Antimicrobial and Anticancer Properties of Synthetic Peptides Derived from the Wasp Parachartergus fraternus.

Agelaia-MPI and protonectin are antimicrobial peptides isolated from the wasp Parachartergus fraternus that show antimicrobial and neuroactive activities. Previously, two analogues of these peptides, neuroVAL and protonectin-F, were designed to reduce nonspecific toxicity and improve potency. Here, the three-dimensional structures of neuroVAL, protonectin and protonectin-F were determined by using circular dichroism and NMR spectroscopy. Antibacterial, antifungal, cytotoxic and hemolytic activities were tested for the parent peptides and analogues. All peptides showed moderate antimicrobial activity against Gram-positive bacteria, with agelaia-MPI being the most active. Protonectin and protonectin-F were found to be toxic to cancerous and noncancerous cell lines. Internalization experiments revealed that these peptides accumulate inside both cell types. By contrast, neuroVAL was nontoxic to all tested cells and was able to enter cells without accumulating. In summary, neuroVAL has potential as a nontoxic cell-penetrating peptide, while protonectin-F needs further modification to realize its potential as an antitumor peptide.

Ceftriaxone pretreatment confers neuroprotection in rats with acute glaucoma and reduces the score of seizures induced by pentylenotetrazole in mice.

ß-Lactam antibiotics such as ceftriaxone, are potent stimulators of the expression of l-glutamate transporter GLT-1 and may exert neuroprotective effects when chronically used in rats and mice. In this study, we used two animal models to test the neurological effect of subchronic treatment with ceftriaxone: experimental acute glaucoma in Wistar rats and induction of acute seizures with pentylenetetrazole in mice. We also assessed the performance of mice in the rotarod to calculate therapeutic indexes and exploratory activity in the open field. Our results showed that subchronic use of ceftriaxone was neuroprotective in both models, reducing injury in acute ischemia and ischemia/reperfusion in specific layers of retina and leading to a decrease in the seizure severity score. In behavioral experiments, we observed that ceftriaxone increased hyperactivity followed by a decrease in exploratory behavior in the open field, and there was no motor impairment in the rotarod test. We conclude that ceftriaxone may be useful as a tool in the development of new neuroprotective drugs targeting diseases which present a possible dysfunction in the balance of glutamatergic neurotransmission.

Alzheimer's Disease: Innovative Therapeutic Approaches Based on Peptides and Nanoparticles.

Alzheimer's disease (AD) is the main cause of dementia in the world and its etiology is not yet fully understood. The pathology of AD is primarily characterized by intracellular neurofibrillary tangles and extracellular amyloid-ß plaques. Unfortunately, few treatment options are available, and most treat symptoms, as is the case of acetylcholinesterase inhibitors (IAChE) and N-methyl-d-aspartate receptor antagonists. For more than 20 years pharmaceutical research has targeted the "amyloid cascade hypothesis," but this has not produced meaningful results, leading researchers to focus now on other characteristics of the disease and on multitarget approaches. This review aims to evaluate some new treatments that are being developed and studied. Among these are new treatments based on peptides, which have high selectivity and low toxicity; however, these compounds have a short half-life and encounter challenges when crossing the blood-brain barrier. The present review discusses up-and-coming peptides tested as treatments and explores some nanotechnological strategies to overcome the downsides. These compounds are promising, as they not only act on the symptoms but also aim to prevent progressive neuronal loss.

Exploring the therapeutic potential of an antinociceptive and anti-inflammatory peptide from wasp venom.

Animal venoms are rich sources of neuroactive compounds, including anti-inflammatory, antiepileptic, and antinociceptive molecules. Our study identified a protonectin peptide from the wasp Parachartergus fraternus' venom using mass spectrometry and cDNA library construction. Using this peptide as a template, we designed a new peptide, protonectin-F, which exhibited higher antinociceptive activity and less motor impairment compared to protonectin. In drug interaction experiments with naloxone and AM251, Protonectin-F's activity was decreased by opioid and cannabinoid antagonism, two critical antinociception pathways. Further experiments revealed that this effect is most likely not induced by direct action on receptors but by activation of the descending pain control pathway. We noted that protonectin-F induced less tolerance in mice after repeated administration than morphine. Protonectin-F was also able to decrease TNF-α production in vitro and modulate the inflammatory response, which can further contribute to its antinociceptive activity. These findings suggest that protonectin-F may be a potential molecule for developing drugs to treat pain disorders with fewer adverse effects. Our results reinforce the biotechnological importance of animal venom for developing new molecules of clinical interest.

