Antitrypanosomal Activity of Isololiolide Isolated from the Marine Hydroid T Macrorhynchia Philippina (Cnidaria, Hydrozoa) Marta L

Bioorganic Chemistry 89 (2019) 103002

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Bioorganic Chemistry

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Antitrypanosomal activity of isololiolide isolated from the marine hydroid T Macrorhynchia philippina (Cnidaria, Hydrozoa) Marta L. Limaa, Maiara M. Romanellia, Samanta E.T. Borboremaa, Deidre M. Johnsb, ⁎ ⁎ ⁎ Alvaro Esteves Migottoc,1, , João Henrique G. Lagod,1, , Andre G. Temponea,1, a Centre for Parasitology and Mycology, Instituto Adolfo Lutz, São Paulo 01246-000, Brazil b Department of Biomedical Sciences, Oregon State University, Oregon 97331, USA c Centro de Biologia Marinha, Universidade de São Paulo, São Sebastião 11600-000, Brazil d Center of Natural Sciences and Humanities, Federal University of ABC (UFABC), Santo Andre 09210-580, Brazil

ARTICLE INFO ABSTRACT

Keywords: Marine invertebrates are a rich source of small antiparasitic compounds. Among them, Macrorhynchia philippina Macrorhynchia philippina is a chemically underexplored marine cnidarian. In the search for candidates against the neglected protozoan Marine invertebrate Chagas disease, we performed a bio-guided fractionation to obtain active compounds. The structural char- Stinging hydroid acterization of the active compound was determined using NMR analysis and MS and resulted in the isololiolide, Isololiolide a compound described for the first time in this species. It showed in vitro activity against both trypomastigote and Therapy intracellular amastigotes of Trypanosoma cruzi, with IC values of 32 µM and 40 µM, respectively, with no Trypanosoma cruzi 50 Mechanism of action mammalian cytotoxicity (> 200 µM). The lethal action was investigated in T. cruzi using different fluorophores to study: (i) mitochondrial membrane potential; (ii) plasma membrane potential and (iii) plasma membrane permeability. Our results demonstrated that isololiolide caused disruption of the plasma membrane integrity and a strong depolarization of the mitochondrial membrane potential, rapidly leading the parasite to death. Despite being considered a possible covalent inhibitor, safety in silico studies of isololiolide also considered neither mutagenic nor genotoxic potential. Additionally, isololiolide showed no resemblance to interference compounds (PAINS), and it succeeded in most filters for drug-likeness. Isololiolide is a promising candidate for futureop- timization against Chagas disease.

1. Introduction The longer the T. cruzi infection endures, lower is the efficacy of the only two drugs available for Chagas treatment, nifurtimox and benz- Chagas disease (CD), also known as American Trypanosomiasis, is nidazole (BZN) [4]. In addition, both drugs require prolonged treat- an infection caused by the protozoan hemoflagellate Trypanosoma cruzi ment, display a wide range of side effects and treatment responsiveness (Kinetoplastida, Trypanosomatidae). Although originally endemic in varies according to the T. cruzi strain [5]. Latin American, population movements have contributed to its Among several lines of research that may lead to the discovery of spreading across the globe raising an emergent worldwide public health new compounds with anti-T. cruzi activity, marine organisms such as issue [1]. According to the World Health Organization, 8 million people sponges [6–8] colonial tunicates [9], brown alga [10] have demon- are infected with T. cruzi and more than 7000 deaths are counted per strated their chemical potential for novel antitrypanosomal compounds. year [2]. Whilst some groups of marine organisms have been fairly well char- Clinically, this disease encompasses an acute phase characterized by acterized [11], others are scarcely investigated. This is the case of the peaks of parasitemia in bloodstream producing unspecific symptoms marine hydroid Macrorhynchia philippina (Cnidaria, Hydrozoa). To date, hardly diagnosed. In absence of an specific treatment, most cases evolve only two reports have chemically examined extracts of M. philippina, to chronic asymptomatic infection or to cardiomyopathy, gastro- being macrophilones the sole compounds described so far in this or- intestinal (megacolon, megaesophagus) and neurological disorders [3]. ganism, or even in the entire genus Macrorhynchia [12,13]. Regarding

