Evaluation of the Antioxidant Activity of the Marine Pyrroloiminoquinone Makaluvamines.

Makaluvamines are pyrroloiminoquinones isolated from Zyzzya sponges. Until now, they have been described as topoisomerase II inhibitors with cytotoxic effects in diverse tumor cell lines. In the present work, seven makaluvamines were tested in several antioxidant assays in primary cortical neurons and neuroblastoma cells. Among the alkaloids studied, makaluvamine J was the most active in all the assays. This compound was able to reduce the mitochondrial damage elicited by the well-known stressor H2O2. The antioxidant properties of makaluvamine J are related to an improvement of the endogenous antioxidant defenses of glutathione and catalase. SHSY5Y assays proved that this compound acts as a Nrf2 activator leading to an improvement of antioxidant defenses. A low concentration of 10 nM is able to reduce the reactive oxygen species release and maintain a correct mitochondrial function. Based on these results, non-substituted nitrogen in the pyrrole plus the presence of a p-hydroxystyryl without a double bond seems to be the most active structure with a complete antioxidant effect in neuronal cells.

Identification of Spongionella compounds as cyclosporine A mimics.

Marine sponges are found to be a wide source of bioactive compounds with different effects such as anti-inflammatory or anticancer actions among others. Cyclophilin A (Cyp A) is a target protein implicated in the mechanism of action of immunosuppressive compounds such as Cyclosporine A (CsA). In the present paper we studied the binding between 4 Spongionella compounds (Gracilins H, A, L and Tetrahydroaplysulphurin-1) and Cyp A immobilized over a CM5 sensor chip. Thus, we found that Spongionella compounds showed to have similar binding affinities than CsA with dissociation equilibrium constant in the range. Next, the effect of these Spongionella isolated compounds was tested over calcineurin phosphatase activity. The same than CsA, Gracilin H, A and Tetrahydroaplysulphurin-1 were able to inhibit phosphatase activity once the complex between Cyp A-CsA/Spongionella compounds was formed. The ability to avoid the dephosphorylation of NFATc1 was also checked in human T cells isolated from peripheral blood. First, cells were pre-treated with Spongionella compounds or CsA following by Concanavalin A (Con A) stimulation. In these conditions nuclear NFATc1 levels were diminished either by CsA or Gracilin A, L, and Tetrahydroaplysulphurin-1 treatment. Moreover, as happens with CsA due to the inhibition of NFATc1, Interleukine-2 (IL-2) released to the culture medium was significantly decreased with all Spongionella compounds. Results conclude that, Spongionella derivatives preserve T lymphocytes from activation modulating the same pathway than CsA. Thus, this mechanism of action suggests that these compounds could be interesting candidates in drug development as immunosuppressive or anti-inflammatory drugs.

Spongionella Secondary Metabolites Regulate Store Operated Calcium Entry Modulating Mitochondrial Functioning in SH-SY5Y Neuroblastoma Cells.

BACKGROUND/AIMS: The effect of four secondary metabolites isolated from sponge Spongionella, gracilins H, A, L and tetrahydroaplysulphurin-1 on Calcium ion (Ca2+) fluxes were studied in SH-SY5Y neuroblastoma cells. METHODS AND RESULTS: These compounds did not modify cytosolic baseline Ca2+-levels. Nevertheless, when cytosolic Ca2+-influx through store operated calcium channels (SOC channels) was stimulated with Thapsigargin (Tg), a strong inhibition was observed in the presence of gracilin A, gracilin L and tetrahydroaplysulphurin-1. Since these compounds were able to protect mitochondria from oxidative stress, the role of this organelle in the Ca2+-influx inhibition was tested. In this sense, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) and Cyclosporine A (CsA) were used. Surprisingly, both the inhibitory effect over Tg-sensitive stores and Ca2+ influx through SOC channels produced by FCCP were abolished with different potencies by Spongionella compounds in a similar way than CsA. CsA is able to avoid Mitochondrial Permeability Transition Pore (mPTP) opening. As well as CsA, Spongionella compounds reverted mPTP opening induced by FCCP. In the case of CsA the mPTP blockade is due to the direct binding to Cyclophilin D (Cyp D), a mitochondrial matrix protein. This association was also observed between gracilin L and tetrahydroaplysulphurin-1 and Cyp D. Therefore, Spongionella compounds modulate mitochondrial activity by preventing mPTP opening by binding to Cyp D. CONCLUSIONS: These effects make Spongionella compounds as new family of compounds with promising activity in human diseases where mitochondrial alterations are implicated.

Gracilins: Spongionella-derived promising compounds for Alzheimer disease.

