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Peptidase inhibitors (PIs) have been broadly studied due to their wide therapeutic potential for human diseases. A potent trypsin inhibitor from Tityus obscurus scorpion venom was characterized and...

Peptides isolated from spider venoms are of pharmacological interest due to their neurotoxic activity, acting on voltage-dependent ion channels present in different types of human body tissues. Three peptide toxins titled as Ap2, Ap3 and Ap5 were purified by RP-HPLC from Acanthoscurria paulensis venom. They were partially sequenced by MALDI In-source Decay method and their sequences were completed and confirmed by transcriptome analysis of the venom gland. The Ap2, Ap3 and Ap5 peptides have, respectively, 42, 41 and 46 amino acid residues, and experimental molecular masses of 4886.3, 4883.7 and 5454.7 Da, with the Ap2 peptide presenting an amidated C-terminus. Amongst the assayed channels – NaV1.1, NaV1.5, NaV1.7, CaV1.2, CaV2.1 and CaV2.2 – Ap2, Ap3 and Ap5 inhibited 20–30 % of CaV2.1 current at 1 μM concentration. Ap3 also inhibited sodium current in NaV1.1, Nav1.5 and Nav1.7 channels by 6.6 ± 1.91 % (p = 0.0276), 4.2 ± 1.09 % (p = 0.0185) and 16.05 ± 2.75 % (p = 0.0282), respectively. Considering that Ap2, Ap3 and Ap5 belong to the 'U'-unknown family of spider toxins, which has few descriptions of biological activity, the present work contributes to the knowledge of these peptides and demonstrates this potential as channel modulators.

Natural inhibitors of proteases have been classified into different families, among them is the Bowman-Birk Inhibitor (BBI) family. Members of BBI have two structurally reactive loops that simultaneously inhibit trypsin and chymotrypsin. Here, we have investigated the binding of bovine trypsin by a cyclic nonapeptide, named PTRY9 (CTKSIPPQC), derived of the black-eyed pea trypsin/chymotrypsin inhibitor (BTCI) from Vigna unguiculata seeds. This peptide was synthetically produced with the disulfide bond restraining its conformation to mimic the reactive loop that inhibits trypsin. PTRY9 complexed to pancreatic bovine trypsin was crystallized in orthorhombic and trigonal space groups, P212121 and P3221, with maximum resolutions of 1.15 and 1.61 A, respectively. The structures presented refinement parameters of Rwork = 14.52 % and Rfree = 15.59 %; Rwork = 15.60 % and Rfree = 18.78 %, and different surface area between the peptide and the enzyme of 1024 A2 and 1070 A2, respectively. The binding site of the PTRY9 is similar to that found for BTCI as shown by a r.m.s.d. of 0.358 A between the superimposed structures and the electrostatic complementary pattern at the enzyme-peptide interface. Additionally, enzyme inhibition assays show that the affinity of trypsin for PTRY9 is smaller than that for BTCI. In vitro assays revealed that, like BTCI, this synthetic peptide is not cytotoxic for normal mammary epithelial MCF-10A cells, but exerts cytotoxic effects on MDA.MB.231 invasive human breast cancer cells.

To1, previously named Tc49b, is a peptide neurotoxin isolated from venom of the scorpion Tityus obscurus that is responsible for lethal human poisoning cases in the Brazilian Amazonian region. Previously, To1 was shown to be lethal to mice and to change Na+ permeation in cerebellum granular neurons from rat brain. In addition, To1 did not affect Shaker B K+ channels. Based on sequence similarities, To1 was described as a β-toxin. In the present work, To1 was purified from T. obscurus venom and submitted to an electrophysiological characterization in human and invertebrate NaV channels. The analysis of the electrophysiological experiments reveal that To1 enhances the open probability at more negative potentials of human NaV 1.3 and 1.6, of the insect channel BgNaV1 and of arachnid VdNaV1 channel. In addition, To1 reduces the peak of Na+ currents in some of the NaVs tested. These results support the classification of the To1 as a β-toxin. A structure and functional comparison to other β-toxins that share sequence similarity to To1 is also presented.

