Echinometra lucunter molecules reduce Aß42-induced neurotoxicity in SH-SY5Y neuron-like cells: effects on disaggregation and oxidative stress.

Background: Echinometra lucunter is a sea urchin commonly found on America's rocky shores. Its coelomic fluid contains molecules used for defense and biological processes, which may have therapeutic potential for the treatment of amyloid-based neurodegenerative diseases, such as Alzheimer's, that currently have few drug options available. Methods: In this study, we incubated E. lucunter coelomic fluid (ELCF) and fractions obtained by solid phase extraction in SH-SY5Y neuron-like cells to evaluate their effect on cell viability caused by the oligomerized amyloid peptide 42 (Aß42o). Moreover, the Aß42o was quantified after the incubation with ELCF fractions in the presence or not of cells, to evaluate if samples could cause amyloid peptide disaggregation. Antioxidant activity was determined in ELCF fractions, and cells were evaluated to check the oxidative stress after incubation with samples. The most relevant fraction was analyzed by mass spectrometry for identification of molecules. Results: ELCF and certain fractions could prevent and treat the reduction of cell viability caused by Aß42o in SH-SY5Y neuron-like cells. We found that one fraction (El50) reduced the oligomerized Aß42 and the oxidative stress caused by the amyloid peptide through its antioxidant molecules, which in turn reduced cell death. Mass spectrometry analysis revealed that El50 comprises small molecules containing flavonoid antioxidants, such as phenylpyridazine and dihydroquercetin, and two peptides. Conclusion: Our results suggest that sea urchin molecules may interact with Aß42o and oxidative stress, preventing or treating neurotoxicity, which may be useful in treating dementia.

Spatial Distribution and Biochemical Characterization of Serine Peptidase Inhibitors in the Venom of the Brazilian Sea Anemone Anthopleura cascaia Using Mass Spectrometry Imaging.

Sea anemones are known to produce a diverse array of toxins with different cysteine-rich peptide scaffolds in their venoms. The serine peptidase inhibitors, specifically Kunitz inhibitors, are an important toxin family that is believed to function as defensive peptides, as well as prevent proteolysis of other secreted anemone toxins. In this study, we isolated three serine peptidase inhibitors named Anthopleura cascaia peptide inhibitors I, II, and III (ACPI-I, ACPI-II, and ACPI-III) from the venom of the endemic Brazilian sea anemone A. cascaia. The venom was fractionated using RP-HPLC, and the inhibitory activity of these fractions against trypsin was determined and found to range from 59% to 93%. The spatial distribution of the anemone peptides throughout A. cascaia was observed using mass spectrometry imaging. The inhibitory peptides were found to be present in the tentacles, pedal disc, and mesenterial filaments. We suggest that the three inhibitors observed during this study belong to the venom Kunitz toxin family on the basis of their similarity to PI-actitoxin-aeq3a-like and the identification of amino acid residues that correspond to a serine peptidase binding site. Our findings expand our understanding of the diversity of toxins present in sea anemone venom and shed light on their potential role in protecting other venom components from proteolysis.

Design, in silico and pharmacological evaluation of a peptide inhibitor of BACE-1.

Introduction: Alzheimer's disease (AD) is the main type of dementia, caused by the accumulation of amyloid plaques, formed by amyloid peptides after being processed from amyloid precursor protein (APP) by γ- and ß-secretases (BACE-1). Although amyloid peptides have been well established for AD, they have been found in other neurodegenerative diseases, such as Parkinson's disease, Lewy body dementia, and amyotrophic lateral sclerosis. Inhibitors of BACE-1 have been searched and developed, but clinical trials failed due to lack of efficacy or toxicity. Nevertheless, it is still considered a good therapeutic target, as it was proven to remove amyloid peptides and improve memory. Methods: In this work, we designed a peptide based on a sequence obtained from the marine fish Merluccius productus and evaluated it by molecular docking to verify its binding to BACE-1, which was tested experimentally by enzymatic kinetics and cell culture assays. The peptide was injected in healthy mice to study its pharmacokinetics and toxicity. Results: We could obtain a new sequence in which the first N-terminal amino acids and the last one bound to the catalytic site of BACE-1 and showed high stability and hydrophobicity. The synthetic peptide showed a competitive inhibition of BACE-1 and Ki = 94 nM, and when injected in differentiated neurons, it could reduce Aß42o production. In plasma, its half-life is ∼1 h, clearance is 0.0015 µg/L/h, and Vss is 0.0015 µg/L/h. The peptide was found in the spleen and liver 30 min after injection and reduced its level after that, when it was quantified in the kidneys, indicating its fast distribution and urinary excretion. Interestingly, the peptide was found in the brain 2 h after its administration. Histological analysis showed no morphological alteration in any organ, as well as the absence of inflammatory cells, indicating a lack of toxicity. Discussion: We obtained a new BACE-1 inhibitor peptide with fast distribution to the tissues, without accumulation in any organ, but found in the brain, with the possibility to reach its molecular target, BACE-1, contributing to the reduction in the amyloid peptide, which causes amyloid-linked neurodegenerative diseases.

