Antiprotozoal Activity Against Plasmodium falciparum. and Trypanosoma cruzi. of Aeroplysinin-1 Isolated from the New Sponge Aplysina chiriquensis.

ABSTRACT

Crude methanol extracts from the recently discovered sponge, Aplysina chiriquensis. and Aplysina gerardogreeni., collected at the southwest Pacific Coast of Panama, showed moderate antiplasmodial activity. Fractionation of the crude extracts from A.. chiriquensis. led to the isolation of the known dibromotyrosine derivatives 1–5. Compound 3, aeroplysinin-1, displayed antiprotozoal activity against chloroquine-resistant Plasmodium falciparum. and Trypanosoma cruzi.. Comparative analysis by HPLC of the five specimens collected showed the presence of the same metabolites but in different relative proportions. Compounds 1–5 are reported for the first time as constituents of A.. chiriquensis. and A.. gerardogreeni.. This is the first report of the antiprotozoal activity of aeroplysinin-1 (3).

Keywords:

• Aeroplysinin-1
• antimalarial
• antitrypanosomal
• Aplysina. chiriquensis
• Aplysina gerardogreeni
• dibromotyrosines
• Plasmodium falciparum
• Trypanosoma cruzi

Introduction

Tropical diseases such as malaria, Chagas disease, leishmaniasis, and dengue fever impose an enormous burden on developing countries, contributing to poverty and underdevelopment (Sachs, 2002). Collectively, the parasitic diseases malaria, Chagas disease, and leishmaniasis affect 3 billion people, most of whom survive on less than $2 a day (Gelb & Hol, 2002). Malaria kills between 1 and 2 million people annually, and half of humanity is at risk (Greenwood, 2004). Approximately 100 million inhabitants of Latin America live in areas considered to present risk of infection by Trypanosoma cruzi., the causative agent of Chagas disease, which is considered one of the largest public health problems in the Western Hemisphere (Lockman et al., 2005).

Given the continuous emergence of drug-resistant Plasmodium falciparum. strains (Greenwood et al., 2002) and the low effectiveness and side effects that present the drugs actually used to treat Chagas disease (Mecca et al., 2002), there is an urgent need for the discovery and development of new antiprotozoal drugs. Marine natural products have proved to be a rich source of bioactive compounds that are being investigated, mainly as anticancer drugs (Newman & Cragg, 2004; Simmons et al., 2005). In the case of tropical diseases, marine natural products have a largely untapped potential as novel antiprotozoal agents.

As a part of a collaborative program between the University of Santiago de Compostela and the Smithsonian Tropical Research Institute through the Panama International Cooperative Biodiversity Groups program (Panama ICBG), we are exploring the biological diversity of the Coiba National Park, the largest marine protected area of the Republic of Panama (Guzmán et al., 2004), using a variety of biological assays including cancer, malaria, leishmaniasis, and Chagas disease. In this communication, we report the antiplasmodial and antitrypanosomal activity of aeroplysinin-1 (3) (Fig. 1), a dibromotyrosine derivative isolated from a new sponge Aplysina chiriquensis. collected in the Coiba National Park and that will be described elsewhere.

Figure 1 Structures of compounds 1–5 isolated from A. chiriquensis. and A. gerardogreeni..

Materials and Methods

General experimental procedures

Optical rotations were measured on a JASCO DIP-360 polarimeter using a 1 dm path-length cell. NMR spectra were recorded at 250, 400, or 500 MHz using a Bruker DPX-250, Varian Inova-400, and Bruker AMX-500, respectively. HREIMS were measured on a Micromass Autospec mass spectrometer. Column chromatography was performed on silica gel 60 (70–230 mesh for VLC and 230–400 mesh for flash columns). HPLC purification was carried out on a Waters apparatus equipped with a refractive index detector and a Spherisorb 10 µm ODS2 semi-prep column (10 × 250 mm).

Animal material

Five sponge specimens (vouchers C1, C3, C10, C11, and C31) were collected by scuba diving in the Coiba National Park, located in the Gulf of Chiriquí, in the southwest Pacific Coast of the Republic of Panama, at 3–10 m depth in August 2002. The samples were frozen immediately after collection and stored at −80°C until extraction. Specimens C1, C10, and C11 were identified by Dr. Maria Cristina Diaz (Museo Marino, Venezuela) and Dr. Rob van Soest (University of Amsterdam, NL) as a new species named Aplysina chiriquensis.. The other specimens, C3 and C31, were identified as Aplysina gerardogreeni. (Gomez & Bakus, 1992). Voucher specimens are maintained for reference at the Smithsonian Tropical Research Institute.

Extraction and isolation

A fresh sample of the sponge A. chiriquensis. (C10, 2.2 kg) was extracted using the same protocol described previously (Gutiérrez et al., 2005). The crude extract (40.8 g) was subjected to solvent partition affording four fractions, (hexane, dichloromethane, n.-butanol, and water). The methylene chloride fraction (2.6 g) was subjected to column chromatography on silica gel eluting with methylene chloride-methanol mixtures (from 2.5% to 50% methanol) affording 11 fractions. Fractions 2 and 4 yielded compounds 1 (7.4 mg) and 2 (15.2 mg), respectively. The other fractions had a mixture of several components and were combined according to their TLC profile. Fractions 8 and 9 were pooled and subjected to HPLC purification (acetonitrile/water 4:6, 0.1% trifluoracetic acid, 1 ml/min, isocratic elution), to afford compounds 3 (10 mg), 4 (10 mg), and 5 (3 mg).

