BotCl, the First Chlorotoxin-like Peptide Inhibiting Newcastle Disease Virus: The Emergence of a New Scorpion Venom AMPs Family.

Newcastle disease virus (NDV) is one of the most serious contagions affecting domestic poultry and other avian species. It causes high morbidity and mortality, resulting in huge economic losses to the poultry industry worldwide. Despite vaccination, NDV outbreaks increase the need for alternative prevention and control means. In this study, we have screened fractions of Buthus occitanus tunetanus (Bot) scorpion venom and isolated the first scorpion peptide inhibiting the NDV multiplication. It showed a dose dependent effect on NDV growth in vitro, with an IC50 of 0.69 µM, and a low cytotoxicity on cultured Vero cells (CC50 > 55 µM). Furthermore, tests carried out in specific pathogen-free embryonated chicken eggs demonstrated that the isolated peptide has a protective effect on chicken embryos against NDV, and reduced by 73% the virus titer in allantoic fluid. The N-terminal sequence, as well as the number of cysteine residues of the isolated peptide, showed that it belongs to the scorpion venom Chlorotoxin-like peptides family, which led us to designate it "BotCl". Interestingly, at 10 µg/mL, BotCl showed an inhibiting effect three times higher than its analogue AaCtx, from Androctonus australis (Aa) scorpion venom, on NDV development. Altogether, our results highlight the chlorotoxin-like peptides as a new scorpion venom AMPs family.

Polylactide Nanoparticles as a Biodegradable Vaccine Adjuvant: A Study on Safety, Protective Immunity and Efficacy against Human Leishmaniasis Caused by Leishmania Major.

Leishmaniasis is the 3rd most challenging vector-borne disease after malaria and lymphatic filariasis. Currently, no vaccine candidate is approved or marketed against leishmaniasis due to difficulties in eliciting broad immune responses when using sub-unit vaccines. The aim of this work was the design of a particulate sub-unit vaccine for vaccination against leishmaniasis. The poly (D,L-lactide) nanoparticles (PLA-NPs) were developed in order to efficiently adsorb a recombinant L. major histone H2B (L. major H2B) and to boost its immunogenicity. Firstly, a study was focused on the production of well-formed nanoparticles by the nanoprecipitation method without using a surfactant and on the antigen adsorption process under mild conditions. The set-up preparation method permitted to obtain H2B-adsorbed nanoparticles H2B/PLA (adsorption capacity of about 2.8% (w/w)) with a narrow size distribution (287 nm) and a positive zeta potential (30.9 mV). Secondly, an in vitro release assay performed at 37 °C, pH 7.4, showed a continuous release of the adsorbed H2B for almost 21 days (30%) from day 7. The immune response of H2B/PLA was investigated and compared to H2B + CpG7909 as a standard adjuvant. The humoral response intensity (IgG) was substantially similar between both formulations. Interestingly, when challenged with the standard parasite strain (GLC94) isolated from a human lesion of cutaneous leishmaniasis, mice showed a significant reduction in footpad swelling compared to unvaccinated ones, and no deaths occurred until week 17th. Taken together, these results demonstrate that PLA-NPs represent a stable, cost-effective delivery system adjuvant for use in vaccination against leishmaniasis.

Functional role of Kv1.1 and Kv1.3 channels in the neoplastic progression steps of three cancer cell lines, elucidated by scorpion peptides.

Voltage-gated potassium (Kv) channels are known to play a pivotal role in the progression of various cancer types and considered as new targets for designing anti-cancer therapy. However, the fact that many Kv channels are expressed in different cell lines makes it difficult to ascribe a functional role for a given Kv channel on a specific aspect of the tumorogenesis. In this work, we showed that although both Kv1.1 and Kv1.3 channels are expressed in U87 (glioblastoma), MDA-MB-231 (breast cancer) and LS174 (colon adenocarcinoma) cells, these respond differently to KAaH1 or KAaH2, two homologous Kv1 blockers from scorpion venom. KAaH1 is active on Kv1.1 and Kv1.3 and was found to inhibit migration and adhesion of U87 cells whereas KAaH2 which is slightly active only on Kv1.1 channel, inhibits their proliferation via the EGFR signaling pathway. The correlation between the electro-physiological activity of the scorpion peptides and their anti-migratory effects suggests the involvement of the Kv1.1 and Kv1.3 channels in the mobility of the three cancer cell lines. Our results showed that besides they can elucidate the implication of Kv1.1 and Kv1.3 channels in molecular mechanisms of neoplastic progression, KAaH1 and KAaH2 may be used as therapeutic tools against glioblastoma.

