV.2, and FIV.3 were determined, resulting in 175.99, 190.07, and 218.16 Da, respectively. Further confirmation through 1H-NMR and 13C-NMR analyses revealed that these molecules were serotonin, N-methylserotonin, and bufote-nidine. These intriguing compounds are speculated to play a pivotal role in self-defense and increasing venom toxicity and could also offer promising biotechnological applications as small bioactive molecules.

The methylotrophic yeast Pichia pastoris has been one of the most widely used organisms in recent years as an expression system for a wide variety of recombinant proteins with therapeutic potential. Its popularity as an alternative system to Escherichia coli is mainly due to the easy genetic manipulation and the ability to produce high levels of heterologous proteins, either intracellularly or extracellularly. Being a eukaryotic organism, P. pastoris carries out post-translational modifications that allow it to produce soluble and correctly folded recombinant proteins. This work, evaluated the expression capacity in P. pastoris of two single-chain variable fragments (scFvs) of human origin, 10FG2 and LR. These scFvs were previously obtained by directed evolution against scorpion venom toxins and are able to neutralize different toxins and venoms of Mexican species. The yield obtained in P. pastoris was higher than that obtained in bacterial periplasm (E. coli), and most importantly, biochemical and functional properties were not modified. These results confirm that P. pastoris yeast can be a good expression system for the production of antibody fragments of a new recombinant antivenom.

Previously, it was demonstrated that from the single chain fragment variable (scFv) 3F it is possible to generate variants capable of neutralizing the Cn2 and Css2 toxins, as well as their respective venoms (Centruroides noxius and Centruroides suffusus). Despite this success, it has not been easy to modify the recognition of this family of scFvs toward other dangerous scorpion toxins. The analysis of toxin-scFv interactions and in vitro maturation strategies allowed us to propose a new maturation pathway for scFv 3F to broaden recognition toward other Mexican scorpion toxins. From maturation processes against toxins CeII9 from C. elegans and Ct1a from C. tecomanus, the scFv RAS27 was developed. This scFv showed an increased affinity and cross-reactivity for at least 9 different toxins while maintaining recognition for its original target, the Cn2 toxin. In addition, it was confirmed that it can neutralize at least three different toxins. These results constitute an important advance since it was possible to improve the cross-reactivity and neutralizing capacity of the scFv 3F family of antibodies.

A novel peptide, Cm39, was identified in the venom of the scorpion Centruroides margaritatus. Its primary structure was determined. It consists of 37 amino acid residues with a MW of 3980.2 Da. The full chemical synthesis and proper folding of Cm39 was obtained. Based on amino acid sequence alignment with different K+ channel inhibitor scorpion toxin (KTx) families and phylogenetic analysis, Cm39 belongs to the α-KTx 4 family and was registered with the systematic number of α-KTx 4.8. Synthetic Cm39 inhibits the voltage-gated K+ channel hKV1.2 with high affinity (Kd = 65 nM). The conductance–voltage relationship of KV1.2 was not altered in the presence of Cm39, and the analysis of the toxin binding kinetics was consistent with a bimolecular interaction between the peptide and the channel; therefore, the pore blocking mechanism is proposed for the toxin–channel interaction. Cm39 also inhibits the Ca2+-activated KCa2.2 and KCa3.1 channels, with Kd = 502 nM, and Kd = 58 nM, respectively. However, the peptide does not inhibit hKV1.1, hKV1.3, hKV1.4, hKV1.5, hKV1.6, hKV11.1, mKCa1.1 K+ channels or the hNaV1.5 and hNaV1.4 Na+ channels at 1 μM concentrations. Understanding the unusual selectivity profile of Cm39 motivates further experiments to reveal novel interactions with the vestibule of toxin-sensitive channels.

