LyeTxI-b, a Synthetic Peptide Derived From a Spider Venom, Is Highly Active in Triple-Negative Breast Cancer Cells and Acts Synergistically With Cisplatin.

Breast cancer is the most common cancer that affects women globally and is among the leading cause of women's death. Triple-negative breast cancer is more difficult to treat because hormone therapy is not available for this subset of cancer. The well-established therapy against triple-negative breast cancer is mainly based on surgery, chemotherapy, and immunotherapy. Among the drugs used in the therapy are cisplatin and carboplatin. However, they cause severe toxicity to the kidneys and brain and cause nausea. Therefore, it is urgent to propose new chemotherapy techniques that provide new treatment options to patients affected by this disease. Nowadays, peptide drugs are emerging as a class of promising new anticancer agents due to their lytic nature and, apparently, a minor drug resistance compared to other conventional drugs (reviewed in Jafari et al., 2022). We have recently reported the cytotoxic effect of the antimicrobial peptide LyeTx I-b against glioblastoma cells (Abdel-Salam et al., 2019). In this research, we demonstrated the cytotoxic effect of the peptide LyeTx I-b, alone and combined with cisplatin, against triple-negative cell lines (MDA-MD-231). LyeTx-I-b showed a selectivity index 70-fold higher than cisplatin. The peptide:cisplatin combination (P:C) 1:1 presented a synergistic effect on the cell death and a selective index value 16 times greater than the cisplatin alone treatment. Therefore, an equi-effective reduction of cisplatin can be reached in the presence of LyeTx I-b. Cells treated with P:C combinations were arrested in the G2/M cell cycle phase and showed positive staining for acridine orange, which was inhibited by bafilomycin A1, indicating autophagic cell death (ACD) as a probable cell death mechanism. Furthermore, Western blot experiments indicated a decrease in P21 expression and AKT phosphorylation. The decrease in AKT phosphorylation is indicative of ACD. However, other studies are still necessary to better elucidate the pathways involved in the cell death mechanism induced by the peptide and the drug combinations. These findings confirmed that the peptide LyeTx I-b seems to be a good candidate for combined chemotherapy to treat breast cancer. In addition, in vivo studies are essential to validate the use of LyeTx I-b as a therapeutic drug candidate, alone and/or combined with cisplatin.

Tityus serrulatus (Scorpion): From the Crude Venom to the Construction of Synthetic Peptides and Their Possible Therapeutic Application Against Toxoplasma gondii Infection.

Toxoplasmosis, caused by Toxoplasma gondii, is a major public concern owing to its neurotropic nature and high morbidity and mortality rates in immunocompromised patients and newborns. Current treatment for this disease is inefficient and produces side effects. Inflammatory mediators produced during T. gondii infection (e.g., cytokines and nitric oxide) are crucial in controlling parasite replication. In this context, Tityus serrulatus venom (TsV) induces the production of inflammatory mediators by immune cells. Thus, this study aimed to isolate and identify the components of TsV with potential anti-T. gondii activity. TsV was extracted from scorpions and lyophilized or loaded onto a column to obtain its fractions. TsV subfractions were obtained using chromatography, and its amino acid sequence was identified and applied to peptide design using bioinformatics tools. The C57BL/6 mice and their harvested macrophages were used to test the anti-Toxoplasma activity of TsV components and peptides. TsV and its fraction F6 attenuated the replication of tachyzoites in macrophages and induced nitric oxide and cytokine (IL-12, TNF, and IL-6) production by infected cells, without host cell toxicity. Moreover, Su6-B toxin, a subfraction of F6, demonstrated anti-T. gondii activity. The partially elucidated and characterized amino acid sequence of Sub6-B demonstrated 93% similarity with T. serrulatus 2 toxin (Ts2). Ts2 mimetic peptides ("Pep1," "Pep2a," and "Pep2b") were designed and synthesized. Pep1 and Pep2a, but not Pep2b, reduced the replication of tachyzoites in macrophages. In vivo, treatment of T. gondii-infected mice with Pep1, Pep2a, or Pep2b decreased the number of cerebral cysts and did not induce hepatotoxicity in the animals. Taken together, our data show promising immunomodulatory and antiparasitic activity of TsV that could be explored and applied in future therapies for treating infectious parasitic diseases such as toxoplasmosis.

