Autumnalamide, a Prenylated Cyclic Peptide from the Cyanobacterium

Article pubs.acs.org/jnp

Autumnalamide, a Prenylated Cyclic Peptide from the Cyanobacterium Phormidium autumnale, Acts on SH-SY5Y Cells at the Mitochondrial Level Coralie Audoin,†,‡,∇ Jon Andoni Sánchez,§,∇ Grégory Genta-Jouve,⊥ Amparo Alfonso,§ Laurent Rios,‡ Carmen Vale,§ Olivier P. Thomas,*,†,∥ and Luis M. Botana*,§ †

Institut de Chimie de Nice-PCRE, UMR 7272 CNRS, Faculty of Science, University of Nice Sophia-Antipolis, Parc Valrose, 06108 Nice, France ‡ GREENSEA SAS, Promenade du Sergent Jean-Louis Navarro, 34140 Mèze, France § Department of Pharmacology, Faculty of Veterinary, University of Santiago de Compostela, 27002 Lugo, Spain ⊥ Laboratoire de Pharmacognosie, UMR 8638 CNRS, Faculté des Sciences Pharmaceutiques et Biologiques, Paris Descartes University, Sorbonne Paris Cité, 4 Avenue de l’Observatoire, 75006 Paris, France ∥ Institut Méditerranéen de Biodiversité et d’Ecologie Marine Et Continentale, UMR 7263 CNRS−IRD−Aix-Marseille Université−UAPV, Station Marine d’Endoume, Rue de la Batterie des Lions, 13007 Marseille, France S Supporting Information *

ABSTRACT: 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 13C 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 Ca2+ 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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isolated from marine organisms and active on ion channels, we focused on a strain of the cyanobacterium Phormidium autumnale exhibiting a promising chemical profile with the presence of nonlipidic compounds. Among the most interesting compounds produced by cyanobacteria, peptides and especially cyclic peptides have attracted much interest.6 Cyclic peptides represent a diverse family of natural products characterized by a large array of bioactivities.7 They are produced by different types of organisms ranging from terrestrial plants8 to marine cyanobacteria.9 A thorough chemical study of a strain of the cyanobacterium P. autumnale, collected in Cumbria and cultivated at the Greensea company, led us to isolate and characterize a prenylated cyclic peptide named autumnalamide (1). While cytotoxicity was most frequently assayed for peptides produced by cyanobacteria, we decided to investigate

icroalgae are undoubtedly lead organisms for the development of marine biotechnologies in the near future. Indeed, several species from diverse groups have been cultivated in closed systems, which opens the way for the production of large quantities of biomass, although this issue is not resolved for all species.1 Microalgae are mostly developed for their ability to store large amounts of energetic lipids that can offer an alternative to biodiesel.2 Less studied is their propensity to produce specialized metabolites that may find applications in the cosmetic or pharmaceutical sectors.3 Among the huge taxonomical diversity of microalgae, cyanobacteria have attracted much attention due to their ability to biosynthesize toxic metabolites with therapeutic potential.4 Ion channels are membrane proteins whose principal function includes gating the flow of ions through the cell membrane. These channels are related to numerous processes in the cell, and they are also an attractive target for the prevention of many diseases.5 During the course of our investigations directed toward the discovery of natural products © XXXX American Chemical Society and American Society of Pharmacognosy

Received: May 1, 2014

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dx.doi.org/10.1021/np500374a | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 1. (a) Structure of autumnalamide (1). (b) Key 2D NMR correlations in DMSO-d6: COSY are in bold, HMBC (H → C) are shown by arrows, and the dotted arrow corresponds to a correlation observed in CD3OD.

Table 1. 1H (500 MHz) and 13C (125 MHz) NMR Data of Autumnalamide (1) in DMSO-d6 residue

position

δC, mult.

Pre-Arg

1 2 3

171.6, C 53.5, CH 28.4, CH2

4

22.9, CH2

5

6 7 8 9 10 11 Phe1

12 13 14 15 16 17 18

Pro

19 20 21

40.6, CH2 HNa 156.1, 38.9, 119.6, 135.3, 25.3, 17.9, HN 169.1, 51.5, 38.6, 136.7, 129.8, 127.9, 126.2, HN 171.2, 53.6, 29.0,

C CH2 CH C CH3 CH3 C CH CH2 C CH CH CH C CH CH2

22

24.5, CH2

23

47.6, CH2

δH, mult. (J in Hz)

residue

position

δC, mult.