Alzheimer disease (AD) is a neurodegenerative pathology that is strongly linked with oxidative stress and mitochondrial dysfunction. The unclear origin of AD lead researchers to study several drug targets and it has been proposed that a multi-target drug would be a more promising candidate. Gracilins are sponge-derived diterpenoid compounds that have been described to act as antioxidants through mitochondrial targeting and through the induction of Nrf2 translocation. In this work gracilin H, A and L and tetrahydroaplysulphurin-1 have been studied in two neuroblastoma cellular models. First the BE(2)-M17 cell line has been used as a model for APP metabolism studies and next, SH-SY5Y-TMHT441 cells were used for AD drugs screening targeting tau phosphorylation. In vitro assays showed that gracilins were able to inhibit BACE1, reduce tau hyperphosphorylation and inhibit ERK. These positive results lead us to test gracilin H and L in 3xTg-AD mice. After chronic intraperitoneal treatments, a preliminary behavioral test pointed a positive trend on learning and spatial memory of mice treated with these compounds. Moreover in vivo assays confirmed the previous results. Amyloid-ß42 and hyperphosphorylated tau levels were decreased after treatments and the ERK inhibition was also observed. This research highlights new bioactivities for gracilins, such as BACE1 and ERK inhibition, and provides more evidence for their potential therapeutic application in neurodegenerative diseases due to their multi-target activities, especially in AD.

Spongionella secondary metabolites protect mitochondrial function in cortical neurons against oxidative stress.

The marine habitat provides a large number of structurally-diverse bioactive compounds for drug development. Marine sponges have been studied over many years and are found to be a rich source of these bioactive chemicals. This study is focused on the evaluation of the activity of six diterpene derivatives isolated from Spongionella sp. on mitochondrial function using an oxidative in vitro stress model. The test compounds include the Gracilins (A, H, K, J and L) and tetrahydroaplysulphurin-1. Compounds were co-incubated with hydrogen peroxide for 12 hours to determine their protective capacities and their effect on markers of apoptosis and Nrf2/ARE pathways was evaluated. Results conclude that Gracilins preserve neurons against oxidative damage, and that in particular, tetrahydroaplysulphurin-1 shows a complete neuroprotective activity. Oxidative stress is linked to mitochondrial dysfunction and consequently to neurodegenerative disorders like Parkinson and Alzheimer diseases, Friedreich ataxia or Amyotrophic lateral sclerosis. This neuroprotection against oxidation conditions suggest that these metabolites could be interesting lead candidates in drug development for neurodegenerative diseases.

Mitigation of ROS insults by Streptomyces secondary metabolites in primary cortical neurons.

Oxidative stress is a common point in neurodegenerative diseases, widely connected with mitochondrial dysfunction. In this study, we screened seven natural products from Streptomyces sources against hydrogen peroxide insult in primary cortical neurons, an oxidative stress in vitro model. We showed the ability of these compounds to inhibit neuronal cytotoxicity and to reduce ROS release after 12 h treatment. Among the tested compounds, the quinone anhydroexfoliamycin and the red pyrrole-type pigment undecylprodigiosin stand out. These two compounds displayed the most complete protection against oxidative stress with mitochondrial function improvement, ROS production inhibition, and increase of antioxidant enzyme levels, glutathione and catalase. Further investigations confirmed that anhydroexfoliamycin acts over the Nrf2-ARE pathway, as a Nrf2 nuclear translocation inductor, and is able to strongly inhibit the effect of the mitochondrial uncoupler FCCP over cytosolic Ca(2+), pointing to mitochondria as a cellular target for this molecule. In addition, both compounds were able to reduce caspase-3 activity induced by the apoptotic enhancer staurosporine, but undecylprodigiosin failed to inhibit FCCP effects and it did not act over the Nrf2 pathway as was the case for anhydroexfoliamycin. These results show that Streptomyces metabolites could be useful for the development of new drugs for prevention of neurodegenerative disorders such as Parkinson's and Alzheimer's diseases and cerebral ischemia.

Sustainable production of biologically active molecules of marine based origin.

The marine environment offers both economic and scientific potential which are relatively untapped from a biotechnological point of view. These environments whilst harsh are ironically fragile and dependent on a harmonious life form balance. Exploitation of natural resources by exhaustive wild harvesting has obvious negative environmental consequences. From a European industry perspective marine organisms are a largely underutilised resource. This is not due to lack of interest but due to a lack of choice the industry faces for cost competitive, sustainable and environmentally conscientious product alternatives. Knowledge of the biotechnological potential of marine organisms together with the development of sustainable systems for their cultivation, processing and utilisation are essential. In 2010, the European Commission recognised this need and funded a collaborative RTD/SME project under the Framework 7-Knowledge Based Bio-Economy (KBBE) Theme 2 Programme 'Sustainable culture of marine microorganisms, algae and/or invertebrates for high value added products'. The scope of that project entitled 'Sustainable Production of Biologically Active Molecules of Marine Based Origin' (BAMMBO) is outlined. Although the Union is a global leader in many technologies, it faces increasing competition from traditional rivals and emerging economies alike and must therefore improve its innovation performance. For this reason innovation is placed at the heart of a European Horizon 2020 Strategy wherein the challenge is to connect economic performance to eco performance. This article provides a synopsis of the research activities of the BAMMBO project as they fit within the wider scope of sustainable environmentally conscientious marine resource exploitation for high-value biomolecules.