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Programa de Pós-Graduação em Biologia Molecular, 2016. O interesse na caracterização de novos inibidores de peptidases (PIs) tem aumentado nas últimas décadas devido a seu potencial uso na prevenção e tratamento de diversas doenças humanas. Muitos PIs já foram caracterizados de animais venenosos e peçonhentos, como anêmonas, serpentes, anuros e, mais recentemente, escorpiões e aranhas. Nesse estudo apresentamos uma nova classe de inibidor de serinopeptidase isolado da peçonha do escorpião Tityus obscurus (Buthidae), responsável por muitos casos de envenenamento na região Amazônica. Denominado ToPI1, ele foi purificado da peçonha bruta por RP-HPLC, e sua sequência foi determinada por MALDI-ISD e pela análise de seu precursor, obtido da biblioteca de cDNA da glândula de peçonha do T. obscurus. O ToPI1 compartilha menos de 45% de similaridade com outros peptídeos descritos em bancos de dados públicos. Ainda assim, todas eles são bloqueadores ou possíveis bloqueadores de canais de K+, não havendo similaridade com nenhum PI descrito até o momento. O peptídeo sintético oxidado (ToPI1s), obtido usando a estratégia Fmoc/t-butila, foi ativo contra tripsina em ensaios cromogênicos, mostrando-se do tipo tight-binding, e também por Ressonância Plasmônica de Superfície (SPR), em um biossensor Biacore 3000, onde apresentou afinidade picomolar por tripsina imobilizada. Os resultados indicaram que a formação do complexo ToPI1:tripsina é espontânea, endotérmica e favorecida por um aumento de entropia. Experimentos em SPR indicaram que o ToPI1s se liga ao sítio ativo da tripsina, semelhante aos inibidores canônicos. O ToPI1s não inibiu quimotripsina mesmo em concentrações elevadas. Também não reduziu a viabilidade de células tumorais HeLa (de câncer cervical humano) e B16F10 (de melanoma murino) nem de fibroblastos murinos não tumorais (NIH-3T3). Seu efeito foi mínimo ou nulo nas correntes de diferentes subtipos de canais de K+. O ToPI1s não causou alterações comportamentais e/ou fisiológicas visíveis em camundongos. A potente atividade inibidora de tripsina do ToPI1s em conjunto com esses resultados negativos o tornam um candidato atrativo para futuras aplicações terapêuticas. Análises por dicroísmo circular (190-260 nm) sugerem ausência de diferenças de estrutura secundária entre o ToPI1 nativo e o ToPI1s, com predominância de estruturas do tipo β (folhas-β e voltas-β) e desordenadas, e menor quantidade de α-hélice. O ToPI1s mostrou-se bastante estável, não desnaturando na faixa de pH 3,0-9,0 mesmo a 95°C. Dois análogos do ToPI1 foram sintetizados, os quais estão sob processo de proteção à propriedade intelectual. The interest in the characterization of new peptidase inhibitors (PIs) has increased in recent decades due to their potential use in the prevention and treatment of several human diseases. Many PIs from poisonous and venomous animals such as sea anemones, snakes, anurans and, more recently, scorpions and spiders have been characterized. In the present study we present a new class of serinopeptidase inhibitor isolated from Tityus obscurus scorpion venom. This species (Buthidae) is responsible for many cases of envenomation in the Amazon region. Named ToPI1, it was purified from the crude venom by RP-HPLC and its sequence was determined by MALDI-ISD and by the analysis of its precursor, obtained from the cDNA library of T. obscurus venom gland. ToPI1 shares less than 45% sequence similarity with other peptides described in public databases. Still, they are all blockers or potential blockers of K+ channels, with no similarity to any PI described so far. The oxidized synthetic peptide (ToPI1s), obtained by using the Fmoc/t-butyl strategy, was active against trypsin in chromogenic assays, displaying tight-binding inhibition, and also by Surface Plasmon Resonance (SPR) in a Biacore 3000 biosensor, where it showed picomolar affinity to immobilized trypsin. The results indicated that the formation of the bimolecular complex ToPI1:trypsin is spontaneous, endothermic and favored by an entropy increase. SPR experiments indicated that ToPI1s binds to the active site of trypsin, similar to the canonical inhibitors. ToPI1s did not inhibit chymotrypsin even at high doses. In addition, it reduced neither the viability of the tumor cells HeLa (from human cervical cancer) and B16F10 (from murine melanoma) nor of non-tumor murine fibroblasts (NIH-3T3). It showed minimal or null effect on the currents of different subtypes of K+ channels. The ToPI1s did not cause any visible behavioral and/or physiological changes in mice. The potent trypsin inhibitory activity of ToPI1s together with these negative results make it an attractive candidate for future therapeutic purposes. Analysis by circular dichroism (190-260 nm) suggests the absence of differences in secondary structures between ToPI1s and the native ToPI1, with predominance of β-type structures (β-sheets and β-turns) and randon coil, and a smaller amount of α-helix. The ToPI1s proved to be very stable, non-denaturing at the range of pH 3.0 to 9.0 even at 95 °C. Two ToPI1 analogues were synthesized, which are under process of intellectual property protection.