Oral delivery of fish oil in oil-in-water nanoemulsion: development, colloidal stability and modulatory effect on in vivo inflammatory induction in mice.

To improve the oral absorption of fish oil and test its anti-inflammatory effect, a fish oil nanoemulsion was developed using cis-4,7,10,13,16,19-docosahexaenoic fatty acid as a biomarker for oral administration. The colloidal stability tests of the fish oil nanoemulsion showed an average size of 155.44 nm ±â€¯6.46 (4 °C); 163.04 nm ±â€¯9.97 (25 °C) and polydispersity index 0.22 ±â€¯0.02 (4 °C), 0.21 ±â€¯0.02 (25 °C), indicating systems with low polydispersity and stable droplets. The fish oil nanoemulsion did not alter the cell viability of the RAW 264.7 macrophages and, at a concentration of 0.024 mg/mL, was kinetically incorporated into the cells after 18 h of contact. The nanoemulsion was maintained in the gastrointestinal region for a significantly shorter period of time (p ≤ 0.05) compared to the intake of fish oil in free form. Inflammatory tests demonstrated that nanoemulsion and fish oil showed less (p ≤ 0.05) neutrophil infiltration after 24h of sepsis induction and there was a significant reduction (p ≤ 0.05) in the volume of paw edema in female adult Balb/c mice who received the nanoemulsion diet compared to the other experimental groups (control, formalin, fish oil and sunflower oil). These results indicate that the fish oil nanoemulsion was significantly effective in the dietary conditions tested here, presenting satisfactory responses in the modulation of inflammatory disorders, demonstrating interesting and beneficial nutraceutical effects.

New insights in the mode of action of (+)-erythravine and (+)-11α-hydroxy-erythravine alkaloids.

Erythrinian alkaloids ((+)-erythravine and (+)-11-α-hydroxy-erythravine) have been pointed as the main responsible agents for the anticonvulsant and anxiolytic properties of Erythrina mulungu Mart ex Benth. The present work provides a new set of information about the mode of action of these alkaloids by the use of a complementary approach of neurochemical and electrophysiological assays. We propose here that the antiepileptic and anxiolytic properties exhibited by both alkaloids appear not to be related to the inhibition of glutamate binding or GABA uptake, or even to the increase of glutamate uptake or GABA binding, as investigated here by the use of rat cortical synaptosomes. Similarly, and even in a high concentration, (+)-erythravine and (+)-11-α-hydroxy-erythravine did not modulate the main sodium and potassium channel isoforms checked by the use of voltage-clamp studies on Xenopus laevis oocytes. However, unlike (+)-11-α-hydroxy-erythravine, which presented a little effect, it was possible to observe that the (+)-erythravine alkaloid produced a significant inhibitory modulation on α4ß2, α4ß4 and α7 isoforms of nicotinic acetylcholine receptors also checked by the use of voltage-clamp studies, which could explain at least partially its anxiolytic and anticonvulsant properties. Since (+)-11-α-hydroxy-erythravine and (+)-erythravine modulated nicotinic acetylcholine receptors to different extents, it is possible to reinforce that small differences between the chemical structure of these alkaloids can affect the selectivity and affinity of target-ligand interactions, conferring distinct potency and/or pharmacological properties to them, as previously suggested by differential experimental comparison between different erythrinian alkaloids.

Animal venoms: therapeutic tools for tackling Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative pathology of the central nervous system, mainly involving the selective and progressive loss of dopaminergic neurons from the substantia nigra, resulting in motor and non-motor symptoms. PD remains an incurable ailment; thus, treatments are limited to symptom alleviation. With long-term use, conventional treatments can become inefficient, often triggering possible side effects. Considering these drawbacks, drug discovery constantly turns to nature as a source of efficient therapeutics. Thus, this review explores animal venoms as a rich source of bioactive compounds with potent neuropharmacological profiles for the development of effective adjuvant treatments with fewer side effects, ultimately aiming for the neuroprotection of dopaminergic neurons and the symptomatic relief of PD.