Lebecetin, a C-type lectin, inhibits choroidal and retinal neovascularization.

Angiogenesis is a cause of visual impairment and blindness in the wet form of age-related macular degeneration and in ischemic retinopathies. Current therapies include use of anti-VEGF agents to reduce choroidal neovascularization (CNV) and edema. These treatments are effective in most cases, but spontaneous or acquired resistance to anti-VEGF and possible adverse effects of long-term VEGF inhibition in the retina and choroid highlight a need for additional alternative therapies. Integrins αvß3 and αvß5, which regulate endothelial cell proliferation and stabilization, have been implicated in ocular angiogenesis. Lebecetin (LCT) is a 30-kDa heterodimeric C-type lectin that is isolated from Macrovipera lebetina venom and interacts with α5ß1- and αv-containing integrins. We previously showed that LCT inhibits human brain microvascular endothelial cell adhesion, migration, proliferation, and tubulogenesis. To evaluate the inhibitory effect of LCT on ocular angiogenesis, we cultured aortic and choroidal explants in the presence of LCT and analyzed the effect of LCT on CNV in the mouse CNV model and on retinal neovascularization in the oxygen-induced retinopathy model. Our data demonstrate that a single injection of LCT efficiently reduced CNV and retinal neovascularization in these models.-Montassar, F., Darche, M., Blaizot, A., Augustin, S., Conart, J.-B., Millet, A., Elayeb, M., Sahel, J.-A., Réaux-Le Goazigo, A., Sennlaub, F., Marrakchi, N., Messadi, E., Guillonneau, X. Lebecetin, a C-type lectin, inhibits choroidal and retinal neovascularization.

In Silico prediction of the molecular basis of ClTx and AaCTx interaction with matrix metalloproteinase-2 (MMP-2) to inhibit glioma cell invasion.

Glioblastoma is the deadliest type of brain cancer. Treatment could target the Matrix metalloproteinase-2 (MMP-2), which is known to be involved in the invasion process of glioblastoma cells. But current available inhibitors are not selective to MMP-2 due to their interaction with the catalytic binding site, which is highly conserved in all MMPs structures. Interestingly, members of the chloride channel blocker scorpion toxins, such as chlorotoxin (ClTx) and AaCTx, inhibit glioblastoma cell invasion and show a promising therapeutic potential. Indeed, it has been shown that CITx inhibits selectively MMP-2 and was also able to cross the blood brain and tissue barriers. Although ClTx and AaCTx show high sequence similarity, AaCTx is ten times less active than ClTx. By using molecular modeling, molecular dynamics and MM-PB(GB)SA free energy estimation, we present the first computational study reporting the interaction mode of ClTx/AaCTx with MMP-2. We found that the two peptides probably act on an exosite of MMP-2 comprising mainly residues from the collagen binding domain, a feature that could be exploited to enhance the selectivity toward MMP-2. van der Waals and hydrophobic forces are the primary mediators of this interaction. The N- and C-termini of the two peptides harbor the key residues of the interaction spread across a conserved amino acid patch. In particular, F6 contributes mostly to the binding free energy in ClTx. We also suggest that the lack of the C-terminal arginine and the residues P10 and R24, might be responsible for altering the activity of AaCTx toward glioblastoma cells compared to ClTx.

Lebetin 2, a Snake Venom-Derived Natriuretic Peptide, Attenuates Acute Myocardial Ischemic Injury through the Modulation of Mitochondrial Permeability Transition Pore at the Time of Reperfusion.

Cardiac ischemia is one of the leading causes of death worldwide. It is now well established that natriuretic peptides can attenuate the development of irreversible ischemic injury during myocardial infarction. Lebetin 2 (L2) is a new discovered peptide isolated from Macrovipera lebetina venom with structural similarity to B-type natriuretic peptide (BNP). Our objectives were to define the acute cardioprotective actions of L2 in isolated Langendorff-perfused rat hearts after regional or global ischemia-reperfusion (IR). We studied infarct size, left ventricular contractile recovery, survival protein kinases and mitochondrial permeability transition pore (mPTP) opening in injured myocardium. L2 dosage was determined by preliminary experiments at its ability to induce cyclic guanosine monophosphate (cGMP) release without changing hemodynamic effects in normoxic hearts. L2 was found to be as effective as BNP in reducing infarct size after the induction of either regional or global IR. Both peptides equally improved contractile recovery after regional IR, but only L2 increased coronary flow and reduced severe contractile dysfunction after global ischemia. Cardioprotection afforded by L2 was abolished after isatin or 5-hydroxydecanote pretreatment suggesting the involvement of natriuretic peptide receptors and mitochondrial KATP (mitoKATP) channels in the L2-induced effects. L2 also increased survival protein expression in the reperfused myocardium as evidenced by phosphorylation of signaling pathways PKCε/ERK/GSK3ß and PI3K/Akt/eNOS. IR induced mitochondrial pore opening, but this effect was markedly prevented by L2 treatment. These data show that L2 has strong cardioprotective effect in acute ischemia through stimulation of natriuretic peptide receptors. These beneficial effects are mediated, at least in part, by mitoKATP channel opening and downstream activated survival kinases, thus delaying mPTP opening and improving IR-induced mitochondrial dysfunction.