⁎ Corresponding authors at: Center for Parasitology and Mycology, Instituto Adolfo Lutz, Avenida Dr. Arnaldo, 351, 8° andar, 01246-000 São Paulo, Brazil (A.G. Tempone). E-mail addresses: [email protected] (A.E. Migotto), [email protected] (J.H.G. Lago), [email protected] (A.G. Tempone). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.bioorg.2019.103002 Received 11 December 2018; Received in revised form 16 May 2019; Accepted 19 May 2019 Available online 20 May 2019 0045-2068/ © 2019 Elsevier Inc. All rights reserved. M.L. Lima, et al. Bioorganic Chemistry 89 (2019) 103002 the biological potential, macrophilones were found cytotoxicity in 10% FBS supplementation (RPMI-1640-10% FBS). Bone marrow-de- cancer cell lines via complex mechanisms involving, in some manner, rived macrophages (BMM) were obtained in vitro from BALB/c bone the disruption of SUMO-protein regulation, oxidative stress, and low- marrow precursor cells in the presence of the growth factor M-CSF ering the cellular levels of specific kinases of the ERK signal cascade secreted by L929 cells as described elsewhere[14]. Macrophages were [12,13]. In the search for new candidates for CD, in this work, we in- collected from the peritoneal cavity of BALB/c mice by washing with vestigated the potential of M. philippina to provide active compounds RPMI-1640 medium supplemented with 10% FBS and were maintained against Trypanosoma cruzi parasites. Additionally, we studied the pre- at 37 °C in a 5% CO2-humidified incubator. vailing lethal mechanisms in T. cruzi, investigating by different fluori- metric assays, the affected cellular targets. 2.5. Cytotoxicity in bone-marrow derivate macrophages (BMM)

2. Material and methods To determine the 50% cytotoxic concentration (CC50), BMM cells were seeded at 1 × 105 cells/well in 96-well microplates and incubated 2.1. General experimental procedures with isololiolide during 48 h at 37 °C in 5% CO2-humidified incubator. The isololiolide was dissolved in DMSO and serially diluted (2-fold) NMR spectra were recorded at 500 in a Bruker Ultrashield 500 from the highest concentrations of 200 μM in RPMI-1640-10% FBS. spectrometer. CDCl3 (Aldrich) was used as the solvent with TMS as the Benznidazole was used as the standard drug for reference. Controls internal standard. HRESIMS spectrum was measured on a Bruker wells were established by the absence of compound (100% viability) Daltonics MicroTOF QII spectrometer. High performance liquid chro- and medium free of cells (blank). Cell viability was determined by the matography (HPLC) analysis was performed in a Dionex Ultimate 3000 MTT assay [15]. The final concentration of DMSO never exceeded 0.5% chromatograph, using a Luna Phenomenex RP-18 columns (5 μm, assuring non-toxic concentrations. 150 × 4.6 mm to analytical and 250 × 10 mm to semi-preparative) and an UV-diode array detector (DAD). Silica gel (Merck, 230–400 mesh) 2.6. Antitrypanosomal activity and Sephadex LH-20 (Sigma-Aldrich) were used for column chromatography (CC). For all extraction and chromatography procedures, To determine the 50% inhibitory concentration (IC50) against T. analytical grade solvents (Labsynth Ltda) were used. cruzi trypomastigotes 1 × 106 cells/well were seeded in 96-well mi- Benznidazole (BZN) [N-benzyl-2-(2-nitro-1H-imidazol-1-yl) acet- croplates and incubated with the compound isololiolide serially diluted amide] was donated by Pharmaceutical Laboratory of the State of (2-fold, highest concentration of 100 μM) in medium RPMI-1640-2%