To1, previously named Tc49b, is a peptide neurotoxin isolated from venom of the scorpion Tityus obscurus that is responsible for lethal human poisoning cases in the Brazilian Amazonian region. Previously, To1 was shown to be lethal to mice and to change Na

Many scorpion toxins that act on sodium channels (NaScTxs) have been characterized till date. These toxins may act modulating the inactivation or the activation of sodium channels and are named α- or β-types, respectively. Some venom toxins from Tityus obscurus (Buthidae), a scorpion widely distributed in the Brazilian Amazon, have been partially characterized in previous studies; however, little information about their electrophysiological role on sodium ion channels has been published. In the present study, we describe the purification, identification and electrophysiological characterization of a NaScTx, which was first described as Tc54 and further fully sequenced and renamed To4. This toxin shows a marked β-type effect on different sodium channel subtypes (hNa v 1.1–hNa v 1.7) at low concentrations, and has more pronounced activity on hNa v 1.1, hNa v 1.2 and hNa v 1.4. By comparing To4 primary structure with other Tityus β-toxins which have already been electrophysiologically tested, it is possible to establish some key amino acid residues for the sodium channel activity. Thus, To4 is the first toxin from T. obscurus fully electrophysiologically characterized on different human sodium channel isoforms.

The Kunitz-type protease inhibitors are the best-characterized family of serine protease inhibitors, probably due to their abundance in several organisms. These inhibitors consist of a chain of ~60 amino acid residues stabilized by three disulfide bridges, and was first observed in the bovine pancreatic trypsin inhibitor (BPTI)-like protease inhibitors, which strongly inhibit trypsin and chymotrypsin. In this review we present the protease inhibitors (PIs) described to date from marine venomous animals, such as from sea anemone extracts and Conus venom, as well as their counterparts in terrestrial venomous animals, such as snakes, scorpions, spiders, Anurans, and Hymenopterans. More emphasis was given to the Kunitz-type inhibitors, once they are found in all these organisms. Their biological sources, specificity against different proteases, and other molecular blanks (being also K+ channel blockers) are presented, followed by their molecular diversity. Whereas sea anemone, snakes and other venomous animals present mainly Kunitz-type inhibitors, PIs from Anurans present the major variety in structure length and number of Cys residues, with at least six distinguishable classes. A representative alignment of PIs from these venomous animals shows that, despite eventual differences in Cys assignment, the key-residues for the protease inhibitory activity in all of them occupy similar positions in primary sequence. The key-residues for the K+ channel blocking activity was also compared.

In the present study we conducted proteomic and pharmacological characterizations of the venom extracted from the Brazilian tarantula Acanthoscurria paulensis, and evaluated the cardiotoxicity of its two main fractions. The molecular masses of the venom components were identified by mass spectrometry (MALDI-TOF-MS) after chromatographic separation (HPLC). The lethal dose (LD50) was determined in mice. Nociceptive behavior was evaluated by intradermal injection in mice and the edematogenic activity by the rat hind-paw assay. Cardiotoxic activity was evaluated on in situ frog heart and on isolated frog ventricle strip. From 60 chromatographic fractions, 97 distinct components were identified, with molecular masses between 601.4 and 21,932.3 Da. A trimodal molecular mass distribution was observed: 30% of the components within 500-1999 Da, 38% within 3500–5999 Da and 21% within 6500–7999 Da. The LD50 in mice was 25.4 ± 2.4 μg/g and the effects observed were hypoactivity, anuria, constipation, dyspnea and prostration until death, which occurred at higher doses. Despite presenting a dose-dependent edematogenic activity in the rat hind-paw assay, the venom had no nociceptive activity in mice. Additionally, the venom induced a rapid blockage of electrical activity and subsequent diastolic arrest on in situ frog heart preparation, which was inhibited by pretreatment with atropine. In the electrically driven frog ventricle strip, the whole venom and its low molecular mass fraction, but not the proteic one, induced a negative inotropic effect that was also inhibited by atropine. These results suggest that despite low toxicity, A. paulensis venom can induce severe physiological disturbances in mice.