δH, mult. (J in Hz) 3.14, m

3.96, 1.79, 1.15, 1.29, 1.05, 2.99, 2.90, 7.97,

m m m m m br s m br s

Gly

Leu

Asp

1.65, s 1.63, s 8.78, br d (8.8) 4.79, br q (6.3) 2.83, dd (13.0, 6.5) 2.75, dd (13.0, 7.7) d (7.5) m m br s

4.13, 2.06, 1.73, 1.88, 1.79, 3.60,

t (7.5) dd (11.6, 7.0) m m m m

168.0, C 42.4, CH2

26 27 28

HN 170.1, C 53.0, CH 42.6, CH2

29 30 31

3.74, br s 5.19, t (6.1)

7.34, 7.17, 7.17, 7.79,

24 25

32 33 34 35

Phe2

36 37 38 39 40 41 42

23.8, 22.1, 22.2, HN 171.7, 50.9, 37.7,

CH CH3 CH3

174.4, HN 170.6, 56.7, 35.3,

C

138.2, 128.9, 128.2, 126.2, HN

C CH CH CH

C CH CH2

4.04, dd (17.3, 7.5) 3.33, d (17.3) 8.85, dd (7.5, 5.0) 4.49, 1.59, 1.38, 1.51, 0.75, 0.75, 8.11,

dt (10.2, 7.5) dt (13.0, 7.5) dd (13.0, 7.5) hept (6.5) d (6.5) d (6.5) d (10.2)

4.12, m 3.01, m 2.81, m 8.14, d (5.5)

C CH CH2

4.17, dt (8.7, 4.0) 3.06, dd (14.5, 4.0) 3.02, m 7.26, 7.26, 7.17, 8.18,

m m m br s

a

This NH was located between C-5 and C-6 due to a H-5/H−N COSY correlation. The other H−N signals of the guanidine were not observed due to broadening. All other H−N signals of 1 were assigned to peptidic nitrogens.

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the effect of 1 on Na+ and Ca2+ ion fluxes in SH-SY5Y and hNav1.6 HEK cells. Indeed, as important examples, the lipopeptides kalkitoxin, jamaicamide, and antillatoxins isolated from cyanobacteria showed potent neurotoxicities by virtue of their modulation of ion channels.10 Additionally, several neurotoxins have shown promising activities as potential lead compounds for the treatment of neurodegenerative diseases.11−15

RESULTS AND DISCUSSION A strain of the cyanobacterium P. autumnale was identified by 16S rRNA analysis and cultured in a closed system. The biomass was then filtered, freeze-dried, and extracted by a mixture of the solvents CH2Cl2/MeOH (1:1) under ultrasound to yield an organic extract. The oil was first fractionated by reversed-phase vacuum liquid chromatography. The CH2Cl2/ MeOH (1:1) fraction was purified by reversed-phase semipreparative HPLC using a PhenylHexyl column, and the pure B



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Figure 2. Selected fragments observed for 1 by MSn.

H-5/N−H COSY correlation and the absence of the H-7/C-5 and H-5/C-7 HMBC correlations. The connections between all residues were first inferred from COSY and HMBC couplings involving the H−N protons of the peptidic bonds (Figure 1b). However, frequent overlapping of the HMBC correlations did not allow us to fully ascertain the primary structure of this peptide, and we decided to use HRMS fragmentation data as a final confirmation. Cyclic peptides are known to produce a high number of fragments in MS analysis due to the initial cleavage of an amide bond, which leads to several linear b-ions having a free N-terminus and a C-terminus oxazolone.16,17 Acquisition of the MS2 spectrum of the protonated molecule ([M + H]+ 901.49300) using collisioninduced dissociation (CID) led to the identification of several key fragments that confirmed the primary structure obtained after inspection of the NMR spectra. The most intense fragment at m/z 816.40448 ([M − C5H10N + H]+) could be attributed to the loss of an N-prenyl moiety, in accordance with the location of the prenyl on a secondary amine (or a terminal guanidine). This result confirmed the location of the prenyl moiety on the guanidine of the arginine residue and not on the nitrogen of a peptide bond. Two possible initial breakups of the peptide are represented in Figure 2 in order to obtain two linear b-ions. Cleavage of the amide bond between Pro and Phe-1 gave important fragments, among them a fragment at m/ z 383.19272 ([M + H]+) assigned to the Pro-Gly-Leu-Asp sequence. This assumption was confirmed by another fragment at m/z 773.39858 ([M + H]+) indicating a cleavage of the C-α and C-β of the Gly residue resulting in the loss of the Pro Nterminus. Another fragment at m/z 617.30866 ([M + H]+) was observed corresponding to the loss of the Asp residue. The other half of the peptide was also observed at m/z 519.30815 ([M + H]+) associated with the remaining Phe-2-PreArg-Phe-1 residues. A key fragment at m/z 530.26131 ([M + H]+) confirmed the Asp-Phe-2 connection, while the last connection