Shortened derivatives from native antimicrobial peptide LyeTx I: In vitro and in vivo biological activity assessment.

In the continuing search for novel antibiotics, antimicrobial peptides are promising molecules, due to different mechanisms of action compared to classic antibiotics and to their selectivity for interaction with microorganism cells rather than with mammalian cells. Previously, our research group has isolated the antimicrobial peptide LyeTx I from the venom of the spider Lycosa erythrognatha. Here, we proposed to synthesize three novel shortened derivatives from LyeTx I (LyeTx I mn; LyeTx I mnΔK; LyeTx I mnΔKAc) and to evaluate their toxicity and biological activity as potential antimicrobial agents. Peptides were synthetized by Fmoc strategy and circular dichroism analysis was performed, showing that the three novel shortened derivatives may present membranolytic activity, like the original LyeTx I, once they folded as an alpha helix in 2.2.2-trifluorethanol and sodium dodecyl sulfate. In vitro assays revealed that the shortened derivative LyeTx I mnΔK presents the best score between antimicrobial (↓ MIC) and hemolytic (↑ EC50) activities among the synthetized shortened derivatives, and LUHMES cell-based NeuriTox test showed that it is less neurotoxic than the original LyeTx I (EC50 [LyeTx I mnΔK] ⋙ EC50 [LyeTx I]). In vivo data, obtained in a mouse model of septic arthritis induced by Staphylococcus aureus, showed that LyeTx I mnΔK is able to reduce infection, as demonstrated by bacterial recovery assay (∼10-fold reduction) and scintigraphic imaging (less technetium-99m labeled-Ceftizoxime uptake by infectious site). Infection reduction led to inflammatory process and pain decreases, as shown by immune cells recruitment reduction and threshold nociception increment, when compared to positive control group. Therefore, among the three shortened peptide derivatives, LyeTx I mnΔK is the best candidate as antimicrobial agent, due to its smaller amino acid sequence and toxicity, and its greater biological activity.

GiTx1(ß/κ-theraphotoxin-Gi1a), a novel toxin from the venom of Brazilian tarantula Grammostola iheringi (Mygalomorphae, Theraphosidae): Isolation, structural assessments and activity on voltage-gated ion channels.

Spider venoms, despite their toxicity, represent rich sources of pharmacologically active compounds with biotechnological potential. However, in view of the large diversity of the spider species, the full potential of their venom molecules is still far from being known. In this work, we report the purification and structural and functional characterization of GiTx1 (ß/κ-TRTX-Gi1a), the first toxin purified from the venom of the Brazilian tarantula spider Grammostola iheringi. GiTx1 was purified by chromatography, completely sequenced through automated Edman degradation and tandem mass spectrometry and its structure was predicted by molecular modeling. GiTx1 has a MW of 3.585 Da, with the following amino acid sequence: SCQKWMWTCDQKRPCCEDMVCKLWCKIIK. Pharmacological activity of GiTx1 was characterized by electrophysiology using whole-cell patch clamp on dorsal root ganglia neurons (DRG) and two-electrode voltage-clamp on voltage-gated sodium and potassium channels subtypes expressed in Xenopus laevis oocytes. GiTx1, at 2 µM, caused a partial block of inward (∼40%) and outward (∼20%) currents in DRG cells, blocked rNav1.2, rNav1.4 and mNav1.6 and had a significant effect on VdNav, an arachnid sodium channel isoform. IC50 values of 156.39 ± 14.90 nM for Nav1.6 and 124.05 ± 12.99 nM for VdNav, were obtained. In addition, this toxin was active on rKv4.3 and hERG potassium channels, but not Shaker IR or rKv2.1 potassium channels. In summary, GiTx1 is a promiscuous toxin with multiple effects on different types of ion channels.

Antinociceptive effect of PnTx4(5-5), a peptide from Phoneutria nigriventer spider venom, in rat models and the involvement of glutamatergic system.