Sponge samples C1, C3, C11, and C31 were extracted and partitioned as described above for A. chiriquensis. (C10). The methylene chloride fractions of all specimens were compared by HPLC. A summary of the relative concentration of compounds 1–5 in each of the sponges used in this study is shown in Table 1.

Table 1.. Relative concentration (% by HPLC) of compounds 1–5 in the dichloromethane extracts of Aplysina. specimens C1, C3, C10, C11, and C31.

Species (specimen)	1	2	3	4	5
A.. chiriquensis. (C1)	2.12	4.24	35.30	22.98	2.01
A.. gerardogreeni. (C3)	1.13	4.84	41.96	28.56	1.52
A.. chiriquensis. (C10)	3.01	4.61	12.05	8.12	0.95
A.. chiriquensis. (C11)	2.06	4.27	58.06	7.47	1.58
A.. gerardogreeni. (C31)	6.76	9.38	9.99	11.07	1.21

Intracellular T. cruzi. Bioassay

The assay is based on the growth inhibition of the parasite and employs a β-galactosidase expressing transgenic T. cruzi. (Talahuen strain, clone C4), and growth of the intracellular form of T. cruzi. was determined from cleavage of chlorophenol red-β.-D-galactoside (CPRG, 570 nm) (Buckner et al., 1996).

Antiplasmodial activity

Antiplasmodial activity was determined in a chloroquine-resistant P. falciparum. strain (W2) using a novel microfluorimetric assay to measure the inhibition of the parasite growth based on the detection of the parasitic DNA by intercalation with PicoGreen (Corbett et al., 2004). P. falciparum. was cultured according the methods described by Trager and Jensen (1976). The parasites were mantained at 2% hematocrit in flat-bottom flasks (75 ml) with RMPI 1640 medium (GibcoBRL) supplemented with 10% human serum.

Results and Discussion

Five sponge specimens were collected in the Coiba National Park during a cruise operated by the Smithsonian Tropical Research Institute (STRI). Two of the specimens were identified as Aplysina gerardogreeni. (Gomez & Bakus, 1992), whereas the other samples represent a new species in the genus Aplysina. named A. chiriquensis. that soon will be described elsewhere.

The crude methanol extract of all five samples were investigated separately and showed moderate in vitro. activity against chloroquine-resistant P. falciparum.. The crude extract of A. chiriquensis. (C10) was subjected to fractionation using solvent partition followed by silica gel column chromatography and semipreparative HPLC purification to yield compounds 1–5. The structures of compounds 1–5 were readily deduced by comparison of their spectroscopic data (optical rotation, ¹³C NMR, ¹H NMR, and MS) with those of the literature (Sharma et al., 1970; Fattorusso et al., 1971; Minale et al., 1972; Ciminiello et al., 1994; Murakata et al., 1997) and shown to be metabolites found in other Aplysina. species.

A comparison by HPLC of the methylene chloride fractions of the five sponge specimens showed the presence of compounds 1–5 in each sponge, albeit in significantly different proportions (Table 1). Although A.. gerardogreeni. has been previously subjected to chemical studies, (Encarnación-Dimayuga et al., 2000) compounds 1–5 are described here for the first time as metabolites from this species. In the case of A. chiriquensis., this work constitutes the first chemical study of the sponge.

Compounds 1–5 were tested for their activity against a chloroquine-resistant P. falciparum. strain using a novel fluorescence-based bioassay developed through the Panama-ICBG program (Corbett et al., 2004). Aeroplysinin-1 (3) showed an IC₅₀ of 17.7 µM in the antiplasmodial assay, whereas compounds 1, 2, 4, 5 did not show measurable activity (Table 2). Compound 3 also showed in vitro. activity against intracellular T. cruzi. with an IC₅₀ value of 37.7 µM (Table 2). This is the first report of the antimalarial and the antitrypanosomal activity of compound 3 and although its antiprotozoal activity can be considered as moderate, these results constitute one of the very few examples of marine natural products with activity against T. cruzi..

Table 2.. Activity of compounds 1–5 against chloroquine-resistant Plasmodium falciparum. and intracellular Trypanosoma cruzi..

Compound	P. falciparum. IC50 (µM)	T. cruzi. IC50 (µM)
1	>50	ND
2	>50	ND
3	17.7	37.7
4	>50	ND
5	>50	ND
Chloroquine	0.072	ND
Nifurtimox	ND	11

ND, not determined.

Acknowledgments

We thank the Ministerio de Educación y Ciencia (MEC) and the Xunta de Galicia for financial support for this research (grant nos. BQU2002-01195, SAF2003-08765-C03-01, PGIDT02BTF20902PR, PGIDT03PXIC20908PN, PGIDIT04PXIC20903PN). Funding was also provided by the Smithsonian Tropical Research Institute, the AVINA Foundation, and the International Cooperative Biodiversity Groups program (grant nos. 1U01 TW01021-01 and 1U01 TW006634-01) from the National Institutes of Health, National Science Foundation, and the U.S. Department of Agriculture. We thank Mr. Carlos Guevara for field assistance, Dr. Maria Cristina Diaz for identifying the sponges, and the crew members of the R/V Urracá. for logistical support.