compound 1 was obtained after a subsequent purification by C18 semipreparative HPLC. The molecular formula C46H64N10O9 was deduced from HRESIMS data ([M + H]+ 901.49300, Δ +1.015). The 1H NMR spectrum of 1 in DMSO-d6 displayed signals that were reminiscent of a peptide, like H−N signals around 8 ppm and signals of the α-protons that resonate between 4 and 5 ppm. A combination of TOCSY, HSQC, and HMBC spectra interpretation allowed us to identify the seven residues of 1, mass spectrometry data confirming the cyclic nature of this peptide (Figure 1).The proteinogenic residues were identified as Arg, Phe1, Pro, Gly, Leu, Asp, and Phe2 (Table 1). An unusual feature of this compound was detected in the 1H NMR spectrum, where one trisubstituted double bond was evidenced by the signal at δH 5.19 (t, J = 6.1 Hz, H-8) and the corresponding ethylenic signals at δC 119.6 (C-8) and 135.3 (C-9) in the 13C NMR spectrum. Both additional signals at δH 1.65 (s, H3-10) and 1.63 (s, H3-11) suggested that the double bond was substituted by two methyls, while the last nonpeptidic signal at δH 3.74 (br s, H2-7) suggested the presence of a prenyl unit linked to the cyclic peptide. The issue of its location could not be resolved using NMR data in DMSO-d6, as no HMBC correlation was evidenced starting from the H2-7 methylene. Nevertheless, the chemical shift of the signal at δC 38.9 (C-7) was consistent with a substitution of the prenyl on a nitrogen, and because all peptidic nitrogens belonged to nonsubstituted secondary amides (with the exception of proline) as shown by the presence of six peptidic H−N bonds, we first assumed a substitution of this prenyl unit on the guanidine moiety of the arginine. Fortunately, a small H-7/C-6 HMBC correlation was observed when the NMR experiments were performed in CD3OD, thus confirming the location of the prenyl on the arginine side chain (Figure 1). Prenylation on the terminal nitrogen of the guanidine was inferred from the presence of a C



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Figure 3. Differences of 13C NMR chemical shifts between the calculated values for the four possible diastereomers and the experimental values for the 42 carbons (numbering following Table 1).

chemical shifts are the following: s-trans δCβ 28−30, δCγ 24− 25.5 and s-cis δCβ 31−32, δCγ 20−23.23 Prenylation of peptides is not a rare phenomenon, as exemplified for other cyclic peptides produced by cyanobacteria, like the cyanobactins.24 Even if prenyl transferases have been observed acting on the alcohols of serine or threonine or on the aromatic tryptophan, the most described prenylated amino acid is tyrosine when a spontaneous Claisen rearrangement has been invoked to explain prenylation on the phenol ring.25 For arginine, prenylation has been rarely encountered, and it has only been found for stictamides A and C, highly modified acyclic peptides isolated from a lichen.26 In this case, it is interesting to notice that a D configuration was also deduced for the prenylated arginine. First the effect of 1 on cell viability in SH-SY5Y cells was checked. The treatment with 1 did not affect cell viability after 1 h incubation with concentrations ranging from 0.1 to 20 μM. Because 25 μM induced a 42% decrease in cell viability (data not shown), 10 μM was selected to check biological activity. Sodium and calcium movements through the cytosolic membrane are fundamental steps in cellular activation. Hence the effect of 1 on these ion fluxes was investigated. Voltagegated sodium channels (VGSC) are necessary for the production and propagation of action potentials, and therefore these channels play an important role in regulating many cellular processes.27 However, no change in the total sodium current in SH-SY5Y was observed when the effect of 1 was tested in a concentration range from 0.1 nM to 10 μM, even at the highest concentration used. The same lack of effect was observed when the current through the Nav 1.6 channel was studied in the cellular line hNav1.6 HEK. The conclusion to these experiments is that 1 does not interact with sodium currents. Ca2+ homeostasis in animal cells is strictly controlled. It regulates a wide range of physiological processes in eukaryotic cells and plays an important role in different cellular processes such as cellular growth, exocytosis, gene transcription, apoptosis, or cell proliferation, among others.28 Then, the