Pernambuco – LAFEPE. Roswell Park Memorial Institute (RPMI) 1640 FBS during 24 h at 37 °C in a 5% CO2-humidified incubator. without phenol red, Hank’s Balanced Salt Solution (HBSS), fetal bovine Benznidazole was used as the standard drug. The parasite viability was serum (FBS), Triton X-100, FCCP (carbonyl cyanide 4-(tri- determined using the resazurin assay (0.011% in PBS) [16]. Internal fluoromethoxy)phenylhydrazone) were purchased from Sigma Aldrich, controls consisted of untreated trypomastigote (100% viability) and USA. Alamar blue (resazurin), SYTOX Green, rhodamine 123, medium free of cells (blank). The optical density was read at 570 nm DiSBAC2(3) (bis-(1,3-diethylthiobarbituric acid) trimethine oxonol) using a FilterMax F5Multi-Mode Microplate Reader (Molecular De- were purchased from Molecular Probes® (Thermo Fisher Scientific). vices). To determine the IC50 against T. cruzi intracellular amastigotes, BMM cells were seeded into 16-well chamber slides (NUNC, Thermo, 2.2. Marine invertebrate collection USA) at 1 × 105 cells/well. After 24 h, non-adherent macrophages were removed by two-step washings and the infection performed at the ratio

The colonies of the cnidarian Macrorhynchia philippina were col- 1:5 (macrophage: trypomastigotes) for 4 h at 37 °C in a 5% CO2 humi- lected by scuba diving at a depth of 1 to 14 m, in May 2007, at several dified incubator [17]. Subsequently, infected cells were incubated with localities in the São Sebastião Channel, near the Centre for Marine the isololiolide during 48 h. Finally, the slides were fixed with me- Biology of the University of Sao Paulo, São Sebastião, São Paulo State, thanol, stained with Giemsa, and observed in light microscopy. The Brazil. The material was brought to the laboratory, washed several parasite load was defined by counting 200 macrophages/well by eval- times with filtered seawater to remove the epifauna, identified, and uating the infection index (infected macrophage × amastigotes/total immediately frozen. macrophage). Benznidazole was used as standard drug.

2.3. Extraction and Isolation 2.7. Effects on mitochondrial membrane potential (ΔΨm) in T. cruzi

The cnidarian (102 g) was exhaustively extracted with MeOH Changes in the ΔΨm were determined using the mitochondrial specific (10 × 200 mL) and filtered. After partial evaporation of the solvent at dye, rhodamine 123 (Rd123) by flow cytometry. Briefly, trypomastigotes 6 reduced pressure, an equal volume of H2O was added. The MeOH:H2O (2 × 10 /well) were incubated in HBSS supplemented with 10 mM glu- 1:1 solution was sequentially partitioned against hexane (4 × 100 mL) cose (HBSS + Glu) with isololiolide at IC50 and IC99 values during and EtOAc (5 × 100 mL) to afford, respectively, 158 mg and 347 mgof 120 min. Afterwards, parasites were washed and stained with Rd123 at each phase. As bioactivity was detected in the EtOAc phase, part of this 0.3 µg/mL during 15 min, at 37 °C, in the dark. The washed parasites were material (300 mg) was subjected to Sephadex LH-20 column chroma- resuspended in PBS and propidium iodide (PI) at 5 µg/mL were added tography (30 × 3 cm) eluted with MeOH to afford 15 fractions. As immediately before acquisition to exclude non-viable cells from Rd123 bioactivity was detected on fraction 6 (12 mg), it was purified over si- measurements. Untreated trypomastigotes and treated with oligomycin lica gel column chromatography (12 × 2 cm) eluted with n-hexane- (20 μM) were used to established minimal and maximal mitochondrial EtOAc (8:2, 7:3 and 1:1) to afford 1.2 mg of pure isololiolide. depolarization, respectively. Flow cytometry was performed using an Attune NxT Acoustic Focusing Cytometer (Thermo Fisher Scientific) by 2.4. Parasites and mammalian cells analyzing 10,000 gated events using BL1-A filter 530/30 nm for Rd123 and BL2-A filter 574/26 nm for PI, as previously described [16]. Trypanosoma cruzi (Y strain) were maintained in Rhesus-monkey kidney cells (LLC-MK2, ATCC CCL-7) using RPMI-1640 medium sup- 2.8. Effects on the plasma membrane in T. cruzi plemented with 2% FBS (RPMI-1640-2% FBS) at 37 °C in 5% CO2 humidified incubator. LLC-MK2 and murine conjunctive cells (NCTC clone Permeabilization of the plasma membrane was evaluated by spec- 929, ATCC CCL-1) were maintained in the same conditions, except for trofluorimetry through monitoring the fluorescence of Sytox Green dye,

