Search

Snake venom C-type lectin-like proteins (CLPs) belong to the nonenzymatic proteins. To date, no CLP with both platelet and coagulation factors activating activities has been reported. In this study, a novel CLP, termed protocetin, with molecular weight of 29.986 kDa, was purified from the Protobothrops mucrosquamatus venom (PMV). It consists of α- and β-chains, with 67% similarity in their N-terminal sequence. Protocetin activates glycoprotein Ib (GPIb) by binding to von Willebrand factor (vWF), inducing platelet aggregation. It also activates the intrinsic coagulation pathway by binding to coagulation factor IX. After injection of protocetin into mice at dose of 0.5 µg/g or 1.5 µg/g, it resulted in activation of platelets, a notable reduction in platelet count and prolonged tail bleeding time. Additionally, the plasma activated partial thromboplastin time (APTT) was significantly extended, and the fibrinogen concentration was markedly reduced. Thrombelastogram comfirmed the anticoagulation effect of protocetin. Notably, no microthrombosis was observed in tissues of lung, liver and kidney within 1 h after injection of protocetin into the mice at dose of 0.5 µg/g. This study revealed protocetin as a novel CLP from PMV that has dual functions in activating platelet and coagulation factor IX, thereby modulates coagulation in vivo. This work contributes to a better understanding of the structure and function of snake venom CLP., (© 2024. The Author(s).)

Metastatic melanoma, a deadly form of skin cancer, often develops resistance to the BRAF inhibitor drug vemurafenib, highlighting the need for understanding the underlying mechanisms of resistance and exploring potential therapeutic strategies targeting integrins and TGF-β signalling. In this study, the role of integrins and TGF-β signalling in vemurafenib resistance in melanoma was investigated, and the potential of combining vemurafenib with cilengitide as a therapeutic strategy was investigated. In this study, it was found that the transcription of PAI1 and p21 was induced by acquired vemurafenib resistance, and ITGA5 levels were increased as a result of this resistance. The transcription of ITGA5 was mediated by the TGF-β pathway in the development of vemurafenib resistance. A synergistic effect on the proliferation of vemurafenib-resistant melanoma cells was observed with the combination therapy of vemurafenib and cilengitide. Additionally, this combination therapy significantly decreased invasion and colony formation in these resistant cells. In conclusion, it is suggested that targeting integrins and TGF-β signalling, specifically ITGA5 , ITGB3 , PAI1 , and p21 , may offer promising approaches to overcoming vemurafenib resistance, thereby improving outcomes for metastatic melanoma patients.

The interactions between specific snake venom toxins and muscle constituents are the major cause of severe muscle damage that often result in amputations and subsequent socioeconomic ramifications for snakebite victims and/or their families. Therefore, improving our understanding of venom-induced muscle damage and determining the underlying mechanisms of muscle degeneration/regeneration following snakebites is critical to developing better strategies to tackle this issue. Here, we analysed intramuscular bleeding and thrombosis in muscle injuries induced by two different snake venom toxins (CAMP- Crotalus atrox metalloprotease (a PIII metalloprotease from the venom of this snake) and a three-finger toxin (CTX, a cardiotoxin from the venom of Naja pallida )). Classically, these toxins represent diverse scenarios characterised by persistent muscle damage (CAMP) and successful regeneration (CTX) following acute damage, as normally observed in envenomation by most vipers and some elapid snakes of Asian, Australasian, and African origin, respectively. Our immunohistochemical analysis confirmed that both CAMP and CTX induced extensive muscle destruction on day 5, although the effects of CTX were reversed over time. We identified the presence of fibrinogen and P-selectin exposure inside the damaged muscle sections, suggesting signs of bleeding and the formation of platelet aggregates/microthrombi in tissues, respectively. Intriguingly, CAMP causes integrin shedding but does not affect any blood clotting parameters, whereas CTX significantly extends the clotting time and has no impact on integrin shedding. The rates of fibrinogen clearance and reduction in microthrombi were greater in CTX-treated muscle compared to CAMP-treated muscle. Together, these findings reveal novel aspects of venom-induced muscle damage and highlight the relevance of haemostatic events such as bleeding and thrombosis for muscle regeneration and provide useful mechanistic insights for developing better therapeutic interventions.

Inflammation is associated with many pathology disorders and the malignant progression of most cancers. Therefore, targeting inflammatory pathways could provide a promising strategy for disease prevention and treatment. In this study, we experimentally investigated the anti-inflammatory effect of CC5 and CC8, two disintegrin isoforms isolated from Cerastes cerastes snake venom, on LPS-stimulated macrophages, both on human THP-1 and mouse RAW264.7 cell adherence and their underlying mechanisms by measuring cytokine release levels and Western blot assay. Equally, both molecules were evaluated on a carrageenan-induced edema rat model. Our findings suggest that CC5 and CC8 were able to reduce adhesion of LPS-stimulated macrophages both on human THP-1 and mouse RAW264.7 cells to fibrinogen and vitronectin through the interaction with the αvβ3 integrin receptor. Moreover, CC5 and CC8 reduced the levels of reactive oxygen species (ROS) mediated by the NF-κB, MAPK and AKT signaling pathways that lead to decreased production of the pro-inflammatory cytokines TNF-α, IL-6 and IL-8 and increased secretion of IL-10 in LPS-stimulated THP-1 and RAW264.7 cells. Interestingly, both molecules potently exhibited an anti-inflammatory effect in vivo by reducing paw swelling in rats. In light of these results, we can propose the CC5 and CC8 disintegrins as interesting tools to design potential candidates against inflammatory-related diseases.

Snakebite is a globally neglected tropical disease, with coagulation disturbances being the primary pathology of many deadly snake venoms. Age-related differences in human plasma have been abundantly reported, yet the effect that these differences pose regarding snakebite is largely unknown. We tested for differences in coagulotoxic effects (via clotting time) of multiple snake venoms upon healthy human adult (18+) and paediatric (median 3.3 years old) plasma in vivo and compared these effects to the time it takes the plasmas to clot without the addition of venom (the spontaneous clotting time). We tested venoms from 15 medically significant snake species (from 13 genera) from around the world with various mechanisms of coagulotoxic actions, across the three broad categories of procoagulant, pseudo-procoagulant, and anticoagulant, to identify any differences between the two plasmas in their relative pathophysiological vulnerability to snakebite. One procoagulant venom ( Daboia russelii , Russell's Viper) produced significantly greater potency on paediatric plasma compared with adult plasma. In contrast, the two anticoagulant venoms ( Pseudechis australis , Mulga Snake; and Bitis cornuta , Many-horned Adder) were significantly more potent on adult plasma. All other procoagulant venoms and all pseudo-procoagulant venoms displayed similar potency across both plasmas. Our preliminary results may inform future studies on the effect of snake venoms upon plasmas from different age demographics and hope to reduce the burden of snakebite upon society.

Snake toxins are widely studied owing to their importance in snakebite accidents, a serious public health issue in tropical countries, and their broad therapeutic potential. Isolated fractions from venom produced by snakes of the genus Crotalus sp. present a wide variety of pharmacological uses such as antifungal, antiviral, antibacterial, and antitumor properties, among other therapeutic potentialities. Given the direct effect of this venom on tumor cells, isolation of its compounds is important for the characterization of its anticarcinogenic actions. Crotalus durissus terrificus venom and its toxins have been widely evaluated as potential candidates for the development of new antineoplastic therapies that are efficient against different tumor lines and cellular targets. This review highlights the venom toxins of this species, with a focus on their antineoplastic properties.