between PreArg and Phe-1 was unambiguously attributed by the fragment at m/z 754.42515 ([M + H]+). Because the cyclic peptide was refractory to Marfey’s analysis, certainly due to the presence of the prenyl on the arginine, we turned to enantioselective GC analysis of the free amino acids in order to assess the absolute configuration of 1. Thus, the peptide was hydrolyzed under acidic conditions, and a derivation protocol with heptafluorobutanol and ethyl chloroformate was applied prior to the enantioselective GC×GC/ MS analysis of the mixture. Comparison with the data obtained with the enantiopure derivatives enabled us to identify L-Phe, LAsp, and L-Leu. However, our analytical conditions proved unsuccessful to separate both enantiomers of derivatized proline, and the substitution on the arginine hampered any conclusion on this amino acid. In order to complete the assignment of the absolute configuration of autumnalamide (1), 13 C NMR calculations were undertaken as previously applied with success for other families of natural products.18−20 Indeed, 13 C NMR chemical shifts are highly sensitive to conformational changes, and it has been demonstrated that comparisons of experimental chemical shifts with calculated values have been successful for the determination of the relative configuration of natural products. Theoretical calculations of the magnetic properties were then realized for the four diastereomers of 1 using density functional theory (DFT) at the B3LYP/6-31(g) level (Supporting Information). In order to assign the absolute configurations for the prenylarginine and the proline, theoretical chemical shifts of each carbon were compared with experimental ones for the whole compound. The lowest energy conformer obtained for Darginine and D-proline led to 13C NMR chemical shifts in almost complete agreement with the experimental ones (Figure 3). We were then very confident to assume these two absolute configurations for both amino acids. Even if this result was unexpected, D-amino acids are not rare in cyclic peptides produced by microalgae.21,22 The s-trans conformation of the Dproline obtained for the lowest energy conformer was in accordance with the empirical rules described where the D



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Figure 4. Effect of 1 on cytosolic Ca2+ profile in SH-SY5Y neuroblastoma cells. (A) Cytosolic Ca2+ profile in cells treated with 10 μM 1 (open squares) and control (open circles). The first arrow indicates the addition of 1 in a Ca2+-free media, and the second the addition of 1 mM Ca2+ after 5 min incubation with the compound. Means ± SEM of three experiments. (B) Cytosolic Ca2+ profile in cells stimulated with 2 μM Tg and incubated for 5 min with 10 μM (open squares), 5 μM (lines), or 1 μM (black/white squares) 1 and in controls of untreated cells (open circles) and Tg-treated cells (open triangles). The first arrow indicates the addition of Tg, and the second the addition of 1 in a Ca2+-free media. The third arrow indicates the addition of 1 mM Ca2+. Means ± SEM of three experiments.

Figure 5. Effect of 1 and SKF96365 on cytosolic Ca2+ profile in SH-SY5Y neuroblastoma cells. (A) Cytosolic Ca2+ profile in cells stimulated with 2 μM Tg and incubated for 5 min with 30 μM SKF96365 (solid circles) and controls of untreated cells (open circles) and Tg-treated cells (open triangles). The first arrow indicates the addition of Tg, and the second the addition of SKF96365 in a Ca2+-free media. The third arrow indicates the addition of 1 mM Ca2+. Means ± SEM of three experiments. (B) Cytosolic Ca2+ profile in cells stimulated with 2 μM Tg and incubated for 5 min with 30 μM SKF96365 and then for 5 min with 10 μM 1 (solid squares) and controls of untreated cells (open circles) and Tg (open triangles). The first arrow indicates the addition of Tg; the second, the addition of SKF96365; and the third, the addition of 1 in a Ca2+-free media. The fourth arrow indicates the addition of 1 mM Ca2+. Means ± SEM of three experiments.

In these conditions, when 10 μM 1 was added after pool depletion, the Ca2+ influx was totally abolished. This effect is dose dependent: when 5 μM 1 is added, the Ca2+ influx induced by Tg was significantly reduced (33%), while in the presence of 1 μM, no modification in Ca2+ influx was observed compared to the Tg control. From these results a concentration of 10 μM was selected to further study the effects of 1 on Ca2+ influx. Then the effect of 1-[β-(3-(4-methoxyphenyl)propoxy)-4methoxyphenethyl]-1H-imidazole·HCl (SKF-96365), a known SOC channel inhibitor, was checked in SH-SY5Y cells. As Figure 5A shows, the Ca2+ influx induced by Tg was totally inhibited in the presence of 30 μM SKF-96365, as the Ca2+ entry was similar to control cells without treatment. In addition,

effect of 1 on Ca2+ signaling was checked. The incubation of 1 in a Ca2+-free media shows that 1 had no effect on cytoplasmatic levels, not even when 1 mM Ca2+ is restored to the bath solution (Figure 4A). In the same way, the role of 1 on Ca 2+ influx induced by thapsigargin was studied. Thapsigargin (Tg) is a tumor-promoting sesquiterpene lactone that inhibits the endoplasmatic reticulum ATPase (SERCA). As a consequence, this drug depletes the intracellular stores of this organelle and induces Ca2+ entry of Ca2+ influx through store operated calcium (SOC) channels and allows the study in an isolated way.29 In SH-SY5Y cells after 2 μM Tg addition, a significant increase in cytosolic Ca2+ levels was observed, corresponding to depletion of intracellular pools (Figure 4B). Then, when Ca2+ was restored to the medium, a sustained and significant Ca2+ influx through the SOC channels was obtained. E