2 M.L. Lima, et al. Bioorganic Chemistry 89 (2019) 103002 as previously described elsewhere [17]. Briefly, T. cruzi trypomastigotes Table 1 (2 × 106/well) were firstly stained with 1 µM of Sytox Green dye during Antitrypanosomal activity and mammalian cytotoxicity of isololiolide. 5 min in HBSS + Glu in the dark. Then, the isololiolide was added at the Compound IC50 μM ( ± SD) IC50 μM ( ± SD) CC50 μM SI IC50 and IC99 values, and fluorescence monitored up to 120 min. Un- Trypomastigotes Amastigotes treated trypomastigotes or treated with 5% Tx100 detergent solution were used as parameters for minimal and maximal plasma membrane isololiolide 31.9 ± 8.0 40.4 ± 7.4 > 200.0 > 5.0 BZN 16.2 ± 3.7 5.3 ± 0.3 > 200.0 > 38.4 permeabilization, respectively. Changes in the plasma membrane potential (ΔΨp) were evaluated by flow cytometry monitoring the fluor- IC50: 50% inhibitory concentration; CC50: 50% cytotoxic concentration; 6 escence of DiSBAC2(3) dye. Briefly, T. cruzi trypomastigotes (2 × 10 / Selectivity index (SI) calculated from cytotoxicity/amastigote activity. BZN: well) were treated with isololiolide at the IC50 and IC99 values in benznidazole. SD: standard deviation. HBSS + Glu at 37 °C for 60 min. Washed trypomastigotes were resuspended in 0.2 µM DiSBAC2(3) solution prepared in PBS, and the staining was allowed during 10 min in the dark. As controls, trypo- human lung cell lines and demonstrated no cytotoxicity up to 600 µM mastigotes treated with gramicidin D (0.1 µg/mL) or untreated pro- [20]. It is worthy to mention that mammalian toxicity has been the vided information on the maximal or minimal plasma membrane de- major limiting factor among potent anti-T. cruzi compounds isolated polarization, respectively. Flow cytometry was performed using an from other natural sources [6,7,9]. The isololiolide demonstrated se- Attune™ NxT Acoustic Focusing Cytometer (Thermo Fisher Scientific) lectivity against trypomastigotes and amastigotes forms of T. cruzi, by analyzing 10,000 gated events using BL2-A (585/40 nm) channel, as showing IC50 values of 31.9 µM and 40.4 µM, respectively, and SI previously described [18]. higher than 2.5 (Table 1). Despite a moderate in vitro potency against T. cruzi compared to the 2.9. Statistical analysis standard drug BZN, the isololiolide was active against both forms of the parasite involved in the human infection. Such capability is critical, The Inhibitory Concentrations and 50% Cytotoxic Concentration since compounds that only target intracellular amastigotes may leave values were calculated using sigmoidal dose-response curves using the infective trypomastigotes capable of maintaining infection after GraphPad Prism 5.0 software. Unless otherwise stated, the reported treatment [21]. data correspond to the mean ± standard deviation of at least two in- Despite our present work demonstrates for the first time the iso- dependent experiments performed with samples in duplicate. One-way loliolide in M. philippina, its occurrence has been demonstrated among ANOVA of variance with Tukey's Multiple Comparison Test was used other organisms, but with very limited investigations concerning its for significance test (P value). biological/pharmacological potential. Isololiolide was isolated from the brown alga Homoeostrichus formosana and the green alga (sea lettuce), Ulva sp., but no biological activity was attributed to the compound 3. Results and discussion [22]. A new isololiolide derivative, named schiffnerilolide, was isolated from the marine brown alga Cystoseira schiffneri, collected from the 3.1. Chemical characterization Tunisian marine coast [23]. The compound was also found in the brown