Antimicrobial and Chemotactic Activity of Scorpion-Derived Peptide, ToAP2, against Mycobacterium massiliensis.

Mycobacterium massiliense is a rapid growing, multidrug-resistant, non-tuberculous mycobacteria that is responsible for a wide spectrum of skin and soft tissue infections, as well as other organs, such as the lungs. Antimicrobial peptides had been described as broad-spectrum antimicrobial, chemotactic, and immunomodulator molecules. In this study we evaluated an antimicrobial peptide derived from scorpion Tityus obscurus as an anti-mycobacterial agent in vitro and in vivo. Bioinformatics analyses demonstrated that the peptide ToAP2 have a conserved region similar to several membrane proteins, as well as mouse cathelicidin. ToAP2 inhibited the growth of four M. massiliense strains (GO01, GO06, GO08, and CRM0020) at a minimal bactericidal concentration (MBC) of 200 µM. MBC concentration used to treat infected macrophages was able to inhibit 50% of the bacterial growth of all strains. ToAP2 treatment of infected mice with bacilli reduced the bacterial load in the liver, lung, and spleen, similarly to clarithromycin levels (90%). ToAP2 alone recruited monocytes (F4/80low Gr1), neutrophils (F4/80- Gr1), and eosinophils (F4/80+ Gr1+). ToAP2, together with M. massiliense infection, was able to increase F4/80low and reduce the percentage of F4/80high macrophages when compared with infected and untreated mice. ToAP2 has in vitro anti-microbial activity that is improved in vivo due to chemotactic activity.

Label-free quantitative proteomics of rat hypothalamus under fever induced by LPS and PGE2.

Fever is a brain-mediated increase in body temperature mainly during inflammatory or infectious challenges. Although there is considerable data regarding the inflammation pathways involved in fever, metabolic alterations necessary to orchestrate the complex inflammatory response are not totally understood. We performed proteomic analysis of rat hypothalamus using label-free LC-MS/MS in a model of fever induced by lipopolysaccharide (LPS) or prostaglandin E2 (PGE2). In total, 7021 proteins were identified. As far as we know, this is the largest rat hypothalamus proteome dataset available to date. Pathway analysis showed proteins from both stimuli associated with inflammatory and metabolic pathways. Concerning metabolic pathways, rats exposed to LPS or PGE2 presented lower relative abundance of proteins involved in glycolysis, pentose phosphate pathway and tricarboxylic acid cycle. Mitochondrial function may also be altered by both stimuli because significant downregulation of several proteins was found, mainly in complexes I and IV. LPS was able to induce downregulation of important proteins in the enzymatic antioxidant system, thereby contributing to oxidative stress. The results offered comprehensive information about fever responses and helped to reveal new insights into proteins potentially involved in inflammatory signaling and metabolic changes in the hypothalamus during systemic LPS and central PGE2 administration. SIGNIFICANCE: The evolutionary persistence of fever, despite the elevated cost for maintenance of this response, suggests that elevation in core temperature may represent an interesting strategy for survival. Fever response is achieved through the integrated behavioral, physiological, immunological and biochemical processes that determine the balance between heat generation and elimination. The development of such complex response arouses interest in studying how the cell metabolism responds or even contributes to promote fever. Our results offered comprehensive information about fever responses, including metabolic and inflammatory pathways, providing new insights into candidate proteins potentially involved in inflammatory signaling and metabolic changes in the hypothalamus during fever induced by systemic LPS and central PGE2 perturbation.

Non-disulfide-Bridge Peptide 5.5 from the Scorpion Hadrurus gertschi Inhibits the Growth of Mycobacterium abscessus subsp. massiliense.