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Figure 6. Effect of 1 and FCCP on cytosolic Ca2+ profile in SH-SY5Y neuroblastoma cells. (A) Cytosolic Ca2+ profile in cells stimulated with 2 μM Tg and incubated for 5 min with 10 μM FCCP (solid triangles) and controls of untreated cells (open circles) and Tg-treated cells (open triangles). The first arrow indicates the addition of Tg, and the second the addition of FCCP in a Ca2+-free media. The third arrow indicates the addition of 1 mM Ca2+. Means ± SEM of three experiments. (B) Cytosolic Ca2+ profile in cells stimulated with 2 μM Tg and incubated with 10 μM FCCP for 5 min and then with 10 μM 1 for 5 min (crosses) and controls of untreated cells (open circles) and Tg-treated cells (open triangles). The first arrow indicates the addition of Tg; the second, the addition of FCCP; and the third, the addition of 1 in a Ca2+-free media. The fourth arrow indicates the addition of 1 mM Ca2+. Means ± SEM of three experiments.

the same blockade was observed when SKF-96365 and 1 were applied together (Figure 5B). Therefore, the effect of 1 seems to mimic the SKF-96365 effect, inducing full inhibition of the Tg-dependent Ca2+ entry. However, the total abolition of the Ca2+ entry can be explained as a direct action of 1 at SOC channels, just like SKF-96365, or as an indirect inhibition of SOC channels through a receptor targeted by 1. In this sense, mitochondria have an irreplaceable role in the regulation of Ca2+ entry through the SOC channels.30,31 Such regulation depends on the metabolic state of this organelle.32,33 To understand the role of mitochondria in the effect of 1, the protonophoric uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) was used (Figure 6). It is well known that FCCP produces an indirect blockade of SOC channels in different cellular models.34−37 After incubation in the presence of 10 μM FCCP for 5 min, a reduction of 51% in Ca2+ influx induced by Tg was observed (Figure 6A). However, in the presence of both FCCP and 1, a 72% reduction in Ca2+ entry was observed (Figure 6B). The small Ca2+ entry, 28%, observed under those conditions could point to the same cellular target for both compounds.38 Therefore, the effect of 1 on SOC channels could be due to an indirect inhibition through the state of the mitochondria. The next step was to determine if 1 directly acts by disrupting the mitochondrial membrane potential. Tetramethylrhodamine ethyl ester (TMRE) is a cell-permeable dye, positively charged, that readily accumulates in active mitochondria due to its relative negative charge. Thereafter, depolarized or altered mitochondria fail to sequester TMRE.39 This happens in the presence of the mitochondrial uncoupler FCCP, which is often used as a positive control in TMRE experiments.34 FCCP produces a significant reduction, 68%, of TMRE signal, which means a loss of mitochondrial membrane potential (Figure 7). This effect was partially blocked in the presence of 0.2 μM cyclosporine A (CsA). This drug prevents the formation of the mitochondrial permeability transition pore (mPTP) by binding to cyclophilin D, a mitochondrial matrix protein.40,41 In the same way, a 36% reduction of TMRE

Figure 7. Effect of 1 on mitochondrial membrane potential in SHSY5Y neuroblastoma cells. TMRE fluorescence of cells incubated with 10 μM 1 (white column), 10 μM FCCP (black column), or 0.2 μM CsA (squares column) for 10 min. When CsA is combined with 1 (oblique lines column) or with FCCP (lines column), cells are first incubated 10 min with CsA and then 1 or FCCP is added. Means ± SEM of three experiments. Significant differences: (*) with respect to control cells, (+) with respect to CsA plus FCCP, and (#) with respect to CsA plus 1.

fluorescence was obtained in the presence of 1. This effect was totally abolished with the co-incubation of the cells with CsA. Therefore, the suppression of mPTP by CsA inhibits the alteration of mitochondrial membrane potential induced by FCCP and by 1.42 However, the effects are different. While FCCP does not directly modulate the mPTP,43 and its effect on TMRE is not totally blocked by CsA treatment, the effect of 1 on TMRE is totally inhibited by CsA. Therefore, an effect of 1 on mPTP can be suggested from these data. To clarify if 1 modulates the mPTP, the dye calcein, often employed to check the mPTP opening, was used.44 Confocal data show (Figure 8) that after incubation with FCCP a 20% decrease in calcein fluorescence was observed. On the contrary, F