Venom conjugated polylactide applied as biocompatible material for passive and active immunotherapy against scorpion envenomation.

Scorpion envenoming represents a public health issue in subtropical regions of the world. Treatment and prevention need to promote antitoxin immunity. Preserving antigenic presentation while removing toxin effect remains a major challenge in toxin vaccine development. Among particulate adjuvant, particles prepared with poly (D,L-lactide) polymer are the most extensively investigated due to their excellent biocompatibility and biodegradability. The aim of this study is to develop surfactant-free PLA nanoparticles that safely deliver venom toxic fraction to enhance specific immune response. PLA nanoparticles are coated with AahG50 (AahG50/PLA) and BotG50 (BotG50/PLA): a toxic fraction purified from Androctonus australis hector and Buthus occitanus tunetanus venoms, respectively. Residual toxicities are evaluated following injections of PLA-containing high doses of AahG50 (or BotG50). Immunization trials are performed with the detoxified fraction administered alone without adjuvant. A comparative study of the effect of Freund is also included. The neutralizing capacity of sera is determined in naive mice. Six months later, immunized mice are challenged subcutaneously with increased doses of AahG50. Subcutaneous lethal dose 50 (LD50) of AahG50 and BotG50 is of 575 µg/kg and 1300 µg/kg respectively. By comparison, BotG50/PLA is totally innocuous while 50% of tested mice survive 2875 µg AahG50/kg. Alhydrogel and Freund are not able to detoxify such a high dose. Cross-antigenicity between particulate and soluble fraction is also, ensured. AahG50/PLA and BotG50/PLA induce high antibody levels in mice serum. The neutralizing capacity per mL of anti-venom was 258 µg/mL and 186 µg/mL calculated for anti-AahG50/PLA and anti-BotG50/PLA sera, respectively. Animals immunized with AahG50/PLA are protected against AahG50 injected dose of 3162 µg/kg as opposed all non-immunized mice died at this dose. We find that the detoxification approach based PLA nanoparticles, benefit the immunogenicity and protective efficacy of venom immunogen.

CC5 and CC8, two homologous disintegrins from Cerastes cerastes venom, inhibit in vitro and ex vivo angiogenesis.

Angiogenesis constitutes a fundamental step in tumor progression. Thus, targeting tumour angiogenesis has been identified to be promising in cancer treatment. In this work, CC5 and CC8, two highly homologous disintegrins isolated from the venom Cerastes cerastes viper from the south of Tunisia, were assessed for their anti-angiogenic effect by testing their ability to interfere with viability, adhesion, migration and angiogenesis of Human Microvascular Endothelial Cells, HMEC-1 and HBMEC. We found that CC5 and CC8 displayed pro-apoptotic potential in HMEC-1 cells. Anoïkis like induced by these two disintegrins was evidenced by cell detachment, down regulation of FAK/AKT/PI3K axis and caspase activation. In addition, both CC5 and CC8 exhibited in vitro anti-adhesive, anti-migratory and anti-proliferative effects on endothelial cells HBMEC. These effects appeared to require RGD and/or WGD loops disintegrin. CC5 and CC8 also inhibited tube-formation on matrigel and displayed potent anti-angiogenic activities as assessed ex vivo, using both the embryo chick chorioallantoic membrane model (CAM) and rat aortic ring assay. Altogether our results demonstrate that CC5 and CC8, are potent inhibitors of angiogenesis, by disrupting αvß3 and α5ß1 binding. The use of CC5 and/or CC8 could provide a beneficial tool to inhibit abnormal angiogenesis and to induce cancer regression.

Kbot55, purified from Buthus occitanus tunetanus venom, represents the first member of a novel α-KTx subfamily.