Background and Purpose: Venomous animals express numerous Kunitz-type peptides. The mambaquaretin-1 (MQ1) peptide identified from the Dendroaspis angusticeps venom is the most selective antagonist of the arginine-vasopressin V2 receptor (V2R) and the only unique Kunitz-type peptide active on a GPCR. We aimed to exploit other mamba venoms to enlarge the V2R-Kunitz peptide family and gain insight into the MQ1 molecular mode of action., Experimental Approach: We used a bio-guided screening assay to identify novel MQs and placed them phylogenetically. MQs were produced by solid-phase peptide synthesis and characterized in vitro by binding and functional tests and in vivo by diuresis measurement in rats., Key Results: Eight additional MQs were identified with nanomolar affinities for the V2R, all antagonists. MQs form a new subgroup in the Kunitz family, close to the V2R non-active dendrotoxins and to two V2R-active cobra toxins. Sequence comparison between active and non-active V2R Kunitz peptides highlighted five positions, among which four are involved in V2R interaction and belong to the two large MQ1 loops. We finally determined that eight positions, part of these two loops, interact with the V2R. The variant MQ1-K39A showed a higher affinity for the hV2R, but not for the rat V2R., Conclusions and Implications: A new function and mode of action is associated with the Kunitz peptides. The number of MQ1 residues involved in V2R binding is large and may explain its absolute selectivity. MQ1-K39A represents the first step in the improvement of the MQ1 design from a medicinal perspective., (© 2022 The British Pharmacological Society.)

The growing use of phytotherapy in clinical practice arouses interest in studies using medicinal plants as active ingredients for new medicines. Ipomoea pes-caprae has a wide medicinal use in the treatment of inflammatory disorders, skin wounds, stings, and painful rheumatic processes. Assayed in this study are the physicochemical characterization of a gel developed with this extract and the evaluation of its anti-inflammatory and healing efficacy, in addition to its antiedematogenic action on Bothrops snake envenoming in mice. The qualitative and quantitative analyses of the hydroethanolic extract by mass spectrometry showed 18 phenolic compounds, highlighting a high content of chlorogenic acid (0.92 µg/g), neochlorogenic acid (6.07 µg/g), and isochlorogenic acid (0.80 µg/g) compounds. The formulation was stable in relation to the physical-chemical characteristics during the time of analysis and was considered safe for topical treatment in animals, causing no skin irritation. Although the results have shown an absence of activity in the model of ear edema induced by croton oil (acute inflammation), the herbal gel efficiently inhibited carrageenan paw edema and chronic ear edema induced by multiple applications of croton oil, which may indicate the possible performance under the kinin pathway such as bradykinin, histamine, and serotonin. Wound healing in the group treated with the I. pes-caprae gel was accelerated compared with the placebo group, also confirmed through histological data. Edema induced by Bothrops erythromelas snake venom was efficiently reduced in the treatment with I. pes-caprae gel associated with the antibothropic-crotalic serum, whereas the antivenom alone was not effective. This approach presents a promising formulation based on I. pes-caprae with potential therapeutic use for inflammatory disorders., (Copyright © 2022. Published by Elsevier Masson SAS.)

Nanomedicine has led to the development of new biocompatible and biodegradable materials able to improve the pharmaceutical effect of bioactive components, broadening the options of treatment for several diseases, including cancer. Additionally, some snake venom toxins have been reported to present cytotoxic activity in different tumor cell lines, making them an auspicious option to be used as cancer drugs. The present study aims to evaluate the cytotoxic activity of the northern black-tailed rattlesnake (Crotalus molossus molossus) venom-loaded chitosan nanoparticles (Cs-Venom NPs) against the T-47D breast carcinoma cell line. To do so, we first identified the significant proteins composing the venom; afterward, hemocompatibility and cytotoxic activity against tumoral cells were evaluated. The venom was then loaded into chitosan nanoparticles through the ionotropic gelation process, obtaining particles of 415.9±21.67 nm and ζ-potential of +28.3±1.17 mV. The Cs-Venom complex delivered the venom into the breast carcinoma cells, inhibiting their viability and inducing morphological changes in the T-47D cells. These features indicate that these nanoparticles are suitable for the potential use of C. m. molossus venom toxins entrapped within polymer nanoparticles for the future development and research of cancer drugs.

Integrins play an important role in multiple stages of tumor progression and metastasis. Previous studies have shown synergistic effects of combined αvβ6-integrin and αvβ8-integrin inhibitors with immunotherapy. However, the role of αvβ3-integrin inhibitor in tumor immunity is still unclear. In this study, we aimed to elucidate the impact of the αvβ3-integrin inhibitor on PD-L1 expression and sensitivity to immune checkpoint blockade in melanoma. We investigated the effects of cilengitide, an αvβ3-integrin inhibitor, on cell viability and apoptosis of melanoma cell lines. And we explored how cilengitide regulated the expression of PD-L1 in melanoma cells in vitro and in vivo, using immunofluorescence, flow cytometry, Western blotting, and immunohistochemistry. A subcutaneous B16 murine melanoma model was utilized to determine whether combining cilengitide with anti-PD1 therapy inhibited tumor growth and positively regulated tumor microenvironment (TME). Our results showed that cilengitide inhibited cell viability and induced apoptosis in B16 and A375 cell lines. Furthermore, cilengitide decreased PD-L1 expression by reducing STAT3 phosphorylation in two melanoma cell lines. Cilengitide also reduced subcutaneous tumor PD-L1 expression in the B16 murine melanoma model. Accordingly, cilengitide positively regulated antitumor immune responses and provided durable therapy when combined with anti-PD1 monoclonal antibody in the murine melanoma model. This combination therapy reduced tumor growth and extended survival. Our study highlights that cilengitide enhances the efficacy of anti-PD1 therapy and produces a stronger antitumor immune response. This combination therefore represents a novel therapeutic regimen that may improve immunotherapy treratment.

Background: Cilengitide is a selective α v β 3 and α v β 5 integrin inhibitor. We sought to investigate the effect of cilengitide on the neovascularization of abdominal aortic plaques in rabbits and explore its underlying antiangiogenic mechanism on human umbilical vein endothelial cells (HUVECs)., Materials and Methods: For the in vivo experiment, the abdominal aortic plaque model of rabbits was established and injected with different doses of cilengitide or saline for 14 consecutive days. Conventional ultrasound (CUS) and contrast-enhanced ultrasound (CEUS) were applied to measure the vascular structure and blood flow parameters. CD31 immunofluorescence staining was performed for examining neovascularization. Relative expressions of vascular endothelial growth factor (VEGF) and integrin of the plaque were determined. For in vitro experiments, HUVECs were tested for proliferation, migration, apoptosis, and tube formation in the presence of different doses of cilengitide. Relative expressions of VEGF, integrin, and Ras/ERK/AKT signaling pathways were determined for the exploration of underlying mechanism., Results: CEUS showed modestly increased size and eccentricity index (EI) of plaques in the control group. Different degrees of reduced size and EI of plaques were observed in two cilengitide treatment groups. The expressions of VEGF and integrin in the plaque were inhibited after 14 days of cilengitide treatment. The neovascularization and apoptosis of the abdominal aorta were also significantly alleviated by cilengitide treatment. For in vitro experiments, cilengitide treatment was found to inhibit the proliferation, migration, and tube formation of HUVECs. However, cilengitide did not induce the apoptosis of HUVECs. A higher dose of cilengitide inhibited the mRNA expression of VEGF-A, β 3 , and β 5 , but not α V . Lastly, cilengitide treatment significantly inhibited the Ras/ERK/AKT pathway in the HUVECs . Conclusions . This study showed that cilengitide effectively inhibited the growth of plaque size by inhibiting the angiogenesis of the abdominal aortic plaques and blocking the VEGF-mediated angiogenic effect on HUVECs., Competing Interests: The authors have no conflicts of interest to declare., (Copyright © 2021 Fawang Zhu et al.)