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stores, and after 5 min, 1 was added to the bath solution. Surprisingly, the effect of 1 over Tg Ca2+ entry was totally abolished by CsA incubation (Figure 9A), while the FCCP effect on Ca2+ influx was only partially affected by CsA incubation (Figure 9B). Therefore, based on the experiments performed in SH-SY5Y cells, we can conclude that 1 indirectly blocks SOC channel Ca2+ influx by acting at the mitochondrial level, probably through the inhibition of the formation of mPTP, in such a way that when this structure is inhibited, the effect over Ca2+ influx is suppressed. Hence, the action of 1 opens several interesting possibilities to its use as a modulatory drug in calcium and mitochondrial metabolism and to its potential use in pathologies related to these biochemical signals.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were determined at 20 °C using a Jasco T2000 polarimeter. UV spectrum was obtained on an Agilent Cary 300 UV−vis spectrophotometer. IR spectra were obtained with a Perkin−Elmer Paragon 1000 FT-IR spectrometer. NMR spectra were measured on a Bruker Avance 500 MHz spectrometer with pulsed field gradients and referenced to residual solvent signals (DMSO-d6 at δH 2.50 and δC 39.5 ppm, CD3OD at δH 3.31 and δC 49.0 ppm). All MS spectra were acquired on a hybrid 7-T linear ion trap FT-ICR mass spectrometer (LTQ FT Ultra, Thermo Fisher Scientific). Fragmentation was realized at a normalized 35 eV collision energy. HPLC purification was carried out on a Waters 600 system equipped with a Waters 717 Plus autosampler, a Waters 996 photodiode array detector, and a Sedex 55 evaporative light-scattering detector (Sedere). Thapsigargin, ionomycin, and SKF-96365 were from Alexis Corporation. FURA-2AM was obtained from Molecular Probes. Cyclosporine A, FCCP, sodium dodecyl sulfate (SDS), tetramethylrhodamine ethyl ester (TMRE), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich, and (2-[2-[4-(4-nitrobenzyloxy)-

Figure 8. Effect of 1 on mPTP opening in SH-SY5Y neuroblastoma cells. Changes in calcein fluorescence in the presence of 10 μM 1 (white column), 10 μM FCCP (black column), 0.2 μM CsA (squares column), 0.2 μM CsA plus 10 μM FCCP (lines column), or 0.2 μM CsA plus 1 (oblique lines column). Means ± SEM of three experiments. Significant differences: (*) with respect to control cells, (+) with respect to CsA plus FCCP, and (#) with respect to CsA plus 1.

when cells were pretreated with CsA before FCCP addition, the reduction of calcein fluorescence was suppressed. In the same way, the incubation with 1 produced a significant and higher decrease of 30% in calcein fluorescence levels that can be eliminated by co-incubation with CsA. Finally, to clarify the role of mPTP on Ca2+ influx, the effect of 1 on Tg-induced Ca2+ entry was checked in the presence of CsA. Cells were incubated with CsA after depleting intracellular

Figure 9. Effect of 1 or FCCP and CsA on cytosolic Ca2+ profile in SH-SY5Y neuroblastoma cells. (A) Cytosolic Ca2+ profile in cells stimulated with 2 μM Tg and incubated with 0.2 μM CsA for 5 min and then with 10 μM 1 for 5 min (solid triangles), with 10 μM 1 for 5 min (open squares), or with 0.2 μM CsA for 5 min (×) and controls of untreated cells (open circles) and Tg-treated cells (open triangles). The first arrow indicates the addition of Tg; the second, the addition of CsA; and the third, the addition of 1 in a Ca2+-free media. The fourth arrow indicates the addition of 1 mM Ca2+. Means ± SEM of three experiments. (B) Cytosolic Ca2+ profile in cells stimulated with 2 μM Tg and incubated with 0.2 μM CsA for 5 min and then with 10 μM FCCP for 5 min (squares), with 10 μM FCCP for 5 min (solid rhombus) or with 0.2 μM CsA for 5 min (×) and controls of untreated cells (open circles) and Tg-treated cells (open triangles). The first arrow indicates the addition of Tg; the second, the addition of CsA; and the third, the addition of FCCP in a Ca2+-free media. The fourth arrow indicates the addition of 1 mM Ca2+. Means ± SEM of three experiments. G



