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Previous works with autumnalamide reported that Store Operated Calcium (SOC) channels were blocked through mitochondrial modulation. In the present paper we studied the effect of autumnalamide on ionomycin Ca2+ fluxes. Thus, autumnalamide did not modify ionomycin-sensitive intracellular pools while the ionomycin-induced Ca2+ influx was blocked with similar potency whether the incubation was done before or after ionomycin-sensitive pools depletion. Nevertheless, autumnalamide was not able to inhibit ionomycin-induced Ca2+ influx once the membrane channels were activated. Moreover, the compound efficiently inhibited flufenamic acid (FFA) Ca2+ release induced in this organelle but no the next influx. Since in previous work the effect of autumnalamide was inhibited by cyclosporine A (CsA), structures that target this drug were studied. Therefore, the affinity of autumnalamide for cyclophilin D (Cyp D) was examined. The KD obtained for Cyp D- autumnalamide was 1.51±1.399. Moreover, the KD for Cyp A- autumnalamide was calculated. The peptide had a similar order of Cyp A binding affinity than CsA (8.08±1.23 and 6.85±1.1µM respectively). After testing autumnalamide-binding capacity for Cyp A, the activity of this compound on Cyp A pathway was tested. Thus, the effect on interleukin (IL)-2 release on activated T-lymphocytes was checked. Autumnalamide was able to reduce IL-2 levels near to T cells in resting conditions. Next, the effect over calcineurin and NFATc1 was also evaluated. While CsA inhibits both calcineurin and NFATc1, autumnalamide did not produce any effect. From these results we can conclude that, autumnalamide targeted mitochondrion and prevent T-cells from IL-2 production through the modulation of SOC Ca2+ channels.

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The modulation of the immune system can have multiple applications such as cancer treatment, and a wide type of processes involving inflammation where the potent chemotactic agent cyclophilin A (Cyp A) is implicated. The Porifera phylum, in which Spongionella is encompassed, is the main producer of marine bioactive compounds. Four secondary metabolites obtained from Spongionella (Gracilin H, A, L, and Tetrahydroaplysulphurin-1) were described to hit Cyp A and to block the release of inflammation mediators. Based on these results, some role of Spongionella compounds on other steps of the signaling pathway mediated by this chemotactic agent can be hypothesized. In the present paper, we studied the effect of these four compounds on the surface membrane CD147 receptor expression, on the extracellular levels of Cyp A and on the ability to migrate of concanavalin (Con A)-activated T lymphocytes. Similar to a well-known immunosuppressive agent cyclosporine A (CsA), Gracilin H, A, L, and tetrahydroaplysulphurin-1 were able to reduce the CD147 membrane expression and to block the release of Cyp A to the medium. Besides, by using Cyp A as chemotactic agent, T cell migration was inhibited when cells were previously incubated with Gracilin A and Gracilin L. These positive results lead us to test the in vivo effect of Gracilin H and L in a mouse ear delayed hypersensitive reaction. Thus, both compounds efficiently reduce the ear swelling as well as the inflammatory cell infiltration. These results provide more evidences for their potential therapeutic application in immune-related diseases of Spongionella compounds.

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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.

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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.

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Filamentous cyanobacteria of the genus Phormidium have been rarely studied for their chemical diversity. For the first time, the cultivable Phormidium autumnale was shown to produce a prenylated cyclic peptide named autumnalamide (1). The structure of this peptide was fully determined after a deep exploration of the spectroscopic data, including NMR and HRMS. Interestingly, a prenyl moiety was located on the guanidine end of the arginine amino acid. The absolute configurations of most amino acids were assessed using enantioselective GC/MS analysis, with (13)C NMR modeling being used for the determination of d-arginine and d-proline. The effects of 1 on sodium and calcium fluxes were studied in SH-SY5Y and hNav 1.6 HEK cells. When the Ca(2+) influx was stimulated by thapsigargin, strong inhibition was observed in the presence of 1. As a consequence, this compound may act by disrupting the normal calcium uptake of this organelle, inducing the opening of the mitochondrial permeability transition pore, which results in the indirect blockade of store-operated channels.

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