Venoms are a rich source of potential lead compounds for drug discovery, and descriptive studies of venom form the first phase of the biodiscovery process. In this study, we investigated the pharmacological potential of crude Pseudocerastes and Eristicophis snake venoms in haematological disorders and cancer treatment. We assessed their antithrombotic potential using fibrinogen thromboelastography, fibrinogen gels with and without protease inhibitors, and colourimetric fibrinolysis assays. These assays indicated that the anticoagulant properties of the venoms are likely induced by the hydrolysis of phospholipids and by selective fibrinogenolysis. Furthermore, while most fibrinogenolysis occurred by the direct activity of snake venom metalloproteases and serine proteases, modest evidence indicated that fibrinogenolytic activity may also be mediated by selective venom phospholipases and an inhibitory venom-derived serine protease. We also found that the Pseudocerastes venoms significantly reduced the viability of human melanoma (MM96L) cells by more than 80%, while it had almost no effect on the healthy neonatal foreskin fibroblasts (NFF) as determined by viability assays. The bioactive properties of these venoms suggest that they contain a number of toxins suitable for downstream pharmacological development as candidates for antithrombotic or anticancer agents.

The unprecedented global COVID-19 pandemic has prompted a desperate international effort to accelerate the development of anti-viral candidates. For unknown reasons, COVID-19 infections are associated with adverse cardiovascular complications, implicating that vascular endothelial cells are essential in viral propagation. The etiological pathogen, SARS-CoV-2, has a higher reproductive number and infection rate than its predecessors, indicating it possesses novel characteristics that infers enhanced transmissibility. A unique K403R spike protein substitution encodes an Arg-Gly-Asp (RGD) motif, introducing a potential role for RGD-binding host integrins. Integrin αVβ3 is widely expressed across the host, particularly in the endothelium, which acts as the final barrier before microbial entry into the bloodstream. This mutagenesis creates an additional binding site, which may be sufficient to increase SARS-CoV-2 pathogenicity. Here, we investigate how SARS-CoV-2 passes from the epithelium to endothelium, the effects of αVβ3 antagonist, Cilengitide, on viral adhesion, vasculature permeability and leakage, and also report on a simulated interaction between the viral and host protein in-silico., Competing Interests: The authors have declared that no competing interests exist.

The venom of the Deinagkistrodon acutus snake is composed of numerous bioactive proteins and peptides. In this study, we report the antithrombotic and anticoagulant activities of one of such proteins, herein known as SLPC. This novel protein was isolated and purified via multi-gel chromatography. Its amino acid sequence, structure and function were then determined. This protein was found to exhibit defibration, anticoagulation and general antithrombotic effects based on the results of both in vitro and in vivo studies. Based on same studies, it was found to cleave the α, β, γ chains of fibrinogen and generally improved antiplatelet aggregation and blood rheology. A metabolomic insight of the antithrombotic effects of SLPC was found to be mainly linked to perturbations in the synthesis of unsaturated fatty acids, glycerophospholipid metabolism, arachidonic acid metabolism and other metabolic pathways. In summary, the novel protein SLPC, elicits its antithrombotic effects via degradation of fibrinogen and regulation of various thrombogenic factors in multiple metabolic pathways., (Copyright © 2021. Published by Elsevier Masson SAS.)

Procoagulant snake venoms have been inhibited by the ruthenium containing compounds CORM-2 and RuCl 3 separately, presumably by interacting with critical histidine or other sulfur-containing amino acids on key venom enzymes. However, combinations of these and other platinoid containing compounds could potentially increase, decrease or not affect the procoagulant enzyme function of venom. Thus, the purpose of this investigation was to determine if formulations of platinoid compounds could inhibit venom procoagulant activity and if the formulated compounds interacted to enhance inhibition. Using a human plasma coagulation kinetic model to assess venom activity, six diverse venoms were exposed to various combinations and concentrations of CORM-2, CORM-3, RuCl 3 and carboplatin (a platinum containing compound), with changes in venom activity determined with thrombelastography. The combinations of CORM-2 or CORM-3 with RuCl 3 were found to enhance inhibition significantly, but not in all venoms nor to the same extent. In sharp contrast, carboplatin-antagonized CORM-2 mediated the inhibition of venom activity. These preliminary results support the concept that platinoid compounds may inhibit venom enzymatic activity at the same or different molecular sites and may antagonize inhibition at the same or different sites. Further investigation is warranted to determine if platinoid formulations may serve as potential antivenoms.

Cardiovascular diseases (CVDs) are considered as a major cause of death worldwide. Therefore, identifying and developing therapeutic strategies to treat and reduce the prevalence of CVDs is a major medical challenge. Several drugs used for the treatment of CVDs, such as captopril, emerged from natural products, namely snake venoms. These venoms are complex mixtures of bioactive molecules, which, among other physiological networks, target the cardiovascular system, leading to them being considered in the development and design of new drugs. In this review, we describe some snake venom molecules targeting the cardiovascular system such as phospholipase A2 (PLA2), natriuretic peptides (NPs), bradykinin-potentiating peptides (BPPs), cysteine-rich secretory proteins (CRISPs), disintegrins, fibrinolytic enzymes, and three-finger toxins (3FTXs). In addition, their molecular targets, and mechanisms of action-vasorelaxation, inhibition of platelet aggregation, cardioprotective activities-are discussed. The dissection of their biological effects at the molecular scale give insights for the development of future snake venom-derived drugs.

Objectives: Vitronectin (VTN) has been widely used for the maintenance and expansion of human pluripotent stem cells (hPSCs) as feeder-free conditions. However, the effect of VTN on hPSC differentiation remains unclear. Here, we investigated the role of VTN in early haematopoietic development of hPSCs., Materials and Methods: A chemically defined monolayer system was applied to study the role of different matrix or basement membrane proteins in haematopoietic development of hPSCs. The role of integrin signalling in VTN-mediated haematopoietic differentiation was investigated by integrin antagonists. Finally, small interfering RNA was used to knock down integrin gene expression in differentiated cells., Results: We found that the haematopoietic differentiation of hPSCs on VTN was far more efficient than that on Matrigel that is also often used for hPSC culture. VTN promoted the fate determination of endothelial-haematopoietic lineage during mesoderm development to generate haemogenic endothelium (HE). Moreover, we demonstrated that the signals through αvβ3 and αvβ5 integrins were required for VTN-promoted haematopoietic differentiation. Blocking αvβ3 and αvβ5 integrins by the integrin antagonists impaired the development of HE, but not endothelial-to-haematopoietic transition (EHT). Finally, both αvβ3 and αvβ5 were confirmed acting synergistically for early haematopoietic differentiation by knockdown the expression of αv, β3 or β5., Conclusion: The established VTN-based monolayer system of haematopoietic differentiation of hPSCs presents a valuable platform for further investigating niche signals involved in human haematopoietic development., (© 2021 The Authors. Cell Proliferation Published by John Wiley & Sons Ltd.)

Acid-sensing ion channels (ASICs) are expressed in the nervous system, activated by acidosis, and implicated in pain pathways. Mambalgins are peptide inhibitors of ASIC1 and analgesic in rodents via inhibition of centrally expressed ASIC1a and peripheral ASIC1b. This activity has generated interest in mambalgins as potential therapeutics. However, most mechanism and structure-activity relationship work on mambalgins has focused on ASIC1a, and neglected the peripheral analgesic target ASIC1b. Here, we compare mambalgin potency and mechanism of action at heterologously expressed rat and human ASIC1 variants. Unlike the nanomolar inhibition at ASIC1a and rodent ASIC1b, we find mambalgin-3 only weakly inhibits human ASIC1b and ASIC1b/3 under severe acidosis, but potentiates currents under mild/moderate acidosis. Our data highlight the importance of understanding the activity of potential ASIC-targeting pharmaceuticals at human channels., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Ctn[15-34], the C-terminal fragment of crotalicidin, an antimicrobial peptide from the South American rattlesnake Crotalus durissus terrificus venom, displays remarkable anti-infective and anti-proliferative activities. Herein, its activity on Candida albicans biofilms and its interaction with the cytoplasmic membrane of the fungal cell and with a biomembrane model in vitro was investigated. A standard C. albicans strain and a fluconazole-resistant clinical isolate were exposed to the peptide at its minimum inhibitory concentration (MIC) (10 µM) and up to 100 × MIC to inhibit biofilm formation and its eradication. A viability test using XTT and fluorescent dyes, confocal laser scanning microscopy, and atomic force microscopy (AFM) were used to observe the antibiofilm effect. To evaluate the importance of membrane composition on Ctn[15-34] activity, C. albicans protoplasts were also tested. Fluorescence assays using di-8-ANEPPS, dynamic light scattering, and zeta potential measurements using liposomes, protoplasts, and C. albicans cells indicated a direct mechanism of action that was dependent on membrane interaction and disruption. Overall, Ctn[15-34] showed to be an effective antifungal peptide, displaying antibiofilm activity and, importantly, interacting with and disrupting fungal plasma membrane.