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washed twice with saline solution. MTT (500 μg/mL) dissolved in saline solution was added to the 96-well microplate for 1 h at 37 °C (300 rpm) in the dark. Finally the MTT incubation media was removed and cells were washed once before the addition of SDS at 5%. Colored formazan salt aggregates were measured at 595 nm in a spectrophotometer plate reader. Measurements of Mitochondrial Membrane Potential. Changes in mitochondrial membrane potential were measured with the fluorescent probe TMRE. SH-SY5Y cells were seeded at 200 000 cells per well. TMRE was used at 200 nM. After incubation with drugs, the mitochondrial membrane potential was measured. Cells were incubated for 30 min at 37 °C and 300 rpm. Finally, the cells were washed once with calcium saline solution. The loaded fluorescent neuroblastoma cells were measured in a microplate reader at 549 nm (exc) and 575 nm (em) in saline solution plus 0.2% BSA. Mitochondrial Permeability Transition Pore Opening Measurements. Calcein-AM was used to evaluate mPTP opening. Mitochondria were loaded over 1 h with 10 nM calcein/acetoxymethyl ester at 37 °C and 300 rpm in the presence of 1 mM CoCl2 to quench calcein fluorescence except in the mitochondrial matrix. The solution employed for cell charging was Umbreit saline solution plus BSA (1 mg/mL). Once the cells were loaded, they were washed with saline solution that contained Ca2+ (1 mM) and kept on ice until use. Calcein excitation and emission are 494/517 nm, respectively. Lasers used for excitation and emission were 476 and 510 nm with the confocal laser scanning microscope Nikon D-Eclipse C1. The images were collected using a 40× oil inmersion objective (Nikon). Clusters of mitochondria were selected as regions of interest. Sequential digital images were acquired every 30 s over 15 min. All experiments were carried out in triplicate. Measurements of Cytosolic Free Calcium. Cells were seeded onto 18 mm glass coverslips and used between 48 and 72 h after plating at a density of 120 000 cells/glass coverslip. For calcium measurements, cells were washed twice with saline solution supplemented with 0.1% BSA. Physiological saline solution (Umbreit) was composed of the following (mM): NaCl 119, Mg(SO4) 1.2, NaH2PO4 1.2, NaHCO3 22.85, KCl 5.94, CaCl2 1. Glucose (1 g/L) was added to the medium. In all the assays the solutions were equilibrated with CO2 before being used, adjusting the final pH between 7.2 and 7.4. The cells were loaded with the calcium-sensitive fluorescent dye FURA-2AM (0.5 μM). Neuroblastoma cells were plated on coverslips and shaken for 6.5 min at 37 °C and 300 rpm in a saline solution plus 0.1% BSA. Loaded cells were washed twice with calcium saline solution, and coverslips were placed in a thermostatic chamber (Life Sciences Resources). Cells were viewed using a Nikon Diphot 200 microscope equipped with epifluorescence optics (Nikon 40× immersion UV-Fluor objective). Addition of drugs was made by aspiration and the addition of a fresh bathing solution to the chamber. Cytosolic calcium ratio was obtained from the images collected by fluorescent equipment (Lambda-DG4). The light source was a xenon arc bulb, and the different wavelengths used were chosen with filters. The FURA-2AM cells were excited at 340 and 380 nm light alternately, and emission was collected at 510 nm. The composition of the bath solution used in these experiments was as follows. Saline solution (mM): NaCl 119, MgSO4 1.2, NaH2PO4 1.2, NaHCO3 22.85, KCl 5.94, glucose 0.1%, CaCl2 1. Calcium-free saline solution (mM): NaCl 119, Mg(SO4) 1.2, NaH2PO4 1.2, NaHCO3 22.85, KCl 5.94, glucose 0.1%. The experiments were carried out at least in triplicate. Statistical Analysis. In each experiment, the intracellular calcium ratios are the average of approximately 30 cells. All experiments were carried out a minimum of three times in duplicate. Results were analyzed using the Student’s t test for unpaired data. A probable level of 0.05 or smaller was used for statistical significance. Results were expressed as the mean ± SEM.

