Bioprospection of rattlesnake venom peptide fractions with anti-adipose and anti-insulin resistance activity in vitro.

Animal venoms are natural products that have served as a source of novel molecules that have inspired novel drugs for several diseases, including for metabolic diseases such as type-2 diabetes and obesity. From venoms, toxins such as exendin-4 (Heloderma suspectum) and crotamine (Crotalus durissus terrificus) have demonstrated their potential as treatments for obesity. Moreover, other toxins such as Phospholipases A2 and Disintegrins have shown their potential to modulate insulin secretion in vitro. This suggests an unexplored diversity of venom peptides with a potential anti-obesogenic in Mexican rattlesnake venoms. For that reason, this study explored the in vitro effect of Crotalus venom peptide-rich fractions on models for insulin resistance, adipocyte lipid accumulation, antioxidant activity, and inflammation process through nitric oxide production inhibition. Our results demonstrated that the peptide-rich fractions of C. aquilus, C. ravus, and C. scutulatus scutulatus were capable of reverting insulin resistance, enhancing glucose consumption to normal control; C. culminatus, C. molossus oaxacus, and C. polystictus diminished the lipid accumulation on adipocytes by 20%; C. aquilus, C. ravus, and C. s. salvini had the most significant cellular antioxidant activity, having nearly 80% of ROS inhibition. C. aquilus, C. pyrrhus, and C. s. salvini inhibited nitric oxide production by about 85%. We demonstrated the potential of these peptides from Crotalus venoms to develop novel drugs to treat type-2 diabetes and obesity. Moreover, we described for the first time that Crotalus venom peptide fractions have antioxidant and inflammatory properties in vitro models.

Crotamine derived from Crotalus durissus terrificus venom combined with drugs increases in vitro antibacterial and antifungal activities.

This study investigated crotamine (CTA), a peptide derived from the venom of the South American rattlesnake Crotalus durissus terrificus, known for its exceptional cell penetration potential. The objective was to explore the antibacterial and antifungal activity of CTA, its ability to inhibit efflux pumps and evaluate the effectiveness of its pharmacological combination with antibiotics and antifungals. In microbiological assays, CTA in combination with antibiotics was tested against strains of S. aureus and the inhibition of NorA, Tet(K) and MepA efflux pumps was also evaluated. CTA alone did not present clinically relevant direct antibacterial action, presenting MIC > 209.7 µM against strains S. aureus 1199B, IS-58, K2068. The standard efflux pump inhibitor CCCP showed significant effects in all negative relationships to assay reproducibility. Against the S. aureus 1199B strain, CTA (20.5 µM) associated with norfloxacin diluted 10 × (320.67 µM) showed a potentiating effect, in relation to the control. Against the S. aureus IS-58 strain, the CTA associated with tetracycline did not show a significant combinatorial effect, either with 2304 or 230.4 µM tetracycline. CTA at a concentration of 2.05 µM associated with ciprofloxacin at a concentration of 309.4 µM showed a significant potentiating effect. In association with EtBr, CTA at concentrations of 2.05 and 20.5 µM potentiated the effect in all strains tested, reducing the prevention of NorA, Tet(K) and MepA efflux pumps. In the C. albicans strain, a potentiating effect of fluconazole (334.3 µM) was observed when combined with CTA (2.05 µM). Against the C. tropicalis strain, a significant effect was also observed in the association of fluconazole 334.3 µM, where CTA 2.05 µM considerably reduced fungal growth and decreased the potentiation of fluconazole. Against the C. krusei strain, no significant potentiating effect of fluconazole was obtained by CTA. Our results indicate that CTA in pharmacological combination potentiates the effects of antibiotics and antifungal. This represents a new and promising antimicrobial strategy for treating a wide variety of infections.

Insights into the mechanism of crotamine and potential targets involved in obesity-related metabolic pathways.