The venom of the boomslang (Dispholidus typus) has potent effects on the coagulation system. It is known to produce a venom-induced consumptive coagulopathy (VICC) through the proposed activation of clotting factor II (prothrombin), factor X, and possibly factor IX. Warfarin, an anticoagulant medication, decreases the circulating vitamin K-dependent clotting factors II, VII, IX and X. We report a unique case of a boomslang bite in a patient on warfarin therapy. During the patient's hospital stay he developed abnormal clotting profiles indicating an underlying VICC, but without major bleeding. He received monovalent antivenom and recovered with no complications. We discuss two possible outcomes of a boomslang bite in a patient on warfarin therapy, exploring the underlying pathophysiology that could lead to the presentation of a reduced risk of overall bleeding or, alternatively, that the bleeding could be compounded and exacerbated. It is possible that in our case the anticoagulant effect of warfarin was wholly obscured by the VICC of the boomslang venom. The composition of the snake venom may have been a contributory factor in the reduced clinical bleeding observed.

Rationale: MQ1, a snake toxin which targets with high nanomolar affinity and absolute selectivity for the type 2 vasopressin receptor (V2R), is a drug candidate for renal diseases and a molecular probe for imaging cells or organs expressing V2R. Methods: MQ1's pharmacological properties were characterized and applied to a rat model of hyponatremia. Its PK/PD parameters were determined as well as its therapeutic index. Fluorescently and radioactively labeled MQ1 were chemically synthesized and associated with moderate loss of affinity. MQ1's dynamic biodistribution was monitored by positron emission tomography. Confocal imaging was used to observe the labeling of three cancer cell lines. Results: The inverse agonist property of MQ1 very efficiently prevented dDAVP-induced hyponatremia in rats with low nanomolar/kg doses and with a very large therapeutic index. PK (plasma MQ1 concentrations) and PD (diuresis) exhibited a parallel biphasic decrease. The dynamic biodistribution showed that MQ1 targets the kidneys and then exhibits a blood and kidney biphasic decrease. Whatever the approach used, we found a T1/2α between 0.9 and 3.8 h and a T1/2β between 25 and 46 h and demonstrated that the kidneys were able to retain MQ1. Finally, the presence of functional V2R expressed at the membrane of cancer cells was, for the first time, demonstrated with a specific fluorescent ligand. Conclusion: As the most selective V2 binder, MQ1 is a new promising drug for aquaresis-related diseases and a molecular probe to visualize in vitro and in vivo V2R expressed physiologically or under pathological conditions., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

There is currently no therapy to limit the development of cardiac fibrosis and consequent heart failure. We have recently shown that cardiac fibrosis post-myocardial infarction (MI) can be regulated by resident cardiac cells with a fibrogenic signature and identified by the expression of PW1 (Peg3). Here we identify αV-integrin (CD51) as an essential regulator of cardiac PW1 + cells fibrogenic behavior. We used transcriptomic and proteomic approaches to identify specific cell-surface markers for cardiac PW1 + cells and found that αV-integrin (CD51) was expressed in almost all cardiac PW1 + cells (93% ± 1%), predominantly as the αVβ1 complex. αV-integrin is a subunit member of the integrin family of cell adhesion receptors and was found to activate complex of latent transforming growth factor beta (TGFβ at the surface of cardiac PW1 + cells. Pharmacological inhibition of αV-integrin reduced the profibrotic action of cardiac PW1 + CD51 + cells and was associated with improved cardiac function and animal survival following MI coupled with a reduced infarct size and fibrotic lesion. These data identify a targetable pathway that regulates cardiac fibrosis in response to an ischemic injury and demonstrate that pharmacological inhibition of αV-integrin could reduce pathological outcomes following cardiac ischemia.

The continued evolution of antibiotic resistance has increased the urgency for new antibiotic development, leading to exploration of non-traditional sources. In particular, snake venom has garnered attention for its potent antibacterial properties. Numerous studies describing snake venom proteomic composition as well as antibiotic efficacy have created an opportunity to synthesize relationships between venom proteomes and their antibacterial properties. Using literature reported values from peer-reviewed studies, our study generated models to predict efficacy given venom protein family composition, snake taxonomic family, bacterial Gram stain, bacterial morphology, and bacterial respiration strategy. We then applied our predictive models to untested snake species with known venom proteomic compositions. Overall, our results provide potential protein families that serve as accurate predictors of efficacy as well as promising organisms in terms of antibacterial properties of venom. The results from this study suggest potential future research trajectories for antibacterial properties in snake venom by offering hypotheses for a variety of taxa., Competing Interests: The authors have declared that no competing interests exist.

Objective : Integrin β3 is one of the main integrin heterodimer receptors on the surface of cardiac myocytes. Our previous studies showed that hypoxia induces apoptosis and increases integrin β3 expression in cardiomyocytes. However, the exact mechanism by which integrin β3 protects against apoptosis remains unclear. Hence, the present investigation aimed to explore the mechanism of integrin β3 in cardiomyocyte proliferation and hypoxia-induced cardiomyocyte apoptosis. Methods : Stable cells and in vivo acute and chronic heart failure rat models were generated to reveal the essential role of integrin β3 in cardiomyocyte proliferation and apoptosis. Western blotting and immunohistochemistry were employed to detect the expression of integrin β3 in the stable cells and rat cardiac tissue. Flow cytometer was used to investigate the role of integrin β3 in hypoxia-induced cardiomyocyte apoptosis. Confocal microscopy was used to detect the localization of integrin β3 and integrin αv in cardiomyocytes. Results : A cobaltous chloride-induced hypoxic microenvironment stimulated cardiomyocyte apoptosis and increased integrin β3 expression in H9C2 cells, AC16 cells, and cardiac tissue from acute and chronic heart failure rats. The overexpression of integrin β3 promoted cardiomyocyte proliferation, whereas silencing integrin β3 expression resulted in decreased cell proliferation in vitro . Furthermore, knocking down integrin β3 expression using shRNA or the integrin β3 inhibitor cilengitide exacerbated cobaltous chloride-induced cardiomyocyte apoptosis, whereas overexpression of integrin β3 weakened cobaltous chloride-induced cardiomyocytes apoptosis. We found that integrin β3 promoted cardiomyocytes proliferation through the regulation of the PTEN/Akt/mTOR and ERK1/2 signaling pathways. In addition, we found that knockdown of integrin αv or integrin β1 weakened the effect of integrin β3 in cardiomyocyte proliferation. Conclusion : Our findings revealed the molecular mechanism of the role of integrin β3 in cardiomyocyte proliferation and hypoxia-induced cardiomyocyte apoptosis, providing new insights into the mechanisms underlying myocardial protection., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Life-threatening thrombocytopenia and bleeding, common side effects of clinically available α IIb β 3 antagonists, are associated with the induction of ligand-induced integrin conformational changes and exposure of ligand-induced binding sites (LIBSs). To address this issue, we examined intrinsic mechanisms and structure-activity relationships of purified disintegrins, from Protobothrops flavoviridis venom (i.e., Trimeresurus flavoviridis ), TFV-1 and TFV-3 with distinctly different pro-hemorrhagic tendencies. TFV-1 with a different α IIb β 3 binding epitope from that of TFV-3 and chimeric 7 × 10 3 Fab, i.e., Abciximab, decelerates α IIb β 3 ligation without causing a conformational change in α IIb β 3 , as determined with the LIBS antibody, AP5, and the mimetic, drug-dependent antibody (DDAb), AP2, an inhibitory monoclonal antibody raised against α IIb β 3 . Consistent with their different binding epitopes, a combination of TFV-1 and AP2 did not induce FcγRIIa-mediated activation of the ITAM-Syk-PLCγ2 pathway and platelet aggregation, in contrast to the clinical antithrombotics, abciximab, eptifibatide, and disintegrin TFV-3. Furthermore, TFV-1 selectively inhibits Gα 13 -mediated platelet aggregation without affecting talin-driven clot firmness, which is responsible for physiological hemostatic processes. At equally efficacious antithrombotic dosages, TFV-1 caused neither severe thrombocytopenia nor bleeding in FcγRIIa-transgenic mice. Likewise, it did not induce hypocoagulation in human whole blood in the rotational thromboelastometry (ROTEM) assay used in perioperative situations. In contrast, TFV-3 and eptifibatide exhibited all of these hemostatic effects. Thus, the α IIb β 3 antagonist, TFV-1, efficaciously prevents arterial thrombosis without adversely affecting hemostasis.