Biological characterization of bristle extract of Lonomia descimoni caterpillar (Lepidoptera, Saturniidae) and effectiveness of Lonomia antivenom to neutralize experimental envenomation in rats.

Contact with Lonomia caterpillars can cause severe envenomation with hemorrhagic syndrome, consumptive coagulopathy, acute renal failure, and death. In Brazil, an antivenom was produced using extracts from L. obliqua caterpillar bristles as antigen and has been used in other countries in South America to treat envenomation caused by distinct species of Lonomia. This study aimed to characterize the activities of toxins from Lonomia descimoni caterpillars found in Colombia and the neutralization of these toxins by the Brazilian Lonomia antivenom. The protein composition and coagulant, phospholipase A2, hyaluronidase, and defibrinogenating activities were evaluated and compared with the same parameters of the L. obliqua bristle extract. Immune recognition and the neutralizing ability of Lonomia antivenom were also determined. The results showed that the L. descimoni bristle extract presented marked differences in electrophoretic and mass spectrometry profiles and had coagulant, phospholipase A2, and hyaluronidase activities significantly less intense than those of the L. obliqua extract. In rats, L. descimoni extract induced coagulopathy and hemoglobinuria when injected by intravenous or intraperitoneal routes. The Lonomia antivenom recognized the toxins in the extract of L. descimoni and reversed the experimental envenomation in rats. Our results indicate that L. descimoni caterpillars possess toxins with weaker activities than those of L. obliqua but with the potential to cause envenomation. Moreover, the Lonomia antivenom recognized and neutralized the toxins in the L. descimoni bristle extract.

Effects of Myristicin in Association with Chemotherapies on the Reversal of the Multidrug Resistance (MDR) Mechanism in Cancer.

A range of drugs used in cancer treatment comes from natural sources. However, chemotherapy has been facing a major challenge related to multidrug resistance (MDR), a mechanism that results in a decrease in the intracellular concentration of chemotherapeutic agents, resulting in reduced treatment efficacy. The protein most frequently related to this effect is P-glycoprotein (P-gp), which is responsible for promoting drug efflux into the extracellular environment. Myristicin is a natural compound isolated from nutmeg and has antiproliferative activity, which has been reported in the literature. The present study aimed to evaluate the effect of the association between myristicin and chemotherapeutic agents on the NCI/ADR-RES ovarian tumor lineage that presents a phenotype of multidrug resistance by overexpression of P-gp. It was observed that myristicin showed no cytotoxic activity for this cell line, since its IC50 was >1 mM. When myristicin was associated with the chemotherapeutic agents cisplatin and docetaxel, it potentiated their cytotoxic effects, a result evidenced by the decrease in their IC50 of 32.88% and 75.46%, respectively. Studies conducted in silico indicated that myristicin is able to bind and block the main protein responsible for MDR, P-glycoprotein. In addition, the molecule fits five of the pharmacokinetic parameters established by Lipinski, indicating good membrane permeability and bioavailability. Our hypothesis is that, by blocking the extrusion of chemotherapeutic agents, it allows these agents to freely enter cells and perform their functions, stopping the cell cycle. Considering the great impasse in the chemotherapeutic treatment of cancer that is the MDR acquired by tumor cells, investigating effective targets to circumvent this resistance remains a major challenge that needs to be addressed. Therefore, this study encourages further investigation of myristicin as a potential reverser of MDR.