Crotamine (Ctm) is a peptide isolated from Crotalus durissus terrificus venom. This molecule has been demonstrated to diminish body weight gain and enhance browning in adipose tissue, glucose tolerance, and insulin sensitivity; hence, it has been postulated as an anti-obesogenic peptide. However, the mechanism to elicit the anti-obesogenic effects has yet to be elucidated. Thus, we investigated the possible interaction of Ctm with receptors involved in obesity-related metabolic pathways through protein-protein docking and molecular dynamics refinement. To test the anti-obesogenic mechanism of Ctm, we selected and retrieved 18 targets involved in obesity-related drug discovery from Protein Data Bank. Then, we performed protein-protein dockings. The best three Ctm-target models were selected and refined by molecular dynamics simulations. Molecular docking demonstrated that Ctm was able to interact with 13 of the 18 targets tested. Having a better docking score with glucagon-like peptide-1 receptor (GLP-1R) (-1430.2 kcal/mol), DPP-IV (dipeptidyl peptidase-IV) (-1781.7 kcal/mol) and α-glucosidase (-1232.3 kcal/mol). These three models were refined by molecular dynamics. Ctm demonstrated a higher affinity for GLP-1R (ΔG: -41.886 ± 2.289 kcal/mol). However, Ctm interaction was more stable with DPP-IV (RMSD: 0.360 ± 0.015 nm, Radius of gyration: 2.781 ± 0.009 nm). Moreover, the number of interactions and the molecular mechanics energies of Ctm residues suggest that the interaction of Ctm with these receptors is mainly mediated by basic-hydrophobic dyads Y1-K2, W31-R32, and W33-R34. Together, all these results allow elucidating a possible molecular mechanism behind the previously described anti-obesogenic effects.

Venom of the neotropical rattlesnake, Crotalus culminatus: Intraspecific variation, neutralization by antivenoms, and immunogenicity in rabbits.

Crotalus culminatus is a medically significant species of rattlesnake in Mexico [1]. While the proteomic composition of its venom has been previously reported for both juvenile and adult specimens, there has been limited research into its functional properties, with only a few studies, including one focusing on coagulotoxicity mechanisms. In this study, we aimed to compare the biochemical and biological activities of the venom of juvenile and adult snakes. Additionally, we assessed antibody production using the venoms of juveniles and adults as immunogens in rabbits. Our findings reveal lethality and proteolytic activity differences between the venoms of juveniles and adults. Notably, juvenile venoms exhibited high proportions of crotamine, while adult venoms displayed a reduction of this component. A commercially available antivenom demonstrated effective neutralization of lethality of both juvenile and adult venoms in mice. However, it failed to neutralize the paralytic activity induced by crotamine, which, in contrast, was successfully inhibited by antibodies obtained from hyperimmunized rabbits. These results suggest the potential inclusion of C. culminatus venom from juveniles in commercial antivenom immunization schemes to generate antibodies targeting this small myotoxin.

Evaluation of tumor growth remission in a murine model for subcutaneous solid tumors, benefits of associating the antitumor agent crotamine with mesoporous nanosilica particles to achieve improved dosing frequency and efficacy

Crotamine is a highly cationic polypeptide first isolated from South American rattlesnake venom, which exhibits affinity for acidic lysosomal vesicles and proliferating cells. This cationic nature is pivotal for its in vitro cytotoxicity and in vivo anticancer actions. This study aimed to enhance the antitumor efficacy of crotamine by associating it with the mesoporous SBA-15 silica, known for its controlled release of various chemical agents, including large proteins. This association aimed to mitigate the toxic effects while amplifying the pharmacological potency of several compounds. Comprehensive characterization, including transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potential analysis, confirmed the successful association of crotamine with the non-toxic SBA-15 nanoparticles. The TEM imaging revealed nanoparticles with a nearly spherical shape and variations in uniformity upon crotamine association. Furthermore, DLS showed a narrow unimodal size distribution, emphasizing the formation of small aggregates. Zeta potential measurements indicated a distinct shift from negative to positive values upon crotamine association, underscoring its effective adsorption onto SBA-15. Intraperitoneal or oral administration of crotamine:SBA-15 in a murine melanoma model suggested the potential to reduce the frequency of crotamine doses without compromising efficacy. Interestingly, while the oral route enhanced the antitumor efficacy of crotamine, pH-dependent release from SBA-15 was observed. Thus, associating crotamine with SBA-15 could reduce the overall required dose to inhibit solid tumor growth, bolstering the prospect of crotamine as a potent anticancer agent.