phenyl]ethyl]isothiourea methanesulfonate (KB-R7943) was from Calbiochem Corporation. Calcein-AM was purchased from Invitrogen. Collection of Biological Material. The strain of Phormidium autumnale was purchased from the Culture Collection of Algae and Protozoa (UK, CCAP1446/10) and grown at 20 °C in a BG-11 medium for 15 days. The species identification was performed by 16S rRNA analysis by comparison with a 1098 bp fragment (GenBank DQ493874.1). The biomass was then harvested, centrifuged, and freeze-dried to obtain 11.2 g of a dry weight biomass. Extraction and Isolation. The cyanobacterium (11.2 g dry wt) was extracted repeatedly with CH2Cl2/MeOH (1:1) to yield 0.7 g of an organic extract. This extract was then subjected to C18 vacuum liquid chromatography successively eluted by H2O/MeOH (2:1), H2O/MeOH (1:1), H2O/MeOH (1:2), MeOH, and MeOH/CH2Cl2 (1:1). The MeOH/CH2Cl2 (1:1) fraction was purified by semipreparative reversed-phase HPLC (Waters XSelect Phenyl-Hexyl, 250 × 10 mm, 5 μm, eluting from H2O/CH3CN, 70:30 to 0:100) to afford 1. Pure compound 1 (3.8 mg) was obtained after an additional purification step by semipreparative reversed-phase HPLC (Waters Symmetry C18, 300 × 7.8 mm, 7 μm eluting with isocratic conditions H2O/CH3CN, 62:38). Autumnalamide (1): colorless, amorphous solid; [α]20D = −15 (c 0.27, MeOH); UV (MeOH) λmax (log ε) 258 nm (2.89); IR (ATR) νmax 3310, 2960, 2930, 2870, 1650, 1620, 1590, 1520, 1450, 1400, 1350, 1310, 700 cm−1; for 1H and 13C NMR data (DMSO-d6) see Table 1; HRESIMS m/z [M + H]+ 901.49300 (calcd for C46H64N10O9, 901.4936). Computational Details. All calculations were conducted using Gaussian 09, Rev D.01.45 Geometry optimization was performed at the B3LYP/6.31 level. Magnetic properties were computed at a higher level of theory, i.e., B3LYP/6-31(g), in order to obtain better estimates of the chemical shifts. The difference between calculated and experimental chemical shifts was determined using the best estimation as reference (Δppm − min(Δppm)). Cell Culture. The neuroblastoma cell line SH-SY5Y was purchased from ATCC (CRL-2266). Cells were plated in a 25 cm3 flask at a cultivation ratio of 1:10. The cells were maintained in Eagle’s minimum essential medium from ATCC and F12 medium (Invitrogen) in 1:1 proportion supplemented with 10% fetal bovine serum from PAA Laboratories, 100 UI/mL penicillin, and 100 μg/mL streptomycin. The neuroblastoma cells were resuspended weekly using 0.05% trypsin/EDTA (1×) (Invitrogen). Whole-Cell Patch-Clamp Electrophysiology. Cells were seeded onto 18 mm glass coverslips and used between 48 and 72 h after plating at a density of 120 000 cells/glass coverslip. Whole cell patchclamp recordings were reached by careful mechanical suction of the membrane. The experiments were performed at room temperature (20−25 °C). To obtain the maximum homogeneity, only cells with minimal or no processes were used for the experiments. The electrode resistance was 5−7 Ω, and the pipet was filled with an internal solution containing the following (mM): Cs-gluconate 110, NaCl 3.7, MgCl2 5, HEPES 10, EGTA 5, Na2ATP 5, pH 7.2 (adjusted with CsOH), and the osmolarity was adjusted to 270 mOsmol. Cells were bathed with extracellular solution containing the following (mM): NaCl 137, KCl 4, MgCl2 1, HEPES 10, TEA-Cl 10 (tetraethylammonium chloride), glucose 10, pH 7.4 (adjusted with NaOH), and adjusted to 300 mOsmol. Electrophysiological recordings were done with a computercontrolled current and voltage clamp amplifier (Multiclamp 700B, Molecular Devices). Signals were recorded and analyzed using a Pentium computer equipped with a Digidata 1440 data adquisition system, and pClamp software (Molecular Devices) pClamp 10 was used to generate current and voltage- clamp commands and to record the resulting data. Signals were filtered at 10 kHz. Series resistances were compensated at 80%. After whole cell configuration, cells were allowed to stabilize 5 min before current recording to allow the equilibration between the internal pipet solution and the cell cytoplasm. Culture medium was washed twice before the addition of patch extracellular recording solution. Viability Assay. Cells were incubated with different concentrations of 1 ranging from 0.1 to 25 μM for 1 h. After the incubation, cells were H



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ASSOCIATED CONTENT

S Supporting Information *

All NMR and MS spectra for 1. Conformational analyses for the four possible diastereoisomers. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: +33(0)49207-6134. Fax: +33(0)49207-6189. *E-mail: [email protected]. Tel: +34 982 822 233. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Author Contributions ∇

C. Audoin and J. A. Sánchez contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support was given to C.A. through a grant from the French ANRT and through the European Union’s Seventh Framework Program managed by REA (Research Executive Agency) (FP7/2007-2013) under grant agreement 265896 BAMMBO (www.bammbo.eu). The computation facilities were provided by the Institut de Développement et de Ressources en Informatique (IDRIS, Orsay) under the project OUMOLPO N100483. We are grateful to M. Gaysinski (PFTC Nice) and J.-M. Guigonis (PF Bernard Rossi) for NMR and HRMS analyses, respectively. C. Meinert is also deeply thanked for enantioselective GC/MS analysis.

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Autumnalamide, a Prenylated Cyclic Peptide from the Cyanobacterium

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