Caspase-1 and Cathepsin B Inhibitors from Marine Invertebrates, Aiming at a Reduction in Neuroinflammation.

Neuroinflammation is a condition associated with several types of dementia, such as Alzheimer's disease (AD), mainly caused by an inflammatory response to amyloid peptides that induce microglial activation, with subsequent cytokine release. Neuronal caspase-1 from inflammasome and cathepsin B are key enzymes mediating neuroinflammation in AD, therefore, revealing new molecules to modulate these enzymes may be an interesting approach to treat neurodegenerative diseases. In this study, we searched for new caspase-1 and cathepsin B inhibitors from five species of Brazilian marine invertebrates (four cnidarians and one echinoderm). The results show that the extract of the box jellyfish Chiropsalmus quadrumanus inhibits caspase-1. This extract was fractionated, and the products monitored for their inhibitory activity, until the obtention of a pure molecule, which was identified as trigonelline by mass spectrometry. Moreover, four extracts inhibit cathepsin B, and Exaiptasia diaphana was selected for subsequent fractionation and characterization, resulting in the identification of betaine as being responsible for the inhibitory action. Both molecules are already found in marine organisms, however, this is the first study showing a potent inhibitory effect on caspase-1 and cathepsin B activities. Therefore, these new prototypes can be considered for the enzyme inhibition and subsequent control of the neuroinflammation.

Biochemical and Toxinological Characterization of Venom from Macrorhynchia philippina (Cnidaria, Hydrozoa).

Macrorhynchia philippina is a colonial benthic hydroid from the Class Hydrozoa (Phylum Cnidaria) distributed in the tropical and subtropical marine waters from Atlantic Ocean, Indo-Pacific, and Mozambique. Its colonies somewhat resemble plants, causing confusion in the bathers who accidentally touch the animal. Acute burning/local pain, edema, erythema, and pruritus were symptoms already described, but its venom composition is unknown, as well as the participation of toxins for the symptom's development. Thus, herein, we show the biochemical composition and toxic effects of M. philippina venom. Colonies were collected and processed for histological analysis; alternatively, they were immersed into methanol containing 0.1% acetic acid for venom attainment, which was analyzed by mass spectrometry and submitted to edema and nociception evaluation in mice, hemolysis and antimicrobial assays in vitro. Before the molecule's extraction, it was possible to see the inoculation structures (hydrocladiums and hydrotheca) containing venom, which was released after the immersion of the animal in the solvents. The venom was composed mainly by low molecular mass compounds, able to cause significant reduction of the paw withdrawal latency from the hot plate test, 30 minutes after the injection. Moreover, significant edema was observed 10 and 30 minutes after the injection, indicating the activity of at least two inflammatory mediators. The venom caused no hemolytic activity but reduced the growth of A. baumannii and K. pneumoniae strains. This study is the first biochemical description of M. philippina venom, with molecules that cause fast inflammatory and painful effects, characteristic of the envenomation.

Reproductive behaviour, cutaneous morphology, and skin secretion analysis in the anuran Dermatonotus muelleri.

Despite the common poison and mucous glands, some amphibian groups have differentiated glands associated with reproduction and usually present on the male ventral surface. Known as breeding glands or sexually dimorphic skin glands (SDSGs), they are related to intraspecific chemical communication during mating. Until recently, reproduction associated with skin glands was recognized only in salamanders and caecilians and remained unexplored among anurans. The Brazilian microhylid Dermatonotus muelleri (Muller's termite frog) is known for its very toxic skin secretion. Despite the slippery body, the male adheres to the female back during reproduction, as they have differentiated ventral glands. In this paper, we have gathered data proposing an integrative approach correlated with the species' biology and biochemical properties of their skin secretions. Furthermore, we suggest that the adhesion phenomenon is related to arm shortening and rounded body that make amplexus inefficient, although constituting important adaptive factors to life underground.

A new therapeutic approach for bone metastasis in colorectal cancer: intratumoral melittin.