Arsenic (As) and sulfur dioxide (SO 2 ) are two environmental pollutants that have been shown to promote the development of human cancer. In recent years, due to increased pollution, humans are often exposed to SO 2 , in addition to As. Despite the development and implementation of standards for environment and air quality, cases of disease caused by As or SO 2 continue to rise alarmingly. It is currently unknown whether simultaneous exposure to As and SO 2 results in increased cancer promoting activity. In this study, concentrations of As and SO 2 below the limits established by the world health organization (WHO) in force environmental standards (concentrations of As should be lower than 1×10 -2  mg/L and SO 2 should be lower than 50 μg/m 3 ), were employed to investigate possible, long-term, synergistic effects of As and SO 2 , by using cell-based assays. We found that co-exposure to these pollutants significantly promotes HepG2 cancer cell migration, while As or SO 2 alone have no remarkable effects. Integrins αvβ3 play a key role in this process, as cilengitide, an integrin αvβ3 inhibitor, substantially prevented As and SO 2 -induced cell migration. MMPs, IL-8, and TGF-β were also involved in the induced cell migration. In summary, combined exposure to As and SO 2 promotes integrin-dependent cell migration and may be of relevance for the activation of mechanisms underlying liver cancer progression., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Animal venoms are used as defense mechanisms or to immobilize and digest prey. In fact, venoms are complex mixtures of enzymatic and non-enzymatic components with specific pathophysiological functions. Peptide toxins isolated from animal venoms target mainly ion channels, membrane receptors and components of the hemostatic system with high selectivity and affinity. The present review shows an up-to-date survey on the pharmacology of snake-venom bioactive components and evaluates their therapeutic perspectives against a wide range of pathophysiological conditions. Snake venoms have also been used as medical tools for thousands of years especially in tradition Chinese medicine. Consequently, snake venoms can be considered as mini-drug libraries in which each drug is pharmacologically active. However, less than 0.01% of these toxins have been identified and characterized. For instance, Captopril ® (Enalapril), Integrilin ® (Eptifibatide) and Aggrastat ® (Tirofiban) are drugs based on snake venoms, which have been approved by the FDA. In addition to these approved drugs, many other snake venom components are now involved in preclinical or clinical trials for a variety of therapeutic applications. These examples show that snake venoms can be a valuable source of new principle components in drug discovery., Competing Interests: The authors declare no conflict of interest.

Despite aggressive multimodality treatment, the prognosis of glioma, especially malignant glioma, remains very poor. After decades of effort, anti-angiogenic therapy has become an important method of cancer treatment in addition to surgery, radiotherapy and chemotherapy. Although the performance of anti-angiogenic therapy in colorectal cancer is good, its performance in malignant glioma remains unsatisfactory. Several phase III clinical trials showed no overall survival benefits. To solve this problem, the division of patients into groups based on their molecular biomarkers is an important step. This paper provides current insights into anti-angiogenic drugs undergoing clinical trials and discusses the potential of molecular biomarkers to guide glioma diagnosis., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Background: Bothropic venoms cause intense local damage, pain, edema, and myonecrosis. Morus nigra L. (Moraceae) has several uses in folk medicine and can be a promising candidate for the treatment of several inflammatory disorders., Hypothesis/purpose: The present study aims to evaluate the anti-inflammatory and antinociceptive effects of the ethanolic extract of Morus nigra L. (Mn-EtOH) on paw lesions induced by Bothrops jararacussu snake venom (BjcuV) in mice., Methods: UV-vis absorption of BjcuV was evaluated. A phytochemical study was performed, which led to the isolation and characterization of three compounds. These compounds were identified using spectrometric methods, namely LC-MS and NMR (1D and 2D), followed by the validation of their spectra with the data available in the literature. Further, the flavonoids i.e. rutin and quercetin (chemical markers of M. nigra), Mn-EtOH or Mn-EtOH-encapsulated electrospun fibers of Eudragit L100 (FB/Mn-EtOH), and Mn-EtOH-encapsulated microparticles of Eudragit L100 (MP/Mn-EtOH) were evaluated, in paw edema test induced by BjcuV., Results: UV-vis spectra showed the presence of phospholipases A 2 as component of BjcuV. The chemical examination resulted in the isolation of β-sitosterol, quercetin-3-O-glucopyranoside, and kaempferol-3-O-glucopyranoside. Mn-EtOH, FB/Mn-EtOH, MP/Mn-EtOH, rutin, and quercetin reduced the local edema induced by BjcuV. The Mn-EtOH also prevented edema provoked by serotonin and bradykinin. Moreover, it reduced paw edema and peritoneal leukocyte infiltration induced by carrageenan, and decreased the mechanical hypernociception of BjcuV. Mn-EtOH exerted anti-inflammatory and antinociceptive effects, possibly by the inhibition of leukocyte migration and the modulation of serotonin and bradykinin actions. This anti-inflammatory activity was maintained even upon incorporation of the M. nigra extract into the drug delivery systems (i.e., Mn-EtOH-encapsulated FBs and MPs of Eudragit L100)., Conclusion: These results reinforce the therapeutic potential of M. nigra in the treatment of inflammatory conditions, in addition to, its role as a complementary treatment of snakebites., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Background: To date the TGF-β1 activation mediated by integrin ανβ5 during fibrosis is well-known. This process has been shown also in the heart, where cardiac fibroblasts (CF) differentiate into α-smooth muscle actin (α-SMA)-positive myofibroblasts (MyoFB). Here, we studied the effects on CF, isolated by spontaneously hypertensive rats (SHR), of integrin ανβ5 inhibition in MyoFB differentiation., Methods: Staining and immunohistochemistry were performed on rat cardiac tissue. CF were isolated by enzymatic digestion from SHR (SHR-CF) and normotensive WKY (WKY-CF) rat hearts and then treated for in vitro evaluation., Results: SHR heart tissues revealed a higher TGF-β1 expression vs. WKY samples. SHR-CF showed an enhanced SMAD2/3 activation and an up-regulated expression of α-SMA, a typical MyoFB marker, especially after TGF-β1 treatment. Immunostaining on cardiac tissues revealed a higher expression of integrin ανβ5 in SHR vs. WKY rat hearts. In vitro results confirmed the up-regulation of integrin ανβ5 expression in SHR-CF at basal condition and after TGF-β1 treatment, in comparison with WKY-CF. Inhibition of integrin ανβ5 by cilengitide treatment led a decreased expression of ανβ5, collagen I, and α-SMA in SHR-CF vs. WKY-CF, resulting in a diminished differentiation of CF into MyoFB. Taking together, results suggested that SHR-CF are more susceptible to TGF-β1, showing an up-regulated activation of SMAD2/3 signaling, and an increased ανβ5, α-SMA, and collagen I expression. Hypertension stimulus promoted an up-regulation of integrin ανβ5 on SHR cardiac tissue and its in vitro inhibition reverted pro-fibrotic events of SHR-CF., Conclusion: Inhibition of integrin ανβ5 exerted by cilengitide strongly diminished SHR-CF differentiation into detrimental MyoFB. So, integrin ανβ5 might be considered a novel therapeutic target and cilengitide an effective pharmacological tool to limit the progression of hypertension-induced cardiac fibrosis.