Evaluation of tumor growth remission in a murine model for subcutaneous solid tumors, benefits of associating the antitumor agent crotamine with mesoporous nanosilica particles to achieve improved dosing frequency and efficacy

Crotamine is a highly cationic polypeptide first isolated from South American rattlesnake venom, which exhibits affinity for acidic lysosomal vesicles and proliferating cells. This cationic nature is pivotal for its in vitro cytotoxicity and in vivo anticancer actions. This study aimed to enhance the antitumor efficacy of crotamine by associating it with the mesoporous SBA-15 silica, known for its controlled release of various chemical agents, including large proteins. This association aimed to mitigate the toxic effects while amplifying the pharmacological potency of several compounds. Comprehensive characterization, including transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potential analysis, confirmed the successful association of crotamine with the non-toxic SBA-15 nanoparticles. The TEM imaging revealed nanoparticles with a nearly spherical shape and variations in uniformity upon crotamine association. Furthermore, DLS showed a narrow unimodal size distribution, emphasizing the formation of small aggregates. Zeta potential measurements indicated a distinct shift from negative to positive values upon crotamine association, underscoring its effective adsorption onto SBA-15. Intraperitoneal or oral administration of crotamine:SBA-15 in a murine melanoma model suggested the potential to reduce the frequency of crotamine doses without compromising efficacy. Interestingly, while the oral route enhanced the antitumor efficacy of crotamine, pH-dependent release from SBA-15 was observed. Thus, associating crotamine with SBA-15 could reduce the overall required dose to inhibit solid tumor growth, bolstering the prospect of crotamine as a potent anticancer agent.

Biological and Medical Aspects Related to South American Rattlesnake Crotalus durissus (Linnaeus, 1758): A View from Colombia.

In Colombia, South America, there is a subspecies of the South American rattlesnake Crotalus durissus, C. d. cumanensis, a snake of the Viperidae family, whose presence has been reduced due to the destruction of its habitat. It is an enigmatic snake from the group of pit vipers, venomous, with large articulated front fangs, special designs on its body, and a characteristic rattle on its tail. Unlike in Brazil, the occurrence of human envenomation by C. durisus in Colombia is very rare and contributes to less than 1% of envenomation caused by snakes. Its venom is a complex cocktail of proteins with different biological effects, which evolved with the purpose of paralyzing the prey, killing it, and starting its digestive process, as well as having defense functions. When its venom is injected into humans as the result of a bite, the victim presents with both local tissue damage and with systemic involvement, including a diverse degree of neurotoxic, myotoxic, nephrotoxic, and coagulopathic effects, among others. Its biological effects are being studied for use in human health, including the possible development of analgesic, muscle relaxant, anti-inflammatory, immunosuppressive, anti-infection, and antineoplastic drugs. Several groups of researchers in Brazil are very active in their contributions in this regard. In this work, a review is made of the most relevant biological and medical aspects related to the South American rattlesnake and of what may be of importance for a better understanding of the snake C. d. cumanensis, present in Colombia and Venezuela.

Antileishmanial activity, cytotoxicity and cellular response of amphotericin B in combination with crotamine derived from Crotalus durissus terrificus venom using in vitro and in silico approaches.

OBJECTIVE: To investigate the in vitro activity, synergism, cytotoxicity and cellular immunological response, as well as the molecular affinity between amphotericin B (AmB) and crotamine (CTA), derived from Crotalus durissus terrificus venom against Leishmania amazonensis. METHODS: This study performed the inhibition of promastigotes and amastigotes' growth under different concentrations of the drug and pharmacological combinations (AmB + CTA) based on the Berimbaum method (synergism study). The lactate dehydrogenase (LDH) quantification method was used to determine the cytotoxicity of the drug and combinations employing four cell lines (J774, HepG2, VERO, and C2C12). Following, the levels of Tumour Necrose Factor-alpha (TNF-α) and Interleukin-12 (IL-12) cytokines, using enzyme-linked immunosorbent assay (ELISA) and nitrites, as an indirect measure of Nitric Oxide (NO), using the Griess reaction were assessed in the supernatants of infected macrophages. In silico approach (molecular docking and dynamics) and binding affinity (surface plasmon resonance) between the drug and toxin were also investigated. RESULTS: CTA enhanced AmB effect against promastigote and amastigote forms of L. amazonensis, decreased the drug toxicity in different cell lines and induced the production of important Th1-like cytokines and NO by infected macrophages. The pharmacological combination also displayed consistent molecular interactions with low energy of coupling and a concentration-dependent profile. CONCLUSION: Our data suggest that this pharmacological approach is a promising alternative treatment against L. amazonensis infection due to the improved activity (synergistic effect) achieved against the parasites' forms and to the decreased cytotoxic effect.