Background: Melittin has shown antiproliferative effects on tumor cells. Therefore, it comprises a valuable compound for studies on cancer treatment. To the best of our knowledge, no studies have reported melittin effects on bone metastasis. Herein, we propose an approach based on intrametastatic melittin injection to treat bone metastases in colorectal cancer. Methods: Following the characterization of melittin and antiproliferative tests in vitro, a single dose was injected through intrametastatic route into the mouse bone metastasis model. Following treatment, metastasis growth was evaluated. Results: A single dose of melittin was able to inhibit metastasis growth. Histological analysis showed necrosis and inflammatory processes in melittin-treated metastasis. Except by mild weight loss, no other systemic effects were observed. Conclusion: Our data suggest that melittin might be a promising agent for the future development of treatment strategies aiming to reduce the bone metastasis skeletal-related impact in colorectal cancer patients with bone metastasis.

A new therapeutic approach for bone metastasis in colorectal cancer: intratumoral melittin

Background: Melittin has shown antiproliferative effects on tumor cells. Therefore, it comprises a valuable compound for studies on cancer treatment. To the best of our knowledge, no studies have reported melittin effects on bone metastasis. Herein, we propose an approach based on intrametastatic melittin injection to treat bone metastases in colorectal cancer. Methods: Following the characterization of melittin and antiproliferative tests in vitro, a single dose was injected through intrametastatic route into the mouse bone metastasis model. Following treatment, metastasis growth was evaluated. Results: A single dose of melittin was able to inhibit metastasis growth. Histological analysis showed necrosis and inflammatory processes in melittin-treated metastasis. Except by mild weight loss, no other systemic effects were observed. Conclusion: Our data suggest that melittin might be a promising agent for the future development of treatment strategies aiming to reduce the bone metastasis skeletal-related impact in colorectal cancer patients with bone metastasis.(AU)

A new therapeutic approach for bone metastasis in colorectal cancer: intratumoral melittin

Abstract Background: Melittin has shown antiproliferative effects on tumor cells. Therefore, it comprises a valuable compound for studies on cancer treatment. To the best of our knowledge, no studies have reported melittin effects on bone metastasis. Herein, we propose an approach based on intrametastatic melittin injection to treat bone metastases in colorectal cancer. Methods: Following the characterization of melittin and antiproliferative tests in vitro, a single dose was injected through intrametastatic route into the mouse bone metastasis model. Following treatment, metastasis growth was evaluated. Results: A single dose of melittin was able to inhibit metastasis growth. Histological analysis showed necrosis and inflammatory processes in melittin-treated metastasis. Except by mild weight loss, no other systemic effects were observed. Conclusion: Our data suggest that melittin might be a promising agent for the future development of treatment strategies aiming to reduce the bone metastasis skeletal-related impact in colorectal cancer patients with bone metastasis.

Neglected Venomous Animals and Toxins: Underrated Biotechnological Tools in Drug Development.

Among the vast repertoire of animal toxins and venoms selected by nature and evolution, mankind opted to devote its scientific attention-during the last century-to a restricted group of animals, leaving a myriad of toxic creatures aside. There are several underlying and justifiable reasons for this, which include dealing with the public health problems caused by envenoming by such animals. However, these studies became saturated and gave rise to a whole group of animals that become neglected regarding their venoms and secretions. This repertoire of unexplored toxins and venoms bears biotechnological potential, including the development of new technologies, therapeutic agents and diagnostic tools and must, therefore, be assessed. In this review, we will approach such topics through an interconnected historical and scientific perspective that will bring up the major discoveries and innovations in toxinology, achieved by researchers from the Butantan Institute and others, and describe some of the major research outcomes from the study of these neglected animals.

Proteomic characterization of Naja mandalayensis venom.

BACKGROUND: Naja mandalayensis is a spitting cobra from Myanmar. To the best of our knowledge, no studies on this venom composition have been conducted so far. On the other hand, few envenomation descriptions state that it elicits mainly local inflammation in the victims' eyes, the preferred target of this spiting cobra. Symptoms would typically include burning and painful sensation, conjunctivitis, edema and temporary loss of vision. METHODS: We have performed a liquid-chromatography (C18-RP-HPLC) mass spectrometry (ESI-IT-TOF/MS) based approach in order to biochemically characterize N. mandalayensis venom. RESULTS: A wide variety of three-finger toxins (cardiotoxins) and metallopeptidases were detected. Less abundant, but still representative, were cysteine-rich secretory proteins, L-amino-acid oxidases, phospholipases A2, venom 5'-nucleotidase and a serine peptidase inhibitor. Other proteins were present, but were detected in a relatively small concentration. CONCLUSION: The present study set the basis for a better comprehension of the envenomation from a molecular perspective and, by increasing the interest and information available for this species, allows future venom comparisons among cobras and their diverse venom proteins.