Ontogenetic changes in venom composition have important ecological implications due the relevance of venom in prey acquisition and defense. Additionally, intraspecific venom variation has direct medical consequences for the treatment of snakebite. However, ontogenetic changes are not well documented in most species. The Mexican Black-tailed Rattlesnake ( Crotalus molossus nigrescens ) is large-bodied and broadly distributed in Mexico. To document venom variation and test for ontogenetic changes in venom composition, we obtained venom samples from twenty-seven C. m. nigrescens with different total body lengths (TBL) from eight states in Mexico. The primary components in the venom were detected by reverse-phase HPLC, western blot, and mass spectrometry. In addition, we evaluated the biochemical (proteolytic, coagulant and fibrinogenolytic activities) and biological (LD 50 and hemorrhagic activity) activities of the venoms. Finally, we tested for recognition and neutralization of Mexican antivenoms against venoms of juvenile and adult snakes. We detected clear ontogenetic venom variation in C. m. nigrescens . Venoms from younger snakes contained more crotamine-like myotoxins and snake venom serine proteinases than venoms from older snakes; however, an increase of snake venom metalloproteinases was detected in venoms of larger snakes. Venoms from juvenile snakes were, in general, more toxic and procoagulant than venoms from adults; however, adult venoms were more proteolytic. Most of the venoms analyzed were hemorrhagic. Importantly, Mexican antivenoms had difficulties recognizing low molecular mass proteins (<12 kDa) of venoms from both juvenile and adult snakes. The antivenoms did not neutralize the crotamine effect caused by the venom of juveniles. Thus, we suggest that Mexican antivenoms would have difficulty neutralizing some human envenomations and, therefore, it may be necessary improve the immunization mixture in Mexican antivenoms to account for low molecular mass proteins, like myotoxins.

Nature endowed snakes with a lethal secretion known as venom, which has been fine-tuned over millions of years of evolution. Snakes utilize venom to subdue their prey and to survive in their natural habitat. Venom is known to be a very poisonous mixture, consisting of a variety of molecules, such as carbohydrates, nucleosides, amino acids, lipids, proteins and peptides. Proteins and peptides are the major constituents of the dry weight of snake venoms and are of main interest for scientific investigations as well as for various pharmacological applications. Snake venoms contain enzymatic and non-enzymatic proteins and peptides, which are grouped into different families based on their structure and function. Members of a single family display significant similarities in their primary, secondary and tertiary structures, but in many cases have distinct pharmacological functions and different bioactivities. The functional specificity of peptides belonging to the same family can be attributed to subtle variations in their amino acid sequences. Currently, complementary tools and techniques are utilized to isolate and characterize the peptides, and study their potential applications as molecular probes, and possible templates for drug discovery and design investigations.

Cancer represents the disease of the millennium, a major problem in public health. The proliferation of tumor cells, angiogenesis, and the relationship between the cancer cells and the components of the extracellular matrix are important in the events of carcinogenesis, and these pathways are being used as targets for new anticancer treatments. Various venoms and their toxins have shown possible anticancer effects on human cancer cell lines, providing new perspectives in drug development. In this review, we observed the effects of natural toxins from bee and snake venom and the mechanisms through which they can inhibit the growth and proliferation of cancer cells. We also researched how several types of natural molecules from venom can sensitize ovarian cancer cells to conventional chemotherapy, with many toxins being helpful for developing new anticancer drugs. This approach could improve the efficiency of standard therapies and could allow the administration of decreased doses of chemotherapy. Natural toxins from bee and snake venom could become potential candidates for the future treatment of different types of cancer. It is important to continue these studies concerning therapeutic drugs from natural resource and, more importantly, to investigate their mechanism of action on cancer cells., Competing Interests: The authors declare no conflict of interest

While molecular subtypes of glioblastoma (GBM) are defined using gene expression and mutation profiles, we identify a unique subpopulation based on addiction to the high-affinity glucose transporter, Glut3. Although Glut3 is a known driver of a cancer stem cell phenotype, direct targeting is complicated by its expression in neurons. Using established GBM lines and patient-derived stem cells, we identify a subset of tumors within the "proneural" and "classical" subtypes that are addicted to aberrant signaling from integrin αvβ3, which activates a PAK4-YAP/TAZ signaling axis to enhance Glut3 expression. This defined subpopulation of GBM is highly sensitive to agents that disrupt this pathway, including the integrin antagonist cilengitide, providing a targeted therapeutic strategy for this unique subset of GBM tumors., (Published by Elsevier Inc.)

This study was undertaken to examine the activities and levels of major antioxidants/oxidants in cultured human fibroblasts incubated with a sublethal dose of Echis coloratus venom (EcV). Glutathione peroxidase (GPx), catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR) activities and gene expression levels as well as reduced glutathione (GSH) levels, and the concurrent hydrogen peroxide (H 2 O 2 ), superoxide anions (SOA), lipid peroxides (LPO) and oxidized glutathione (GSSG) generation rates were assayed in fibroblast cultures and sonicates incubated with 0.5 µg ml -1 medium EcV for 4 h at 37°C. Data indicated that the activities of all antioxidant enzymes were significantly decreased and their corresponding transcripts downregulated in EcV-incubated cells compared to controls (p < 0.001). In contrast, there were parallel equally significant increases in H 2 O 2, SOA and LPO generation rates in venom-incubated cells compared to controls (p < 0.001). Additionally, GSH levels were significantly decreased and those of GSSG were equally significantly increased in venom-incubated cultures compared to controls (p < 0.001) leading to a lowered GSH/GSSG ratio. In conclusion, incubation of fibroblast cultures with EcV resulted in a shift towards oxidative metabolism causing severe OS. This correlated with significant downregulation in the expression levels of all investigated antioxidant genes.

Metastasis involves the migration of cancer cells from a primary tumor to invade and establish secondary tumors in distant organs, and it is the main cause for cancer-related deaths. Currently, the conventional cytostatic drugs target the proliferation of malignant cells, being ineffective in metastatic disease. This highlights the need to find new anti-metastatic drugs. Toxins isolated from snake venoms are a natural source of potentially useful molecular scaffolds to obtain agents with anti-migratory and anti-invasive effects in cancer cells. While there is greater evidence concerning the mechanisms of cell death induction of several snake toxin classes on cancer cells; only a reduced number of toxin classes have been reported on (i.e., disintegrins/disintegrin-like proteins, C-type lectin-like proteins, C-type lectins, serinproteases, cardiotoxins, snake venom cystatins) as inhibitors of adhesion, migration, and invasion of cancer cells. Here, we discuss the anti-metastatic mechanisms of snake toxins, distinguishing three targets, which involve (1) inhibition of extracellular matrix components-dependent adhesion and migration, (2) inhibition of epithelial-mesenchymal transition, and (3) inhibition of migration by alterations in the actin/cytoskeleton network., Competing Interests: The authors declare no conflict of interest.