BACKGROUND: The venom of Phoneutria nigriventer spider is a source of numerous bioactive substances, including some toxins active in insects. An example is PnTx4(5-5) that shows a high insecticidal activity and no apparent toxicity to mice, although it inhibited NMDA-evoked currents in rat hippocampal neurons. In this work the analgesic activity of PnTx4(5-5) (renamed Γ-ctenitoxin-Pn1a) was investigated. METHODS: The antinociceptive activity was evaluated using the paw pressure test in rats, after hyperalgesia induction with intraplantar injection of carrageenan or prostaglandin E2 (PGE2). RESULTS: PnTx4(5-5), subcutaneously injected, was able to reduce the hyperalgesia induced by PGE2 in rat paw, demonstrating a systemic effect. PnTx4(5-5) administered in the plantar surface of the paw caused a peripheral and dose-dependent antinociceptive effect on hyperalgesia induced by carrageenan or PGE2. The hyperalgesic effect observed in these two pain models was completely reversed with 5 µg of PnTx4(5-5). Intraplantar administration of L-glutamate induced hyperalgesic effect that was significantly reverted by 5 µg of PnTx4(5-5) injection in rat paw. CONCLUSION: The antinociceptive effect for PnTx4(5-5) was demonstrated against different rat pain models, i.e. induced by PGE2, carrageenan or glutamate. We suggest that the antinociceptive effect of PnTx4(5-5) may be related to an inhibitory activity on the glutamatergic system.

Antinociceptive effect of PnTx4(5-5), a peptide from Phoneutria nigriventer spider venom, in rat models and the involvement of glutamatergic system

The venom of Phoneutria nigriventer spider is a source of numerous bioactive substances, including some toxins active in insects. An example is PnTx4(5-5) that shows a high insecticidal activity and no apparent toxicity to mice, although it inhibited NMDA-evoked currents in rat hippocampal neurons. In this work the analgesic activity of PnTx4(5-5) (renamed Γ-ctenitoxin-Pn1a) was investigated. Methods: The antinociceptive activity was evaluated using the paw pressure test in rats, after hyperalgesia induction with intraplantar injection of carrageenan or prostaglandin E2 (PGE2). Results: PnTx4(5-5), subcutaneously injected, was able to reduce the hyperalgesia induced by PGE2 in rat paw, demonstrating a systemic effect. PnTx4(5-5) administered in the plantar surface of the paw caused a peripheral and dose-dependent antinociceptive effect on hyperalgesia induced by carrageenan or PGE2. The hyperalgesic effect observed in these two pain models was completely reversed with 5 µg of PnTx4(5-5). Intraplantar administration of L-glutamate induced hyperalgesic effect that was significantly reverted by 5 μg of PnTx4(5-5) injection in rat paw. Conclusion: The antinociceptive effect for PnTx4(5-5) was demonstrated against different rat pain models, i.e. induced by PGE2, carrageenan or glutamate. We suggest that the antinociceptive effect of PnTx4(5-5) may be related to an inhibitory activity on the glutamatergic system.(AU)

PnTx2-6 (or δ-CNTX-Pn2a), a toxin from Phoneutria nigriventer spider venom, releases l-glutamate from rat brain synaptosomes involving Na+ and Ca2+ channels and changes protein expression at the blood-brain barrier.

PhTx2 is the most toxic fraction from the venom of the spider Phoneutria nigriventer, being responsible to sodium entry into cortical synaptosomes, increasing the release of neurotransmitters, such as l-glutamate (L-Glu) and; acetylcholine. In this study, we investigated the action of a toxin purified from; PhTx2 fraction, called PnTx2-6 or δ-CNTX-Pn2a, on L-Glu release from rat; brain cortex synaptosomes, as well as its ability to induce blood-brain barrier permeability. PnTx2-6 increased L-Glu release from rat cortical brain synaptosomes in a time- and dose-dependent manner (EC50 = ∼20 nM; Tm = 16min), as measured by a fluorimetric method. The increase of L-Glu by PnTx2-6 was inhibited by tetrodotoxin. And partially inhibited by EGTA. Calcium channel blockers ω-conotoxin MVIIC (P/Q-types) and ω-conotoxin GVIA (N-type), were able to reduce the PnTx2-6-induced release of L-Glu, while nifedipine (L-type) did not show any inhibition. These findings suggest that thew release of L-Glu by PnTx2-6 is due its primary action on sodium channels, well-known to be target of this toxin. PnTx2-6 is able to potentiate penile erection and this effect may be related with the release of l-glutamate from the CNS, besides a local effect on corpus carvenosum, as previously shown by our group. If L-Glu release and penile erection potentiation are indeed correlated, then this toxin should be able to cross the blood brain barrier (BBB). Results by immunoblotting assays indicated a change in the expression of proteins associated with the paracellular and transcellular transport at the blood-brain barrier, suggesting a BBB dysfunction mediated by PnTx2-6. Therefore, PnTx2-6 may induce the release l-glutamate in the central nervous system, when injected peripherally.