Cytotoxic activity in basal and tumoral cell lines of the C0K3N3 protein from the snake venom Crotalus durissus collilineatus, variety crotamine negative.

Snake venoms are natural sources of bioactive substances with therapeutic potential. In this work, we evaluated the cytotoxicity of the Crotalus durissus collilineatus, negative crotamine variety and the isolated fraction C0K3N3 in BALB C/3T3 and K562 cell lines. The results indicate that the C0K3N3 protein is more cytotoxic against the K562 tumor cell line than in the 3T3 baseline.

South American rattlesnake cationic polypeptide crotamine trafficking dynamic in Plasmodium falciparum-infected erythrocytes: Pharmacological inhibitors, parasite cycle and incubation time influences in uptake.

Malaria is a parasitic infectious disease caused by Plasmodium sp, which was responsible for about 409 thousand deaths only in 2019. The clinical manifestations in patients with malaria, which may include fever and anemia and that can occasionally lead to the death of the host, are mainly associated to the asexual blood stage of parasite. The discovery of novel compounds active against stages of the intraerythrocytic cell cycle has been the focus of many researches seeking for alternatives to the control of malaria. The antimalarial effect of a native cationic polypeptide from the venom of a South American rattlesnake named crotamine, with ability of targeting and disrupting the acidic compartments of Plasmodium falciparum parasite, was previously described by us. Herein, we extended our previous studies by investigating the internalization and trafficking of crotamine in P. falciparum-infected erythrocytes at different blood-stages of parasites and periods of incubation. In addition, the effects of several pharmacological inhibitors in the uptake of this snake polypeptide with cell-penetrating properties were also assessed, showing that crotamine internalization was dependent on ATP generated via glycolytic pathway. We show here that crotamine uptake is blocked by the glycolysis inhibitor 2-deoxy-D-glucose, and the most efficient internalization is observed at trophozoite stage of parasite after at least 30 min of incubation. The present data provide important insights into biochemical pathway and cellular features determined by the parasite cycle, which may be underlying the internalization and effects of cationic antimalarials as crotamine.

A Native CPP from Rattlesnake with Therapeutic and Theranostic Properties.

The cell-penetrating peptides (CPPs) are characterized by the ability of internalization into cells in vitro and in vivo, and the ability of these peptides can rely on a high content of positive charges, as it is the case of the native CPP crotamine. Crotamine is a polypeptide of about 42 amino acid residues with high content of basic residues as Arg and Lys. Although most of known CPPs are linear peptides, native crotamine from the venom of a South American rattlesnake has a well-defined 3D structure stabilized by three disulfide bonds which guarantee the exposure of side chains of basic amino acids. This 3D structure also protects this amphipathic polypeptide from the degradation even if administered by oral route, therefore, protecting also the biological activities of crotamine. As several different biological properties of crotamine are dependent of cell penetration, the methods mainly employed for analyzing crotamine properties as anthelminthic and antimalarial activities, antimicrobial and antitumor activities, with a unique selective cytotoxic property against actively proliferating cells, as tumor cells, were chosen based on crotamine ability of internalization mediated by its positive charge. This native cationic polypeptide is also able to efficiently carry, with no need of covalent linkage with the cargo, genetic material into cells in vitro and in vivo, suggesting its use in gene therapy. Moreover, the possibility of decorating gold nanoparticles keeping the ability of transfecting cells was demonstrated. More recently, the ability of crotamine to interfere in animal metabolism, inducing browning of adipose tissue and increasing the energy expenditure, and its application in renal therapy was demonstrated. As crotamine also accumulates specifically in tumor cells in vivo, and the potential utility of crotamine as a theranostic agent was then suggested. Therefore, diverse methodologies employed for the characterization and exploration of the therapeutic applications of this promising native CPP for remediation of several pathogenic conditions are presented here.

Intraspecific venom variation of Mexican West Coast Rattlesnakes (Crotalus basiliscus) and its implications for antivenom production.