Multi-drug resistant microorganisms have been a growing concern during the last decades due to their contribution in mortality rates worldwide. Antimicrobial peptides (AMPs) are broad spectrum antimicrobial agents that display potent microbicidal activity against a wide range of microorganisms. AMPs generally have a rapid mode of action that reduces the risk of resistance developing among pathogens. In this study, an AMP derived from scorpion venom, NDBP-5.5, was evaluated against Mycobacterium abscessus subsp. massiliense, a rapidly growing and emerging pathogen associated with healthcare infections. The minimal bactericidal concentration of NDBP-5.5, AMP quantity necessary to stop bacteria visible growth, against M. abscessus subsp. massiliense was 200 µM, a concentration that did not induce hemolysis of human red blood cells. The therapeutic index was 3.05 indicating a drug with low toxicity and therefore good clinical potential. Treatment of infected macrophages with NDBP-5.5 or clarithromycin presented similar results, reducing the bacterial load. M. abscessus subsp. massiliense-infected animals showed a decrease in the bacterial load of up to 70% when treated with NDBP-5.5. These results revealed the effective microbicidal activity of NDBP-5.5 against Mycobacterium, indicating its potential as an antimycobacterial agent.

Evaluation of the antimicrobial activity of the mastoparan Polybia-MPII isolated from venom of the social wasp Pseudopolybia vespiceps testacea (Vespidae, Hymenoptera).

Mastoparans, a class of peptides found in wasp venom, have significant effects following a sting as well as useful applications in clinical practice. Among these is their potential use in the control of micro-organisms that cause infectious diseases with a significant impact on society. Thus, the present study describes the isolation and identification of a mastoparan peptide from the venom of the social wasp Pseudopolybia vespiceps and evaluated its antimicrobial profile against bacteria (Staphylococcus aureus and Mycobacterium abscessus subsp. massiliense), fungi (Candida albicans and Cryptococcus neoformans) and in vivo S. aureus infection. The membrane pore-forming ability was also assessed. The mastoparan reduced in vitro and ex vivo mycobacterial growth by 80% at 12.5 µM in infected peritoneal macrophages but did not affect the shape of bacterial cells at the dose tested (6.25 µM). The peptide also showed potent action against S. aureus in vitro (EC50 and EC90 values of 1.83 µM and 2.90 µM, respectively) and reduced the in vivo bacterial load after 6 days of topical treatment (5 mg/kg). Antifungal activity was significant, with EC50 and EC90 values of 12.9 µM and 15.3 µM, respectively, for C. albicans, and 11 µM and 22.70 µM, respectively, for C. neoformans. Peptides are currently attracting interest for their potential in the design of antimicrobial drugs, particularly due to the difficulty of micro-organisms in developing resistance to them. In this respect, Polybia-MPII proved to be highly effective, with a lower haemolysis rate compared with peptides of the same family.

Anxiolytic-like effect of a novel peptide isolated from the venom of the social wasp Synoeca surinama.

Pathological anxiety is among the most common psychiatric illnesses, but current treatment is highly limited. In this study, we investigated the potential anxiolytic-like effects of a peptide isolated from Synoeca surinama venom. Rats treated with this peptide spent more time exploring the open arms of elevated plus maze, which indicates an anxiolytic-like profile for this peptide. This study is the first to show the pharmacological use of S. surinama venom in the treatment of anxiety.

Activity of Scorpion Venom-Derived Antifungal Peptides against Planktonic Cells of Candida spp. and Cryptococcus neoformans and Candida albicans Biofilms.

The incidence of fungal infections has been increasing in the last decades, while the number of available antifungal classes remains the same. The natural and acquired resistance of some fungal species to available therapies, associated with the high toxicity of these drugs on the present scenario and makes an imperative of the search for new, more efficient and less toxic therapeutic choices. Antimicrobial peptides (AMPs) are a potential class of antimicrobial drugs consisting of evolutionarily conserved multifunctional molecules with both microbicidal and immunomodulatory properties being part of the innate immune response of diverse organisms. In this study, we evaluated 11 scorpion-venom derived non-disulfide-bridged peptides against Cryptococcus neoformans and Candida spp., which are important human pathogens. Seven of them, including two novel molecules, showed activity against both genera with minimum inhibitory concentration values ranging from 3.12 to 200 µM and an analogous activity against Candida albicans biofilms. Most of the peptides presented low hemolytic and cytotoxic activity against mammalian cells. Modifications in the primary peptide sequence, as revealed by in silico and circular dichroism analyses of the most promising peptides, underscored the importance of cationicity for their antimicrobial activity as well as the amphipathicity of these molecules and their tendency to form alpha helices. This is the first report of scorpion-derived AMPs against C. neoformans and our results underline the potential of scorpion venom as a source of antimicrobials. Further characterization of their mechanism of action, followed by molecular optimization to decrease their cytotoxicity and increase antimicrobial activity, is needed to fully clarify their real potential as antifungals.