Moving pieces in a cryptomic puzzle: Cryptide from Tityus serrulatus Ts3 Nav toxin as potential agonist of muscarinic receptors.

Cryptome is as a subset of a given proteome containing bioactive cryptides embedded in larger peptides or proteins. We pinpointed a striking sequence similarity between two peptides from the Tityus serrulatus venom: Ts10 (KKDGYPVEYDRAY) and the N-terminal of Ts3 (KKDGYPVEYDNCAY). Ts3 (former Tityustoxin or TsIV) is an α-neurotoxin acting on voltage-gated sodium channels while Ts10 (former Peptide T) is a bradykinin-potentiating peptide and was originally reported as inhibitor of the angiotensin-converting enzyme (ACEi). Thus, the goal of this study was to evaluate whether such peptide hidden in the N-terminal of Ts3 (Ts31-14[C12S]) was able to mimic known effects of Ts10 as well as to expand the current knowledge of the vascular effects and molecular targets of these peptides. Similar to Ts10, Ts31-14[C12S] was able to potentiate the hypotensive effect of bradykinin (BK). However, none of these peptides was able to induce a long-lasting BK-potentiating effect, suggesting that this effect may not be their main biological outcome. On the other hand, we report that Ts10 and mainly Ts31-14[C12S] induced a strong vasodilation effect depending on the presence of functional endothelium and nitric oxide (NO) production. Unlike previously reported, Ts10 was not able to inhibit ACE activity (similar result was observed for Ts31-14[C12S]). On the other hand, we report that Ts31-14[C12S] induces vasodilation via the activation of muscarinic acetylcholine receptors (mAChRs) M2 and M3 while only the activation of mAChR M2 seems to be required for Ts10-induced vasodilation.

The proteomic profile of Stichodactyla duerdeni secretion reveals the presence of a novel O-linked glycopeptide.

Sea anemones represent one of the emerging groups of interest concerning venomous animals in toxinology and the goal of the present work was the prospection, and the structural and functional characterization of the compounds present in the secretion of the sea anemone Stichodactyla duerdeni from Brazilian coast. We used a combination of offline RPC-MALDI-TOF and online nano-RPC-ESI-LTQ-Orbitrap proteomic techniques as well as functional bioassays. The mucus was milked by electric stimulation and fractionated by gel filtration on Sephadex G-50 yielding 5 main fractions. The low molecular weight fractions were further submitted to RP-HPLC resulting in 35 new subfractions that were subsequently analyzed by offline MALDI-TOF mass spectrometry. MALDI peptide mass fingerprinting yielded up to 134 different molecular masses, ranging from m/z 901 to 10,833. Among these subfractions, a new peptide of 3431Da, named U-SHTX-Sdd1, was purified and completely sequenced by automated Edman's degradation and tandem mass spectrometry. An analysis of U-SHTX-Sdd1 revealed a modified O-HexNAc-Threonine at position 1, which, at the best of our knowledge, constitutes the first sea anemone toxin reported with such post-translational modification. Because of its sequence similarity with other sea anemone toxins, the pharmacological activity of U-SHTX-Sdd1 was assessed by electrophysiological measurements using the two electrode voltage-clamp technique on cloned voltage-gated potassium channel subtypes, expressed in Xenopus laevis oocytes. However, U-SHTX-Sdd1 did not show activity on these channels. A large-scale proteomic approach was also employed to shed lights on the sea anemone compounds, and a total 67 proteins and peptides were identified. BIOLOGICAL SIGNIFICANCE: In this manuscript, we report an extensive characterization of S. duerdeni secretion by means of peptide mass fingerprinting and high-throughput proteome analyses. Also, we report the structure of a new glycopeptide by a combination of biochemical techniques. Despite the previous studies that described proteinaceous compounds present in sea anemone secretions, the number of reported primary sequences is still low. Thus, to access the scenery of protein components from S. duerdeni mucus, including their biological functions, a robust proteomic approach was used together with bioinformatic tools. The demonstrated strategy of analysis is perfectly suitable to other sea anemone secretions and animal venoms. Moreover, new peptide structures can arise contributing to the knowledge of the diversity of these animal peptides.