Effects of Kynurenic Acid on the Rat Aorta Ischemia-Reperfusion Model: Pharmacological Characterization and Proteomic Profiling.

Kynurenic acid (KYNA) is derived from tryptophan, formed by the kynurenic pathway. KYNA is being widely studied as a biomarker for neurological and cardiovascular diseases, as it is found in ischemic conditions as a protective agent; however, little is known about its effect after ischemia-reperfusion in the vascular system. We induced ischemia for 30 min followed by 5 min reperfusion (I/R) in the rat aorta for KYNA evaluation using functional assays combined with proteomics. KYNA recovered the exacerbated contraction induced by phenylephrine and relaxation induced by acetylcholine or sodium nitroprussiate in the I/R aorta, with vessel responses returning to values observed without I/R. The functional recovery can be related to the antioxidant activity of KYNA, which may be acting on the endothelium-injury prevention, especially during reperfusion, and to proteins that regulate neurotransmission and cell repair/growth, expressed after the KYNA treatment. These proteins interacted in a network, confirming a protein profile expression for endothelium and neuron repair after I/R. Thus, the KYNA treatment had the ability to recover the functionality of injured ischemic-reperfusion aorta, by tissue repairing and control of neurotransmitter release, which reinforces its role in the post-ischemic condition, and can be useful in the treatment of such disease.

Neurotoxicity of Olindias sambaquiensis and Chiropsalmus quadrumanus extracts in sympathetic nervous system.

Cnidarians are equipped with nematocysts, which are specialized organelles used to inoculate venom during prey capturing and defense. Their venoms are rich in toxins and a potential source of bioactive compounds, however, poorly explored so far. In this work, the activity of the methanolic extracts from the hydromedusa Olindias sambaquiensis and the cubozoan jellyfish Chiropsalmus quadrumanus were studied in sympathetic neurotransmission. For that, bisected rat vas deferens - a classic model of sympathetic neurotransmission - were incubated with the extracts for further myographic and histopathological analysis. The O. sambaquiensis extract, at 0.1 µg/mL, facilitated the neurogenic contractions of the noradrenergic-rich epididymal portion, while reducing the noradrenaline (NA) potency, which suggests an interaction with postsynaptic α1-adrenoceptors. On the other hand, a higher concentration (1 µg/mL) leads to time- and frequency-dependent blockade of nerve-evoked contractions without significantly changing the response to exogenous NA. In turn, the C. quadrumanus extract at 0.1 µg/mL induced blockade of nerve-evoked noradrenergic contractions while reducing the potency to exogenous NA. Both extracts did not affect the purinergic neurotransmission or induce muscle damages. Our results demonstrate that O. sambaquiensis and C. quadrumanus extracts significantly interfere with the noradrenergic neurotransmission without altering purinergic response or smooth muscle structure on rat vas deferens. Such results bring to light the pharmacological potential of O. sambaquiensis and C. quadrumanus molecules for therapeutics focusing on noradrenergic neurotransmission.

Box Jellyfish (Cnidaria, Cubozoa) Extract Increases Neuron's Connection: A Possible Neuroprotector Effect.