Acute and subchronic toxicity of the antitumor agent rhodium (II) citrate in Balb/c mice after intraperitoneal administration.

This study aimed to investigate potential acute and subchronic toxicity of rhodium (II) citrate in female Balb/c mice after intraperitoneal injections. In the acute test, independent groups received five doses; the highest dose (107.5 mg/kg) was equivalent to 33 times that used in our previous reports. The other doses were chosen as proportions of the highest, being 80.7 (75%), 53.8 (50%), 26.9 (25%) or 13.8 mg/kg (12.5%). Animals were monitored over 38 days and no severe signs of toxicity were observed, according to mortality, monitoring of adverse symptoms, hematological, biochemical and genotoxic parameters. We conclude that the median lethal dose (LD50) could be greater than 107.5 mg/kg. In the subchronic test, five doses of Rh2Cit (80, 60, 40, 20 or 10 mg/kg) were evaluated and injections were conducted on alternate days, totaling five applications per animal. Paclitaxel (57.5 mg/kg) and saline solution were controls. Clinical observations, histopathology of liver, lung and kidneys and effects on hematological, biochemistry and genotoxic records indicated that Rh2Cit induced no severe toxic effects, even at an accumulated dose up to 400 mg/kg.We suggest Rh2Cit has great potential as an antitumor drug without presenting acute and subchronic toxicity.

Parawixin2, a novel non-selective GABA uptake inhibitor from Parawixia bistriata spider venom, inhibits pentylenetetrazole-induced chemical kindling in rats.

The aims of the present work were to investigate the effects of the repeated administration of Parawixin2 (2-amino-5-ureidopentanamide; formerly FrPbAII), a novel GABA and glycine uptake inhibitor, in rats submitted to PTZ-induced kindling. Wistar rats were randomly divided in groups (n=6-8) for different treatments. Systemic injections of PTZ were administered every 48 h in the dose of 33 mg/kg; i.p., that is sufficient to induce fully kindled seizures in saline i.c.v. treated rats in a short period of time (28 days). Treatments in two types of positive controls (diazepam - DZP and nipecotic acid - NA groups) consisted in daily systemic injections of DZP (2mg/kg; i.p.) or i.c.v. injections of NA (12 µg/µL), while in experimental groups in daily i.c.v. injections of different doses of Parawixin2 (0.15; 0.075; 0.015 µg/µL). Seizures were analyzed using the Lamberty & Klitgaard score and kindling was considered as established after at least three consecutive seizures of score 4 or 5. Cumulative seizure scores for each group were analyzed using repeated measures of ANOVA followed by Tukey test. PTZ induced 4 and 5-score seizures after 12 injections in saline treated rats, whereas daily injection of Parawixin2 inhibited the onset of seizures in a dose dependent manner. Also, the challenging administration of PTZ did not raise seizure score in animals treated with the highest dose of Parawixin2 or those treated with DZP or NA. These findings together with previous data from our laboratory show that Parawixin2 could be a useful probe to design new antiepileptic drugs.

Venomic and pharmacological activity of Acanthoscurria paulensis (Theraphosidae) spider venom.