Centruroides possanii is a recently discovered species of "striped scorpion" found in Mexico. Certain species of Centruroides are known to be toxic to mammals, leading to numerous cases of human intoxications in the country. Venom components are thought to possess therapeutic potential and/or biotechnological applications. Hence, obtaining and analyzing the secretory gland transcriptome and venom proteome of C. possanii is relevant, and that is what is described in this communication. Since this is a newly described species, first, its LD50 to mice was determined and estimated to be 659 ng/g mouse weight. Using RNA extracted from this species and preparing their corresponding cDNA fragments, a transcriptome analysis was obtained on a Genome Analyzer (Illumina) using the 76-base pair-end sequencing protocol. Via high-throughput sequencing, 19,158,736 reads were obtained and ensembled in 835,204 sequences. Of them, 28,399 transcripts were annotated with Pfam. A total of 244 complete transcripts were identified in the transcriptome of C. possanii. Of these, 109 sequences showed identity to toxins that act on ion channels, 47 enzymes, 17 protease inhibitors (PINs), 11 defense peptides (HDPs), and 60 in other components. In addition, a sample of the soluble venom obtained from this scorpion was analyzed using an Orbitrap Velos apparatus, which allowed for identification by liquid chromatography followed by mass spectrometry (LC-MS/MS) of 70 peptides and proteins: 23 toxins, 27 enzymes, 6 PINs, 3 HDPs, and 11 other components. Until now, this work has the highest number of scorpion venom components identified through omics technologies. The main novel findings described here were analyzed in comparison with the known data from the literature, and this process permitted some new insights in this field.

Seven new peptides denominated CboK1 to CboK7 were isolated from the venom of the Mexican scorpion Centruroides bonito and their primary structures were determined. The molecular weights ranged between 3760.4 Da and 4357.9 Da, containing 32 to 39 amino acid residues with three putative disulfide bridges. The comparison of amino acid sequences with known potassium scorpion toxins (KTx) and phylogenetic analysis revealed that CboK1 (α-KTx 10.5) and CboK2 (α-KTx 10.6) belong to the α-KTx 10.x subfamily, whereas CboK3 (α-KTx 2.22), CboK4 (α-KTx 2.23), CboK6 (α-KTx 2.21), and CboK7 (α-KTx 2.24) bear > 95% amino acid similarity with members of the α-KTx 2.x subfamily, and CboK5 is identical to Ce3 toxin (α-KTx 2.10). Electrophysiological assays demonstrated that except CboK1, all six other peptides blocked the Kv1.2 channel with Kd values in the picomolar range (24–763 pM) and inhibited the Kv1.3 channel with comparatively less potency (Kd values between 20–171 nM). CboK3 and CboK4 inhibited less than 10% and CboK7 inhibited about 42% of Kv1.1 currents at 100 nM concentration. Among all, CboK7 showed out-standing affinity for Kv1.2 (Kd = 24 pM), as well as high selectivity over Kv1.3 (850-fold) and Kv1.1 (~6000-fold). These characteristics of CboK7 may provide a framework for developing tools to treat Kv1.2-related channelopathies.

In recent years, morbidity caused by scorpion sting of the species Tityus championi has increased in Panama. Therefore, the LD50 was determined by intravenous injection in 2.9 mg/kg and the venom of T. championi was separated using a HPLC system and their fractions were tested for biological activities in mice to identify the most toxic fractions to mammals. In addition, the venom fractions were also tested against invertebrates to look for insect-specific toxin peptides. The most toxic fractions were analyzed by MS/MS spectrometry. The primary structures of T. championi venom peptides with the most relevant activity were obtained, and the primary structure of one of most neurotoxic peptides was found at least in other four species of Tityus from Panama. This neurotoxin is quite important to be used as a protein target to be neutralized if developing antivenoms against the sting of this Panamanian scorpion or other relevant species of genera Tityus in the country.