Neurodegenerative diseases are one of the major causes of death worldwide, characterized by neurite atrophy, neuron apoptosis, and synapse loss. No effective treatment has been indicated for such diseases so far, and the search for new drugs is being increased in the last years. Animal venoms' secretion/venom can be an alternative for the discovery of new molecules, which could be the prototype for a new treatment. Here, we present the biochemical characterization and activity of the extract from the box jellyfish Chiropsalmus quadrumanus (Cq) on neurites. The Cq methanolic extract was obtained and incubated to human SH-SY5Y neurons, and neurite parameters were evaluated. The extract was tested in other cell types to check its cytotoxicity and was submitted to biochemical analysis by mass spectrometry in order to check its composition. We could verify that the Cq extract increased neurite outgrowth length and branching junctions, amplifying the contact between SH-SY5Y neurons, without affecting cell body and viability. The extract action was selective for neurons, as it did not cause any effects on other cell types, such as tumor line, nontumor line, and red blood cells. Moreover, mass spectrometry analysis revealed that there are no proteins but several low molecular mass compounds and peptides. Three peptides, characterized as cryptides, and 14 low molecular mass compounds were found to be related to cytoskeleton reorganization, cell membrane expansion, and antioxidant/neuroprotective activity, which act together to increase neuritogenesis. After this evaluation, we conclude that the Cq extract is a promising tool for neuronal connection recovery, an essential condition for the treatment of neurodegenerative diseases.

Proteomic characterization of Naja mandalayensis venom

Background Naja mandalayensis is a spitting cobra from Myanmar. To the best of our knowledge, no studies on this venom composition have been conducted so far. On the other hand, few envenomation descriptions state that it elicits mainly local inflammation in the victims' eyes, the preferred target of this spiting cobra. Symptoms would typically include burning and painful sensation, conjunctivitis, edema and temporary loss of vision. Methods We have performed a liquid-chromatography (C18-RP-HPLC) mass spectrometry (ESI-IT-TOF/MS) based approach in order to biochemically characterize N. mandalayensis venom. Results A wide variety of three-finger toxins (cardiotoxins) and metallopeptidases were detected. Less abundant, but still representative, were cysteine-rich secretory proteins, L-amino-acid oxidases, phospholipases A2, venom 5'-nucleotidase and a serine peptidase inhibitor. Other proteins were present, but were detected in a relatively small concentration. Conclusion The present study set the basis for a better comprehension of the envenomation from a molecular perspective and, by increasing the interest and information available for this species, allows future venom comparisons among cobras and their diverse venom proteins.(AU)

Reversal of Ovarian Cancer Cell Lines Multidrug Resistance Phenotype by the Association of Apiole with Chemotherapies.

Multidrug resistance (MDR) is the main obstacle in anticancer therapy. The use of drug combinations to circumvent tumor resistance is a well-established principle in the clinic. Among the therapeutic targets, glycoprotein-P (P-gp), an energy-dependent transmembrane efflux pump responsible for modulating MDR, is highlighted. Many pharmacological studies report the ability of calcium channel blockers to reverse tumor resistance to chemotherapy drugs. Isolated for the first time from parsley, the phenylpropanoid apiole is described as a potent calcium channel inhibitor. Taking this into account, herein, the ability of apiole to potentiate the action of well-established chemotherapeutics in the clinic, as well as the compound's relationship with the reversal of the resistance phenomenon by blocking P-gp, is reported. The association of apiole with both chemotherapeutic drugs doxorubicin and vincristine resulted in synergistic effect, in a concentration-dependent manner, as evaluated by the concentration reduction index. Molecular docking analysis demonstrated the affinity between apiole and the active site of P-gp, corroborating the inhibitory effect. Moreover, apiole demonstrated druglikeness, according to ADME analysis. In conclusion, apiole possibly blocks the active P-gp site, with strong binding energy, which, in turn, inhibits doxorubicin and vincristine efflux, increasing the antiproliferative response of these chemotherapeutic agents.

Morphological Evidence for an Oral Venom System in Caecilian Amphibians.

Amphibians are known for their skin rich in glands containing toxins employed in passive chemical defense against predators, different from, for example, snakes that have active chemical defense, injecting their venom into the prey. Caecilians (Amphibia, Gymnophiona) are snake-shaped animals with fossorial habits, considered one of the least known vertebrate groups. We show here that amphibian caecilians, including species from the basal groups, besides having cutaneous poisonous glands as other amphibians do, possess specific glands at the base of the teeth that produce enzymes commonly found in venoms. Our analysis of the origin of these glands shows that they originate from the same tissue that gives rise to teeth, similar to the venom glands in reptiles. We speculate that caecilians might have independently developed mechanisms of production and injection of toxins early in their evolutionary history.