In the present study we conducted proteomic and pharmacological characterizations of the venom extracted from the Brazilian tarantula Acanthoscurria paulensis, and evaluated the cardiotoxicity of its two main fractions. The molecular masses of the venom components were identified by mass spectrometry (MALDI-TOF-MS) after chromatographic separation (HPLC). The lethal dose (LD(50)) was determined in mice. Nociceptive behavior was evaluated by intradermal injection in mice and the edematogenic activity by the rat hind-paw assay. Cardiotoxic activity was evaluated on in situ frog heart and on isolated frog ventricle strip. From 60 chromatographic fractions, 97 distinct components were identified, with molecular masses between 601.4 and 21,932.3 Da. A trimodal molecular mass distribution was observed: 30% of the components within 500-1999 Da, 38% within 3500-5999 Da and 21% within 6500-7999 Da. The LD(50) in mice was 25.4 ± 2.4 µg/g and the effects observed were hypoactivity, anuria, constipation, dyspnea and prostration until death, which occurred at higher doses. Despite presenting a dose-dependent edematogenic activity in the rat hind-paw assay, the venom had no nociceptive activity in mice. Additionally, the venom induced a rapid blockage of electrical activity and subsequent diastolic arrest on in situ frog heart preparation, which was inhibited by pretreatment with atropine. In the electrically driven frog ventricle strip, the whole venom and its low molecular mass fraction, but not the proteic one, induced a negative inotropic effect that was also inhibited by atropine. These results suggest that despite low toxicity, A. paulensis venom can induce severe physiological disturbances in mice.

Towards therapeutic applications of arthropod venom k(+)-channel blockers in CNS neurologic diseases involving memory acquisition and storage.

Potassium channels are the most heterogeneous and widely distributed group of ion channels and play important functions in all cells, in both normal and pathological mechanisms, including learning and memory processes. Being fundamental for many diverse physiological processes, K(+)-channels are recognized as potential therapeutic targets in the treatment of several Central Nervous System (CNS) diseases, such as multiple sclerosis, Parkinson's and Alzheimer's diseases, schizophrenia, HIV-1-associated dementia, and epilepsy. Blockers of these channels are therefore potential candidates for the symptomatic treatment of these neuropathies, through their neurological effects. Venomous animals have evolved a wide set of toxins for prey capture and defense. These compounds, mainly peptides, act on various pharmacological targets, making them an innumerable source of ligands for answering experimental paradigms, as well as for therapeutic application. This paper provides an overview of CNS K(+)-channels involved in memory acquisition and storage and aims at evaluating the use of highly selective K(+)-channel blockers derived from arthropod venoms as potential therapeutic agents for CNS diseases involving learning and memory mechanisms.

Arthropod venoms: a vast arsenal of insecticidal neuropeptides.

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

Neuropharmacological effects in mice of Lychnophora species (Vernonieae, Asteraceae) and anticonvulsant activity of 4,5-di-O-[E]-caffeoylquinic acid isolated from the stem of L. rupestris and L. staavioides.

Aiming at contributing with the search for neuroactive substances from natural sources, we report for the first time antinociceptive and anticonvulsant effects of some Lychnophora species. We verify the protective effects of polar extracts (600 mg/kg, intraperitoneally), and methanolic fractions of L. staavioides and L. rupestris (100 mg/kg, intraperitoneally) in pentylenetetrazole-induced seizures on mice. Previously, a screening was accomplished, evaluating the antinociceptive central activity (hot plate test), with different extracts of L. rupestris, L. staavioides and L. diamantinana. It was possible to select the possible extracts of Lychnophora with central nervous system activity. Some of the active extracts were submitted to fractionation and purification process and the methanolic fractions of L. rupestris (stem) and L. staavioides (stem), with anticonvulsant properties (100 mg/kg, intraperitoneally), yielded 4,5-di-O-[E]-caffeoylquinic acid. This substance was injected intraperitoneally in mice and showed anticonvulsant effect against pentylenetetrazole-induced seizures at doses of 25 and 50 mg/kg. It has often been shown that seizures induced by pentylenetetrazole are involved in inhibition and/or attenuation of GABAergic neurotransmission. However, other systems of the central nervous system such as adenosinergic and glutamatergic could be involved in the caffeoylquinic acid effects. Further studies should be conducted to verify that the target receptor could be participating in this anticonvulsant property. Although other investigations have reported a series of biological activities from Lychnophora species, this is the first report of central analgesic and anticonvulsant activity in species of this genus.