Coagulation assays currently employed are often low throughput, require specialized equipment and/or require large blood/plasma samples. This study describes the development, optimization and early application of a generic low-volume and high-throughput screening (HTS) assay for coagulation activity. The assay is a time-course spectrophotometric measurement which kinetically measures the clotting profile of bovine or human plasma incubated with Ca 2+ and a test compound. The HTS assay can be a valuable new tool for coagulation diagnostics in hospitals, for research in coagulation disorders, for drug discovery and for venom research. A major effect following envenomation by many venomous snakes is perturbation of blood coagulation caused by haemotoxic compounds present in the venom. These compounds, such as anticoagulants, are potential leads in drug discovery for cardiovascular diseases. The assay was implemented in an integrated analytical approach consisting of reversed-phase liquid chromatography (LC) for separation of crude venom components in combination with parallel post-column coagulation screening and mass spectrometry (MS). The approach was applied for the rapid assessment and identification of profiles of haemotoxic compounds in snake venoms. Procoagulant and anticoagulant activities were correlated with accurate masses from the parallel MS measurements, facilitating the detection of peptides showing strong anticoagulant activity., Competing Interests: The authors declare no conflict of interests.

Rhabdophis lateralis , a colubrid snake distributed throughout the continent of Asia, has recently undergone taxonomic revisions. Previously, Rhabdophis lateralis was classified as a subspecies of R. tigrinus (Yamakagashi) until 2012, when several genetic differences were discovered which classified this snake as its own species. To elucidate the toxicity of venom from this poorly studied colubrid, various biological activities were compared between the venom from the two snake species. The components of their venom were compared by the elution profiles of reversed-phase HPLC and SDS-PAGE, and gel filtrated fractions were tested for effects on blood coagulation. Proteolytic activities of these fractions were also assayed by using synthetic substrates, fibrinogen, and matrix proteins. Similar to the R. tigrinus venom, the higher molecular weight fraction of R. lateralis venom contained a prothrombin activator. Both prothrombin time (PT) and activated partial thromboplastin time (APTT) of human plasma were shortened by the addition of R. lateralis and R. tigrinus venom. The thrombin formation was estimated by the uses of SDS-PAGE and chromogenic substrates. These venom fractions also possessed very specific proteinase activity on human fibrinogen, but the substrates for matrix metalloproteinase, such as collagen and laminin, were not hydrolyzed. However, there were some notable differences in reactivity to synthetic substrates for matrix metalloproteinase, and R. tigrinus venom possessed relatively higher activity. Our chemical investigation indicates that the components included in both venoms resemble each other closely. However, the ratio of components and proteolytic activity of some ingredients are slightly different, indicating differences between two closely-related snakes., Competing Interests: The authors declare no conflict of interest.

This study presents an analytical method for the screening of snake venoms for inhibitors of the angiotensin-converting enzyme (ACE) and a strategy for their rapid identification. The method is based on an at-line nanofractionation approach, which combines liquid chromatography (LC), mass spectrometry (MS), and pharmacology in one platform. After initial LC separation of a crude venom, a post-column flow split is introduced enabling parallel MS identification and high-resolution fractionation onto 384-well plates. The plates are subsequently freeze-dried and used in a fluorescence-based ACE activity assay to determine the ability of the nanofractions to inhibit ACE activity. Once the bioactive wells are identified, the parallel MS data reveals the masses corresponding to the activities found. Narrowing down of possible bioactive candidates is provided by comparison of bioactivity profiles after reversed-phase liquid chromatography (RPLC) and after hydrophilic interaction chromatography (HILIC) of a crude venom. Additional nanoLC-MS/MS analysis is performed on the content of the bioactive nanofractions to determine peptide sequences. The method described was optimized, evaluated, and successfully applied for screening of 30 snake venoms for the presence of ACE inhibitors. As a result, two new bioactive peptides were identified: pELWPRPHVPP in Crotalus viridis viridis venom with IC 50  = 1.1 μM and pEWPPWPPRPPIPP in Cerastes cerastes cerastes venom with IC 50  = 3.5 μM. The identified peptides possess a high sequence similarity to other bradykinin-potentiating peptides (BPPs), which are known ACE inhibitors found in snake venoms.

Polycystic kidney diseases (PKDs) are genetic disorders that can cause renal failure and death in children and adults. Lowering cAMP in cystic tissues through the inhibition of the type-2 vasopressin receptor (V2R) constitutes a validated strategy to reduce disease progression. We identified a peptide from green mamba venom that exhibits nanomolar affinity for the V2R without any activity on 155 other G-protein-coupled receptors or on 15 ionic channels. Mambaquaretin-1 is a full antagonist of the V2R activation pathways studied: cAMP production, beta-arrestin interaction, and MAP kinase activity. This peptide adopts the Kunitz fold known to mostly act on potassium channels and serine proteases. Mambaquaretin-1 interacts selectively with the V2R through its first loop, in the same manner that aprotinin inhibits trypsin. Injected in mice, mambaquaretin-1 increases in a dose-dependent manner urine outflow with concomitant reduction of urine osmolality, indicating a purely aquaretic effect associated with the in vivo blockade of V2R. CD1-pcy/pcy mice, a juvenile model of PKD, daily treated with 13 [Formula: see text]g of mambaquaretin-1 for 99 d, developed less abundant (by 33%) and smaller (by 47%) cysts than control mice. Neither tachyphylaxis nor apparent toxicity has been noted. Mambaquaretin-1 represents a promising therapeutic agent against PKDs., Competing Interests: The authors declare no conflict of interest.

Mamba venoms contain a multiplicity of three-finger fold aminergic toxins known to interact with various α-adrenergic, muscarinic and dopaminergic receptors with different pharmacological profiles. In order to generate novel functions on this structural scaffold and to avoid the daunting task of producing and screening an overwhelming number of variants generated by a classical protein engineering strategy, we accepted the challenge of resurrecting ancestral proteins, likely to have possessed functional properties. This innovative approach that exploits molecular evolution models to efficiently guide protein engineering, has allowed us to generate a small library of six ancestral toxin (AncTx) variants and associate their pharmacological profiles to key functional substitutions. Among these variants, we identified AncTx1 as the most α 1A -adrenoceptor selective peptide known to date and AncTx5 as the most potent inhibitor of the three α2 adrenoceptor subtypes. Three positions in the ρ-Da1a evolutionary pathway, positions 28, 38 and 43 have been identified as key modulators of the affinities for the α 1 and α 2C adrenoceptor subtypes. Here, we present a first attempt at rational engineering of the aminergic toxins, revealing an epistasis phenomenon.