1 algae Cystoseira tamariscifolia and demonstrated anticancer activity on H NMR spectrum (500 MHz, CDCl3) of isolated compound dis- hepatocarcinoma HepG2 cells, modulating apoptosis-related proteins played three singlets at δ 1.27 (3H), 1.33 (3H) and 1.59 (3H) assigned [20]. Isololiolide isolated from the aquatic perennial fern Marsilea cre- to methyl groups H-9, H-10 and H-11, respectively, one singlet at δ 5.77 2 nata, demonstrated phytotoxic activity with potential use in organic (1H) assigned to hydrogen H-7 linked to sp carbon, one multiplet at δ agriculture [24]. The compound has also been found in terrestrial 4.10 (1H), attributed to hydrogen H-3 linked to carbinolic carbon, and plants, as Vitex leptobotrys [25], Tussilago farfara [26], Leucas aspera multiplets at δ 2.45 (2H) and 1.99 (2H) corresponding, respectively, to [27], Achillea millefolium [28] and Viburnum dilatatum [29], but no hydrogens H-4 and H-2. These data associated to analysis of HRESIMS, + biological/pharmacological activity was described. in which was observed the [M + Na] peak at m/z 219.0993 (calcd. for Assuming that parasite persistence has been stressed as a primary C11H16O3Na 219.0997), allowed the identification of isololiolide cause of pathogenesis in chronic T. cruzi infections [30,31], and the (Fig. 1) [19]. complete eradication of the parasite may depend on the ability of compounds to disturb vital processes irreversibly, the mechanism of 3.2. In vitro efficacy against T. Cruzi and lethal mechanisms isololiolide was investigated in trypomastigotes of T. cruzi. In contrast, in higher eukaryotes, apoptosis follows a canonical regulated pathway The mammalian cytotoxicity and the antitrypanosomal activity of that still lacks a convincing description in protozoa parasites such as the compound isololiolide obtained from the methanolic extract of M. Trypanosoma spp. and Leishmania spp. (Trypanosomatids) [32]. None- philippina were evaluated by the colorimetric MTT and resazurin assays, theless, the biochemical and morphological hallmarks of apoptosis are respectively. The isololiolide showed no cytotoxicity towards mam- consistently described in these parasites. For instance, the loss of ΔΨm malian BMM cells at 200 µM. Our data corroborate the results obtained induced by bioactive compounds is widely described as a prominent elsewhere, where isololiolide was tested in foreskin fibroblasts and feature also on the apoptosis-like mechanism of Trypanosomatids [33–36]. Considering the higher efficacy of isololiolide in Trypanosoma cruzi, we investigated the potential cellular targets of the compound in the parasite. In our studies, T. cruzi treated for 120 min with isololiolide at

the 99% Inhibitory Concentration (IC99) showed a decrease in 46% of the ΔΨm in viable trypomastigotes (PI-negative) compared to non-depolarized untreated trypomastigotes, but also produced 59.7% of PI- positive cells (Fig. 2A). PI-positive parasites indicate cell death by the loss of plasma membrane integrity. In particular, membrane integrity is conserved in classical apoptosis [37], although changes in the plasma Fig. 1. Structure of isololiolide, isolated from Macrorhynchia philippina membrane potential (ΔΨp) may occur as a very early event [38]. (Cnidaria, Hydrozoa). Conversely, the rupture of the plasma membrane of the trypomastigotes

3 M.L. Lima, et al. Bioorganic Chemistry 89 (2019) 103002

Fig. 2. Effects of isololiolide isolated from methanol extract of the marine hydroid M. philippina on mitochondrion and plasma membrane of T. cruzi trypomastigotes.

(A) Flow cytometry dot-plot graphs showing Rd123 fluorescence (variation of ΔΨm), PI fluorescence (cell viability) and percentages relative to the numbers of cellsin a sub-population. Trypomastigotes treated with isololiolide at IC50 and IC99 or oligomycin (20 µM) during 120 min showed Rd123 fluorescence intensities of 99 ± 5%, 54 ± 2% or 24 ± 1%, respectively, respect to non-depolarized untreated trypomastigotes (100 ± 3%). PI-positive cells were removed from Rd123 relative percentages. (B) Evaluation of plasma membrane integrity by monitoring of Sytox Green fluorescence. Trypomastigotes were previously stained with 1µM

Sytox Green and the fluorescence monitored spectrofluorimetrically up to 120 min in presence of isololiolide atIC50 and IC99 or Triton X-100 used for 100% permeabilization. *p < 0.05 relative to untreated parasites. C) Evaluation of the plasma membrane potential (ΔΨp) by monitoring of DiSBAC2 (3) fluorescence.