Greater binding affinity of trivalent antimony to a CCCH zinc finger domain compared to a CCHC domain of kinetoplastid proteins.

It has been reported recently that Sb(III) competes with Zn(II) for its binding to the CCHC zinc finger domain of the HIV-1 NCp7 protein, suggesting that zinc finger proteins may be molecular targets for antimony-based drugs. Here, the interaction of Sb(III) with a CCCH zinc finger domain, which is considered to play a crucial role in the biology of kinetoplastid protozoa, has been characterized for the first time. The binding characteristics of Sb(III) were compared between a CCCH-type peptide derived from a kinetoplastid protein and two different CCHC-type zinc finger peptides. The formation of 1 : 1 Zn-peptide and Sb-peptide complexes from the different peptides was demonstrated using circular dichroism, UV absorption, fluorescence spectroscopies and ESI-MS. Titration of the Zn-peptide complexes with SbCl(3) was performed at pH 6 and 7, exploiting the intrinsic fluorescence of the peptides. The differential spectral characteristics of the peptides allowed for competition experiments between the different peptides for binding of Zn(II). The present study establishes that Sb(III) more effectively displaces Zn(II) from the CCCH peptide than CCHC ones, as a result of both the greater stability of the Sb-CCCH complex (compared to Sb-CCHC complexes) and the lower stability of the Zn-CCCH complex (compared to Zn-CCHC complexes). Comparison of the binding characteristics of Sb(III) or Zn(II) between the CCHC-type peptides with different amino acid sequences supports the model that not only the conserved zinc finger motif, but also the sequence of non-conserved amino acids determines the binding affinity of Sb(III) and Zn(II). These data suggest that the interaction of Sb(III) with CCCH-type zinc finger proteins may modulate, or even mediate, the pharmacological action of antimonial drugs.

Complete amino-acid sequence, crystallization and preliminary X-ray diffraction studies of leucurolysin-a, a nonhaemorrhagic metalloproteinase from Bothrops leucurus snake venom.

Leucurolysin-a (leuc-a) is a class P-I snake-venom metalloproteinase isolated from the venom of the South American snake Bothrops leucurus (white-tailed jararaca). The mature protein is composed of 202 amino-acid residues in a single polypeptide chain. It contains a blocked N-terminus and is not glycosylated. In vitro studies revealed that leuc-a dissolves clots made either from purified fibrinogen or from whole blood. Unlike some other venom fibrinolytic metalloproteinases, leuc-a has no haemorrhagic activity. Leuc-a was sequenced and was crystallized using the hanging-drop vapour-diffusion technique. Crystals were obtained using PEG 6000 or PEG 1500. Diffraction data to 1.80 and 1.60 A resolution were collected from two crystals (free enzyme and the endogenous ligand-protein complex, respectively). They both belonged to space group P2(1)2(1)2(1), with very similar unit-cell parameters (a = 44.0, b = 56.2, c = 76.3 A for the free-enzyme crystal).

Animal Toxins: state of the art: perspectives in health and biotecnology

Apresenta, pela primeira vez em um único volume, o resultado de diversas pesquisas com diferentes perspectivas sobre as reais possibilidades do uso de venenos e de toxinas de origem animal na indústria biotecnológica. Animal Toxins é composto por 39 artigos, em inglês, assinados por especialistas renomados de várias nacionalidades. O estado da arte em compostos obtidos a partir de venenos de animais marinhos, aranhas e escorpiões, lagartas, serpentes, entre outros, são o foco desta publicação que visa a atender cientistas, estudantes e pesquisadores da universidade, da indústria farmacêutica e de biotecnologia interessados em toxicologia. Os venenos e toxinas de origem animal foram selecionados ao longo de milhões de anos de evolução para atuarem de forma rápida e eficaz no organismo da vítima, o que resultou em um repertorio maciço de moléculas capazes de se ligarem a alvos específicos. A possibilidade de utilização dessas toxinas em processos biotecnológicos fez com que esses venenos e toxinas sejam considerados como uma das mais promissoras fontes de compostos bioativos naturais.