Venoms can deleteriously affect any physiological system reachable by the bloodstream, including directly interfering with the coagulation cascade. Such coagulopathic toxins may be anticoagulants or procoagulants. Snake venoms are unique in their use of procoagulant toxins for predatory purposes. The boomslang ( Dispholidus typus ) and the twig snakes ( Thelotornis species) are iconic African snakes belonging to the family Colubridae. Both species produce strikingly similar lethal procoagulant pathologies. Despite these similarities, antivenom is only produced for treating bites by D. typus , and the mechanisms of action of both venoms have been understudied. In this study, we investigated the venom of D. typus and T. mossambicanus utilising a range of proteomic and bioactivity approaches, including determining the procoagulant properties of both venoms in relation to the human coagulation pathways. In doing so, we developed a novel procoagulant assay, utilising a Stago STA-R Max analyser, to accurately detect real time clotting in plasma at varying concentrations of venom. This approach was used to assess the clotting capabilities of the two venoms both with and without calcium and phospholipid co-factors. We found that T. mossambicanus produced a significantly stronger coagulation response compared to D. typus . Functional enzyme assays showed that T. mossambicanus also exhibited a higher metalloprotease and phospholipase activity but had a much lower serine protease activity relative to D. typus venom. The neutralising capability of the available boomslang antivenom was also investigated on both species, with it being 11.3 times more effective upon D. typus venom than T. mossambicanus . In addition to being a faster clotting venom, T. mossambicanus was revealed to be a much more complex venom composition than D. typus . This is consistent with patterns seen for other snakes with venom complexity linked to dietary complexity. Consistent with the external morphological differences in head shape between the two species, CT and MRI analyses revealed significant internal structural differences in skull architecture and venom gland anatomy. This study increases our understanding of not only the biodiscovery potential of these medically important species but also increases our knowledge of the pathological relationship between venom and the human coagulation cascade., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Aim: To purify the platelet aggregation inhibitor from Echis multisquamatis snake venom (PAIEM) and characterize its effect on platelet aggregation and HeLa cell proliferation., Methods: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) were used for PAIEM identification. Platelet aggregation in the presence of PAIEM was studied on aggregometer Solar-AP2110. The changes of shape and granularity of platelets in the presence of PAIEM were studied on flow cytometer COULTER EPICS XL, and degranulation of platelets was estimated using spectrofluorimetry. Indirect enzyme-linked immunosorbent assay was used for the determination of target of PAIEM on platelet surface. An assay based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was used to evaluate the effect of PAIEM on the proliferation of HeLa cells in cell culture., Results: The molecular weight of the protein purified from Echis multisquamatis venom was 14.9 kDa. Half-maximal inhibitory concentration (IC50) of PAIEM needed to inhibit adenosine diphosphate (ADP)-induced platelet aggregation was 7 μM. PAIEM did not affect thrombin- or ADP-induced platelet activation, but it did prevent binding of the anti-IIb antibody to glycoprotein IIb/IIIa (GPIIbIIIa)-receptor of adhered platelets and inhibited the viability of HeLa cells by 54%., Conclusion: As a member of the disintegrin family, PAIEM inhibited platelet aggregation and cell proliferation possibly by blocking integrin-mediated interactions. However, it did not impair cellular signaling causing any changes in platelet shape and granularity and did not affect ADP-induced platelet degranulation. This disintegrin was shown to be a potent inhibitor of integrin-mediated cellular interactions including platelet aggregation or cancer cell proliferation.

Background: Determining the expression levels of neuroglial antigen 2 (NG2) in glioma cell lines and to evaluate the potential contribution of NG2 to cilengitide response were aimed., Materials and Methods: Endogenous expression level of NG2 was determined using quantitative reverse transcription polymerase chain reaction and immunoblotting. Cilengitide responses of the cells were monitored to determine half maximal inhibitory concentration values. Whether the suppression of NG2 expression alters the response of A172 cells to cilengitide was examined., Results: The effect of cilengitide on inducing apoptosis of the cells was determined by TUNEL staining. High mRNA and protein expression of NG2 was detected in A172 and U-87MG cells, while T98G, M059K and M059J cells demonstrated low levels of NG2. A172, U-87MG and positive control MG-63 were relatively sensitive to cilengitide compared to T98G, M059K and M059J. MG-63, A172 and U-87MG were unexpectedly found to be more susceptible to cilengitide. In addition, NG2 knock-down showed no significant difference in cell death between small interfering RNA (siRNA)-transfected and cilengitide-treated groups. The results showed that cilengitide caused detachment and subsequently initiated apoptosis. Glioma cell lines express variable levels of NG2 and differ in their responses to cilengitide. Although increased numbers of apoptotic cells were found in untransfected cells compared to siRNA-transfected cells upon exposed to cilengitide, the difference was not documented to be significant between two groups., Conclusion: It may be proposed that the combination therapy of NG2 suppression and cilengitide treatment showed no considerable effect on glioblastoma compared to cilengitide therapy alone. Response to therapy may be further improved by targeting other factors act in concert in this signaling pathway.

VEGF and VEGFR antibodies have been used as a therapeutic strategy to inhibit angiogenesis in many diseases; however, frequent and repeated administration of these antibodies to patients induces immunogenicity. In previous studies, we demonstrated that aggretin, a heterodimeric snake venom C-type lectin, exhibits pro-angiogenic activities via integrin α2β1 ligation. We hypothesised that small-mass aggretin fragments may bind integrin α2β1 and act as antagonists of angiogenesis. In this study, the anti-angiogenic efficacy of a synthesised aggretin α-chain C-terminus (AACT, residue 106-136) was evaluated in both in vitro and in vivo angiogenesis models. The AACT demonstrated inhibitory effects on collagen-induced platelet aggregation and HUVEC adhesion to immobilised collagen. These results indicated that AACT may block integrin α2β1-collagen interaction. AACT also inhibited HUVEC migration and tube formation. Aortic ring sprouting and Matrigel implant models demonstrated that AACT markedly inhibited VEGF-induced neovascularisation. In addition, induction of FAK/PI3K/ERK1/2 tyrosine phosphorylation and talin 1/2 associated with integrin β1 which are induced by VEGF were blocked by AACT. Similarly, tyrosine phosphorylation of VEFGR2 and ERK1/2 induced by VEGF was diminished in integrin α2-silenced endothelial cells. Our results demonstrate that AACT is a potential therapeutic candidate for angiogenesis related-diseases via integrin α2β1 blockade.

L-amino acid oxidases from snake venoms have been described to possess various biological functions. In this study, we investigated the inflammatory responses induced in vivo and in vitro by CR-LAAO, an L-amino acid oxidase isolated from Calloselasma rhodostoma venom, and its antitumor potential. CR-LAAO induced acute inflammatory responses in vivo, with recruitment of neutrophils and release of IL-6, IL-1β, LTB 4 and PGE 2 . In vitro, IL-6 and IL-1β production by peritoneal macrophages stimulated with CR-LAAO was dependent of the activation of the Toll-like receptors TLR2 and TLR4. In addition, CR-LAAO promoted apoptosis of HL-60 and HepG2 tumor cells mediated by the release of hydrogen peroxide and activation of immune cells, resulting in oxidative stress and production of IL-6 and IL-1β that triggered a series of events, such as activation of caspase 8, 9 and 3, and the expression of the pro-apoptotic gene BAX. We also observed that CR-LAAO modulated the cell cycle of these tumor cells, promoting delay in the G0/G1 and S phases. Taken together, our results suggest that CR-LAAO could serve as a potential tool for the development of novel immunotherapeutic strategies against cancer, since this toxin promoted apoptosis of tumor cells and also activated immune cells against them.

Background/aim: To analyze ex vivo effects of combined targeting of the epidermal growth factor-receptor (EGFR) by cetuximab (E) plus αVβ3 and αVβ5 integrins by cilengitide (Cil) on colony formation of epithelial cells (CF ec ) and release of pro-angiogenetic and pro-inflammatory cytokines in head and neck squamous cell carcinoma (HNSCC) cells., Materials and Methods: Collagenase-IV digests of 43 histopathological confirmed HNSCC cases were seeded into laminin-coated 96-well plates containing E, Cil, or Cil+E in final concentrations of 66.7 μg/ml, 10 μM, and 10 μM+66.7 μg/ml, respectively. Following the FLAVINO-assay protocol, supernatants were harvested after 3 days and adherent cells fixed in ethanol. Counting of CF ec was facilitated by FITC-labeled pan-cytokeratin antibodies. Out of 43 HNSCC cases, 39 had adherent growth (mean CF ec ≥4/well in triplicate controls). Cytokines in supernatants were measured using ELISA were interleukin 6 (IL-6), monocyte chemoattractant protein 1 (MCP-1) and vascular endothelial growth factor A (VEGFA)., Results: CF ec on laminin was significantly reduced by Cil, E, and Cil+E. Cytokine measurements also revealed significant suppression of MCP-1, IL-6 and VEGFA. The strongest suppression of CF ec , MCP-1 and VEGFA release was exerted by Cil and E combined. Efficacy of Cil+E exceeded those of the solely applied pharmaceutics but failed regarding significant synergism of both treatments as E was unable to significantly boost the effects of Cil. In contrast, IL-6 release was significantly suppressed by E but not by Cil, while their combination strongly reduced it., Conclusion: Combined targeting of EGFR and integrins with E and Cil heightens their suppressive effects regarding CF ec as well as release of pro-angiogenetic and pro-inflammatory cytokines., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)