Intraspecific variation in snake venoms has been widely documented worldwide. However, there are few studies on this subject in Mexico. Venom characterization studies provide important data used to predict clinical syndromes, to evaluate the efficacy of antivenoms and, in some cases, to improve immunogenic mixtures in the production of antivenoms. In the present work, we evaluated the intraspecific venom variation of Crotalus basiliscus, a rattlesnake of medical importance and whose venom is used in the immunization of horses to produce one of the Mexican antivenoms. Our results demonstrate that there is variation in biological and biochemical activities among adult venoms and that there is an ontogenetic change from juvenile to adult venoms. Juvenile venoms were more lethal and had higher percentages of crotamine and crotoxin, while adult venoms had higher percentages of snake venom metalloproteases (SVMPs). Additionally, we documented crotoxin-like PLA2 variation in which specimens from Zacatecas, Sinaloa and Michoacán (except 1) lacked the neurotoxin, while the rest of the venoms had it. Finally, we evaluated the efficacy of three lots of Birmex antivenom and all three were able to neutralize the lethality of four representative venoms but were not able to neutralize crotamine. We also observed significant differences in the LD50 values neutralized per vial among the different lots. Based on these results, we recommend including venoms containing crotamine in the production of antivenom for a better immunogenic mixture and to improve the homogeneity of lots.

Evaluation of crotamine based probes as intracellular targeted contrast agents for magnetic resonance imaging

Crotamine is a lysine and cysteine rich 42 amino acids long bio-active polypeptide, isolated from the venom of a South American rattlesnake, that can also be used as cell penetrating peptide. A facile synthetic scheme for coupling cargo molecules like fluorophores (carboxyfluorescein) or MRI probes (Gd-DO3A-based macrocycle) is presented. The toxicity, cellular internalization and steady-state accumulation after long-term incubation for 18 h, as well as magnetic resonance relaxivities and cellular relaxation rates of crotamine based probes were evaluated and compared to its shorter synthetic fragment CyLoP-1. The longitudinal relaxivity (r1) of the conjugates of CyLoP-1 and crotamine is significantly lower in medium than in water indicating to the lower contrast enhancement efficacy of DO3A-based probes in biological samples. Carboxyfluorescein labeled crotamine did not exhibit toxicity up to a concentration of 2.5 µM. CyLoP-1 accumulated about four times better within the cells compared to crotamine. Fluorescence microscopy suggests different predominant uptake mechanisms for crotamine and CyLoP-1 in 3T3 cells. While crotamine is predominantly localized in vesicular structures (most likely endosomes and lysosomes) within the cell, CyLoP-1 is mainly homogeneously distributed in the cytosol. The cellular relaxation rate (R1, cell) of the crotamine based probe was not significantly increased whereas the corresponding CyLoP-1-derivative showed a slightly elevated R1, cell. This study indicates the potential of crotamine and in particular the shorter fragment CyLoP-1 to be useful for an efficient transmembrane delivery of agents directed to intracellular (cytosolic) targets. However, the applicability of the conjugates synthesized here as contrast agents in MR imaging is limited. Further improvement is needed to prepare more efficient probes for MRI applications, i.e., by replacing the DO3A- with a DOTA-based chelate.

Synthetic polypeptide crotamine: characterization as a myotoxin and as a target of combinatorial peptides

Crotamine is a rattlesnake-derived toxin that causes fast-twitch muscle paralysis. As a cell-penetrating polypeptide, crotamine has been investigated as an experimental anti-cancer and immunotherapeutic agent. We hypothesized that molecules targeting crotamine could be designed to study its function and intervene in its adverse activities. Here, we characterize synthetic crotamine and show that, like the venom-purified toxin, it induces hindlimb muscle paralysis by affecting muscle contraction and inhibits KCNA3 (Kv1.3) channels. Synthetic crotamine, labeled with a fluorophore, displayed cell penetration, subcellular myofiber distribution, ability to induce myonecrosis, and bind to DNA and heparin. Here, we used this functionally validated synthetic polypeptide to screen a combinatorial phage display library for crotamine-binding cyclic peptides. Selection for tryptophan-rich peptides was observed, binding of which to crotamine was confirmed by ELISA and gel shift assays. One of the peptides (CVWSFWGMYC), synthesized chemically, was shown to bind both synthetic and natural crotamine and to block crotamine-DNA binding. In summary, our study establishes a functional synthetic substitute to the venom-derived toxin and identifies peptides that could further be developed as probes to target crotamine.