Kbot55 is a 39 amino acid peptide isolated from the venom of the Tunisian scorpion Buthus occitanus tunetanus. This peptide is cross-linked by 3 disulfide bridges and has a molecular mass of 4128.65Da. Kbot55 is very low represented in the venom and thus represents a challenge for biochemical characterization. In this study, Kbot55 has been subjected to a screening on ion channels expressed in Xenopus laevis oocytes. It was found that Kbot55 targets voltage-gated potassium channels with high affinity. Kbot55 shows very low amino acid identity with other scorpion potassium toxins and therefore was considered a bona fide novel type of scorpion toxin. Sequence alignment analysis indicated that Kbot55 is the first representative of the new α-Ktx31 subfamily and therefore was classified as α-Ktx31.1.

Characterization of Kbot21 Reveals Novel Side Chain Interactions of Scorpion Toxins Inhibiting Voltage-Gated Potassium Channels.

Scorpion toxins are important pharmacological tools for probing the physiological roles of ion channels which are involved in many physiological processes and as such have significant therapeutic potential. The discovery of new scorpion toxins with different specificities and affinities is needed to further characterize the physiology of ion channels. In this regard, a new short polypeptide called Kbot21 has been purified to homogeneity from the venom of Buthus occitanus tunetanus scorpion. Kbot21 is structurally related to BmBKTx1 from the venom of the Asian scorpion Buthus martensii Karsch. These two toxins differ by only two residues at position 13 (R /V) and 24 (D/N).Despite their very similar sequences, Kbot21 and BmBKTx1 differ in their electrophysiological activities. Kbot21 targets KV channel subtypes whereas BmBKTx1 is active on both big conductance (BK) and small conductance (SK) Ca2+-activated K+ channel subtypes, but has no effects on Kv channel subtypes. The docking model of Kbot21 with the Kv1.2 channel shows that the D24 and R13 side-chain of Kbot21 are critical for its interaction with KV channels.

Purification, synthesis and characterization of AaCtx, the first chlorotoxin-like peptide from Androctonus australis scorpion venom.

AaCtx is the first chlorotoxin-like peptide isolated from Androctonus australis scorpion venom. Its amino acid sequence shares 70% similarity with chlorotoxin from Leiurus quinquestriatus scorpion venom, from which it differs by twelve amino acids. Due to its very low concentration in venom (0.05%), AaCtx was chemically synthesized. Both native and synthetic AaCtx were active on invasion and migration of human glioma cells. However, their activity was found to be lower than that of chlorotoxin. The molecular model of AaCtx shows that most of amino acids differing between AaCtx and chlorotoxin are localized on the N-terminal loop and the α-helix. Based on known compounds that block chloride channels, we suggest that the absence of negative charged amino acids on AaCtx structure may be responsible for its weak activity on glioma cells migration and invasion. This finding serves as a starting point for structure-function relationship studies leading to design high specific anti-glioma drugs.

Neurohormonal activation in severe scorpion envenomation: correlation with hemodynamics and circulating toxin.

We studied the effects of scorpion (Androctonus australis hector) venom on hemodynamics and on the release of catecholamines, neuropeptide Y (NPY), endothelin-1 (ET-1) and atrial natriuretic peptide (ANP) in dog model of severe scorpion envenomation. Nine mongrel anesthetized dogs were submitted to mechanical ventilation through intubation and were administered intravenously purified dried scorpion venom (Androctonus autstralis) 0.05 mg/kg. Measurements including pulmonary artery catheter derived parameters, serum toxin levels and humoral variables were performed at baseline (before venom injection) and 5, 15, 30 and 60 min after venom injection. Humoral variables included: serum lactate, epinephrine (EP), norepinephrine (NE), NPY, ET-1 and ANP plasma concentrations. Scorpion venom caused rapid and transient increase of mean arterial pressure (MAP) and PAOP associated with a marked and sustained decline in cardiac output (-55% at 60 min; P < 0.001). Hemodynamic changes were associated with a rapid and significant increase of all measured hormones. The highest increase was for NE (28-fold) and EP (25-fold). MAP was closely correlated with NE and less significantly correlated with toxin levels. Similarly, significant correlation was observed between PAPO and ANP plasma levels. These findings support the implication of excessive catecholamines release in hemodynamic disturbances of severe SE and suggest that NPY and ET-1 could be involved in this process. Serum toxin does not appear to consistently contribute to these effects. Through its correlation with PAOP, ANP could be a reliable and useful marker of cardiac dysfunction in SE.