Staining using 0.2 µM DiSBAC2 were performed after 60 min incubation of trypomastigotes presence of isololiolide at IC50 and IC99 or gramicidin D (0.5 µg/mL) and the fluorescence quantified by flow cytometry. Data represent normalized fluorescence ± SD respect2 toDiSBAC intensity of gramicidin D-treated trypomastigotes (maximal depolarization). *p < 0.05. For all, one representative assay from two performed independent assays is shown.

Table 2 Table 3 Predicted ADMET properties for isololiolide. Physiochemical properties calculated for isololiolide.

Property isololiolide Property Calculated value

I. Toxicity MW 196 AMES mutagenicity2 non-mutagenic (low reliability) logP1 1 Genotoxicity1 no risks identified logD 1.11 hERG inhibition2 non-inhibitor tPSA 46.53 Human hepatotoxicity2 non-toxic Solubility Forecast Index Good Solubility Liver toxicity (DILI) 2 indeterminate Fsp3 0.73 CYP 3A3 inhibition non-inhibitor Endocrine disruptor1 no ERα binding The XlogP3 model in FAFdrugs4 was used. Phospholipidosis3 non-inducer

II. Pharmacokinetic behavior caco-2 permeability2 high permeability (-4.5 cm/s) by isololiolide at IC99 up to 60 min of incubation, as determined by the PGP substrate1 non-substrate potential-sensitive fluorescent probe DiSBAC(3) (Fig. 2C). PGP inhibitor1 non-inhibitor Rupture of the plasma membrane is a characteristic feature usually 3 Solubility good solubility associated with necrotic mechanisms [39], not to mention that sec- CYP1A2 inhibitor 2 non-inhibitor 2 ondary necrosis is considered the typical outcome of apoptosis in uni- CYP1A2 substrate non-substrate 3A4 inhibitor2 non-inhibitor cellular eukaryotes as T. cruzi [40]. Additionally, differences in plasma CYP3A4 substrate 2 indeterminate membrane composition between trypanosomatids and mammalian cells CYP2C9 inhibitor 2 non-inhibitor support drug selectivity [41]. For instance, the higher binding affinity 2 CYP2C9 substrate non-substrate of the antileishmanial approved drug amphotericin B towards the Clearance2 low clearance Pan Assay Inhibitor (PAIN) structure alerts3 None parasite ergosterol, than its mammalian counterpart, the cholesterol, results in channel-like structures (pores), increasing the permeability of Predictions based on ACD labs (1), ADMETlabs (2) and FAF drugs4 (3). the plasma membrane, leading to cellular death [42]. The lethal mechanism of isololiolide in T. cruzi may be comparable to an amphotericin B-like mechanism in Leishmania, associated with plasma mem- treated with isololiolide at IC99 was also confirmed, with an increased brane disruption or an apoptosis-like mechanism, resulting in early fluorescence level (62%) of Sytox Green dye after 120 min of incubation mitochondrial dysfunction and necrosis. However, further experiments

(Fig. 2B). Besides, an early depolarizing effect onp ΔΨ was not triggered must be addressed to confirm this hypothesis. It is important tonote

4 M.L. Lima, et al. Bioorganic Chemistry 89 (2019) 103002 that both mechanisms lead to irreversible parasite death. In contrast to Appendix A. Supplementary material the thousands of mitochondria found in mammalian cells, T. cruzi possesses a single mitochondrion that has been advocated as a drug Supplementary data to this article can be found online at https:// target [43]. doi.org/10.1016/j.bioorg.2019.103002.

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Porcelia macrocarpa (Annonaceae) against trypomastigotes of Trypanosoma cruzi: Effect of octadec-9-ynoic acid in plasma membrane electric potential, Bioorg. Chem. Acknowledgements 78 (2018) 307–311. [19] J. Kimura, N. Maki, New loliolide derivatives from the brown alga Undaria pin- natifida, J. Nat. Prod. 65 (2002) 57–58. The authors thank Ligia F. Martins and Mariana K. Galuppo for [20] C. Vizetto-Duarte, L. Custódio, K.N. Gangadhar, J.H. Lago, C. Dias, A.M. Matos, technical assistance in assays. The authors thank São Paulo State N. Neng, J.M. Nogueira, L. Barreira, F. Albericio, A.P. Rauter, J. 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