Biological and Biochemical Characterization of Coronado Island Rattlesnake (Crotalus helleri caliginis) Venom and Antivenom Neutralization.

The Baja California Peninsula has over 250 islands and islets with many endemic species. Among them, rattlesnakes are the most numerous but also one of the least studied groups. The study of island rattlesnake venom could guide us to a better understanding of evolutionary processes and the description of novel toxins. Crotalus helleri caliginis venom samples were analyzed to determine possible ontogenetic variation with SDS-PAGE in one and two dimensions and with RP-HPLC. Western Blot, ELISA, and amino-terminal sequencing were used to determine the main components of the venom. The biological and biochemical activities demonstrate the similarity of C. helleri caliginis venom to the continental species C. helleri helleri, with both having low proteolytic and phospholipase A2 (PLA2) activity but differing due to the absence of neurotoxin (crotoxin-like) in the insular species. The main components of the snake venom were metalloproteases, serine proteases, and crotamine, which was the most abundant toxin group (30-35% of full venom). The crotamine was isolated using size-exclusion chromatography where its functional effects were tested on mouse phrenic nerve-hemidiaphragm preparations in which a significant reduction in muscle twitch contractions were observed. The two Mexican antivenoms could neutralize the lethality of C. helleri caliginis venom but not the crotamine effects.

Acute toxicity, antinociceptive, and anti-inflammatory activities of the orally administered crotamine in mice.

Crotamine is a polypeptide toxin isolated from rattlesnake venom. Although several studies have been developed identifying many biological effects of isolated crotamine, none of them evaluated its acute toxicity, antinociceptive, and anti-inflammatory activities through oral administration. All in vivo experiments from this study were performed in mice. The up-and-down procedure and hippocratic screening were carried out to evaluate possible pharmacological and toxic effects. Antinociceptive and anti-inflammatory activities of this toxin were evaluated using acetic acid-induced abdominal writhing, formalin-induced pain assays, croton oil-induced ear edema, and carrageenan-induced pleurisy. Crotamine did not cause lethality or signs of intoxication up to the maximum dose tested (10.88 mg/kg). The number of contortions was reduced significantly by 34, 57, and 74% at the oral doses of 0.08, 0.16, and 0.32 mg/kg, respectively. At the dose of 0.16 mg/kg, crotamine decreases pain time-reactivity at neurogenic phase by 45% and at inflammatory phase by 60%. Also, crotamine elicited antiedematogenic activity through the attenuation of the croton oil-induced ear edema by 77%. In the carrageenan-induced pleurisy, the leukocyte, neutrophil, and mononuclear cell migration to the lesion site were reduced by 52%, 46%, and 59%, respectively. Altogether, crotamine demonstrated in vivo antinociceptive and anti-inflammatory effect through acute oral administration, generating an anti-migratory mechanism of action at non-toxic doses.

Neutralization of crotamine by polyclonal antibodies generated against two whole rattlesnake venoms and a novel recombinant fusion protein.

Crotamine is a paralyzing toxin (MW: ~5 kDa) found in different proportions in some rattlesnake venoms (up to 62%). Mexican pit viper antivenoms have shown low immunoreactivity against crotamine, which is an urgent quality to be improved. The objective of this work was to evaluate the ability of a novel recombinant fusion protein composed of sphingomyelinase D and crotamine, and two whole venoms from Crotalus molossus nigrescens and C. oreganus helleri to produce neutralizing antibodies against crotamine. These immunogens were separately used for immunization procedures in rabbits. Then, we generated three experimental antivenoms to test their cross-reactivity via western-blot against crotamine from 7 species (C. m. nigrescens, C. o. helleri, C. durissus terrificus, C. scutulatus salvini, C. basiliscus, C. culminatus and C. tzabcan). We also performed pre-incubation neutralization experiments in mice to measure the neutralizing potency of each antivenom against crotamine induced hind limb paralysis. Our antivenoms showed broad recognition across crotamine from most of the tested species. Also, neutralization against crotamine paralysis symptom was successfully achieved by our three antivenoms, albeit with different efficiencies. Our results highlight the use of crotamine enriched venoms and our novel recombinant fusion protein as promising immunogens to improve the neutralizing potency against crotamine for the improvement of Mexican antivenoms.