Arthropods are the most diverse animal group on the planet, and occupy almost all ecological niches. Venomous arthropods are a rich source of bioactive compounds evolved for prey capture and defense against predators and/or microorganisms. These highly potent chemical arsenals represent an available source for new insecticidal compounds as they act selectively on their molecular targets. These toxins affect the invertebrate nervous system and, until the moment, several insecticidal compounds belonging to the class of peptides or polyamine-like compounds have been purified and characterized from the venom of arachnids and hymenopterans. This review focuses on invertebrate-specific peptide neurotoxins that have been isolated from the venom ofspiders, scorpions, centipedes, ants, and wasps, discussing their potential in pest control and as invaluable tools in neuropharmacology.

Background: Colombia and Brazil are affected by severe cases of scorpionism. In Colombia the most dangerous accidents are caused by Tityus pachyurus that is widely distributed around this country. In the Brazilian Amazonian region scorpion stings are a common event caused by Tityus obscurus. The main objective of this work was to perform the molecular cloning of the putative Na+-channel scorpion toxins (NaScTxs) from T. pachyurus and T. obscurus venom glands and to analyze their phylogenetic relationship with other known NaScTxs from Tityus species. Methodology/Principal Findings: cDNA libraries from venom glands of these two species were constructed and five nucleotide sequences from T. pachyurus were identified as putative modulators of Na+-channels, and were named Tpa4, Tpa5, Tpa6, Tpa7 and Tpa8; the latter being the first anti-insect excitatory b-class NaScTx in Tityus scorpion venom to be described. Fifteen sequences from T. obscurus were identified as putative NaScTxs, among which three had been previously described, and the others were named To4 to To15. The peptides Tpa4, Tpa5, Tpa6, To6, To7, To9, To10 and To14 are closely related to the a-class NaScTxs, whereas Tpa7, Tpa8, To4, To8, To12 and To15 sequences are more related to the b-class NaScTxs. To5 is possibly an arthropod specific toxin. To11 and To13 share sequence similarities with both a and b NaScTxs. By means of phylogenetic analysis using the Maximum Parsimony method and the known NaScTxs from Tityus species, these toxins were clustered into 14 distinct groups. Conclusions/Significance: This communication describes new putative NaScTxs from T. pachyurus and T. obscurus and their phylogenetic analysis. The results indicate clear geographic separation between scorpions of Tityus genus inhabiting the Amazonian and Mountain Andes regions and those distributed over the Southern of the Amazonian rainforest. Based on the consensus sequences for the different clusters, a new nomenclature for the NaScTxs is proposed.

The venom of social wasps has been poorly studied so far, despite the high number of accidents in humans and assessment of the use of these wasps as a biological control of pests. The study of the pharmacological effects of the venom is of great importance since the poisoning is dangerous causing serious systemic effects, including death in the case of multiple attacks. In this study, the pharmacological activities of venom from the social wasp Synoeca cyanea were evaluated by the following assays: LD50 in mice, the behavioural effects and the hemorrhagic activity induced by the venom in mice, the oedematogenic activity in rat, the haemolysis in human blood, the stimulating effect on guinea-pig smooth muscle, and the antimicrobial activity. The aim was to determine the toxic effects of venom and to perform a comparative study with earlier work conducted with venom from other wasp species. Results showed that S. cyanea venom produced a potent dose-dependent oedema, as well as antibacterial and haemolytic activities, suggesting the presence of histamine, serotonin, kinins and other molecules related to increased vascular permeability and cytolytic activity in this venom. Despite previous studies with wasp venoms, S. cyanea venom presented a slight hemorrhagic effect. Data obtained in the smooth muscle assay also suggest the presence of BK or analogues in S. cyanea whole venom. The knowledge of symptoms and effects produced by S. cyanea venom is critical for health organizations, in order to improve clinical treatment in accidents caused by wasp stings.