The first toxic component identified against mammals in the venom from Centruroides tecomanus scorpion from Colima, Mexico was Ct1a toxin, which was neutralized by human single chain variable fragment (scFv) RAS27. Venom characterization from these scorpions collected on the Pacific coast of Colima, enabled the identification of a second component of medical importance named Ct71 toxin. Amino acid sequence of Ct71 shares a high identity with Chui5 toxin from C. huichol scorpion, which was neutralized by scFv HV. For this reason, the kinetic parameters of interaction between Ct71 toxin and scFv HV were determined by surface plasmon resonance. Results showed a significantly higher affinity for Ct71 as compared to Chui5. As expected, this toxin was neutralized by scFv HV. The injection of a mixture of scFvs HV and RAS27, resulted in the neutralization of C. tecomanus venom, corroborating that human recombinant antibody fragments can efficiently contribute to the neutralization of medically important toxins and their respective venoms from Mexican scorpions.

The Cm28 in the venom of Centruroides margaritatus is a short peptide consisting of 27 amino acid residues with a mol wt of 2,820 D. Cm28 has <40% similarity with other known α-KTx from scorpions and lacks the typical functional dyad (lysine-tyrosine) required to block KV channels. However, its unique sequence contains the three disulfide-bond traits of the α-KTx scorpion toxin family. We propose that Cm28 is the first example of a new subfamily of α-KTxs, registered with the systematic number α-KTx32.1. Cm28 inhibited voltage-gated K+ channels KV1.2 and KV1.3 with Kd values of 0.96 and 1.3 nM, respectively. There was no significant shift in the conductance-voltage (G-V) relationship for any of the channels in the presence of toxin. Toxin binding kinetics showed that the association and dissociation rates are consistent with a bimolecular interaction between the peptide and the channel. Based on these, we conclude that Cm28 is not a gating modifier but rather a pore blocker. In a selectivity assay, Cm28 at 150 nM concentration (>100× Kd value for KV1.3) did not inhibit KV1.5, KV11.1, KCa1.1, and KCa3.1 K+ channels; NaV1.5 and NaV1.4 Na+ channels; or the hHV1 H+ channel but blocked ∼27% of the KV1.1 current. In a biological functional assay, Cm28 strongly inhibited the expression of the activation markers interleukin-2 receptor and CD40 ligand in anti-CD3-activated human CD4+ effector memory T lymphocytes. Cm28, due to its unique structure, may serve as a template for the generation of novel peptides targeting KV1.3 in autoimmune diseases.

The venom of scorpions is a mixture of components that constitute a source of bioactive molecules. The venom of the scorpion Centruroides tecomanus contains peptides toxic to insects, however, to date no toxin responsible for this activity has yet been isolated and fully characterized. This communication describes two new peptides Ct-IT1 and Ct-IT2 purified from this scorpion. Both peptides contain 63 amino acids with molecular weight 6857.85 for Ct-IT1 and 6987.77 Da for Ct-IT2. The soluble venom was separated using chromatographic techniques of molecular size exclusion, cationic exchange, and reverse phase chromatography, allowing the identification of at least 99 components of which in 53 the insecticidal activity was evaluated. The LD50 determined for Ct-IT1 is 3.81 μg/100 mg of cricket weight, but low amounts of peptides (0.8 μg of peptide) already cause paralysis in crickets. The relative abundance of these two peptides in the venom is 2.1% for Ct-IT1 and 1% for Ct-IT2. The molecular masses and N-terminal sequences of both insecticidal toxins were determined by mass spectrometry and Edman degradation. The primary structure of both toxins was compared with other known peptides isolated from other scorpion venoms. The analysis of the sequence alignments revealed the position of a highly conserved amino acid residue, Gly39, exclusively present in anti-insect selective depressant β-toxins (DBTXs), which in Ct-IT1 and Ct-IT2 is at position Gly40. Similarly, a three-dimensional structure of this toxins was obtained by homology modeling and compared to the structure of known insect toxins of scorpions. An important similarity of the cavity formed by the trapping apparatus region of the depressant toxin LqhIT2, isolated from the scorpion Leiurus quinquestriatus hebraeus, was found in the toxins described here. These results indicate that Ct-IT1 and Ct-IT2 toxins have a high potential to be evaluated on pests that affect economically important crops to eventually consider them as a potential biological control method.