Biophysical and pharmacological characterization of a full-length synthetic analog of the antitumor polypeptide crotamine.

Crotamine is a polypeptide isolated from the venom of a South American rattlesnake. Among the properties and biological activities of crotamine, the most extraordinary is its ability to enter cells with unique selective affinity and cytotoxic activity against actively proliferating cells, such as tumor cells. This peptide is also a cargo carrier, and anticipating commercial application of this native polypeptide as a potential theranostic compound against cancer, we performed here a side-by-side characterization of a chemically synthesized full-length crotamine compared with its native counterpart. The structural, biophysical, and pharmacological properties were evaluated. Comparative NMR studies showed structural conservation of synthetic crotamine. Moreover, similarly to native crotamine, the synthetic polypeptide was also capable of inhibiting tumor growth in vivo, increasing the survival of mice bearing subcutaneous tumor. We also confirmed the ability of synthetic crotamine to transfect and transport DNA into eukaryotic cells, in addition to the importance of proteoglycans on cell surface for its internalization. This work opens new opportunities for future evaluation of chimeric and/or point-mutated analogs of this snake polypeptide, aiming for improving crotamine properties and applications, as well as possibly diminishing its potential toxic effects. KEY MESSAGES: • Synthetic crotamine showed ex vivo and in vivo activities similar to native peptide. • Synthetic crotamine structure conservation was demonstrated by NMR analysis. • Synthetic crotamine is able to transfect and transport DNA into eukaryotic cells. • Synthetic crotamine shows tumor growth inhibition in vivo. • Synthetic crotamine increases survival of mice bearing tumor.

A natural cell-penetrating nanopeptide combined with pentavalent antimonial as experimental therapy against cutaneous leishmaniasis.

The inadequacy of available treatments for leishmaniasis has presented up to 40% therapeutic failure. This fact suggests an urgency in the discovery of new drugs or alternative approaches for treating this disease. The objective of this study was to evaluate the antileishmanial activity of combined therapy between crotamine (CTA) from Crotalus durissus terrificus and the pentavalent antimonial Glucantime® (GLU). The assays were in vitro performed measuring the inhibition of Leishmania amazonensis amastigotes, followed by the evaluation of cellular production of cytokines and nitrites. After that, analytical methods were performed in order to characterize the molecules involved in the study by Mass Spectrometry, molecular affinity through an in silico assay and Surface Plasmon Resonance. In vivo experiments with BALB/c mice were performed by analyzing parasitemia, lesion size and immunological mediators. In the in vitro experiments, the pharmacological association improved the inhibition of the amastigotes, modulated the production of cytokines and nitric oxide. The therapy improved the effectiveness of the GLU, demonstrating a decreased parasitemia in the infected tissues. Altogether, the results suggest that the combined approach with CTA and GLU may be a promising alternative for the treatment of cutaneous leishmaniasis.

In vivo effects of the association of the psychoactive phenotiazine thioridazine on antitumor activity and hind limb paralysis induced by the native polypeptide crotamine.

Crotamine is a cationic polypeptide composed by 42 amino acid residues with several pharmacological and biological properties, including the selective ability to enter and kill actively proliferating tumour cells, which led us to propose its use as a theranostic agent for cancer therapy. At the moment, the improvement of crotamine antitumoral efficacy by association with chemotherapeutic adjuvants is envisioned. In the present work, we evaluated the association of crotamine with the antitumoral adjuvant phenotiazine thioridazine (THD). In spite of the clear efficacy of these both compounds as anticancer agents in long-term in vivo treatment of animal model bearing implanted xenograph melanoma tumor, the expected mutual potentiation of the antitumor effects was not observed here. Moreover, this association revealed for the first time the influence of THD on crotamine ability to trigger the hind limb paralysis in mice, and this discovery may represent the first report suggesting the potential involvement of the CNS in the action of this snake polypeptide on the skeletal muscle paralysis, which was classically believed to be essentially limited to a direct action in peripheral tissues as the skeletal muscle. This is also supported by the observed ability of crotamine to potentiate the sedative effects of THD which action was consistently demonstrated to be based on its central action. The better characterization of crotamine properties in CNS may certainly bring important insights for the knowledge needed to pave the way toward the use of this molecule as a theranostic compound in human diseases as cancer.