Identification of Renieramycin A as an Antileishmanial Substance in a Marine Sponge Neopetrosia sp.

The newly developed assay system using recombinant Leishmania amazonensis expressing enhanced green fluorescent protein (La/egfp) has been applied to the screening of Japanese marine sponges for antileishmanial activity. Bioassay-guided fractionation of an active sponge Neopetrosia sp. afforded an active compound which was identified as renieramycin A by spectroscopic analysis. It inhibited La/egfp with an IC50 value of 0.2 μg/mL.


Introduction
Leishmaniasis is caused by parasitic protozoans of the genus Leishmania spread by the bite of infected sand flies [1][2][3][4]. It is endemic in subtropical and tropical countries and approximately 2 million cases are estimated every year [5]. There are several forms of leishmaniasis, of which cutaneous and visceral leishmaniases are the most common. Pentavalent antimony compounds have been used for treatment of leishmaniasis since the 1940s, and more recently amphotericin B and other antifungal drugs are used as alternatives. However, these drugs have disadvantages including toxic effects [6][7][8]. Thus, less toxic antileishmanial drugs are urgently required.
In our continuing program on the discovery of drug leads from Japanese marine invertebrates, we screened 120 marine sponges for antileishmanial activity by the newly developed assay system using recombinant Leishmania amazonensis expressing enhanced green fluorescent protein as shown in Figure 1 (La/egfp) [9], and found promising activity in the lipophilic extract of Neospongia sp. collected in southern Japan. Bioassay-guided isolation furnished renieramycin A (1) as an active constituent. Here, we report the isolation, identification and antileishmanial activity of 1.

Results and Discussion
Since this sponge was known to contain highly cytotoxic renieramycin P (2: IC 50 0.53 nM against P388 cells) [10][11][12], bioassay-guided fractionation was carried out monitoring both leishmanicidal and cytotoxic activities to distinguish less toxic antileishmanial compounds from those with high toxicity. The organic layer of the extract was fractionated by the modified Kupchan procedure [13] to yield hexane, CHCl 3 , and 60 % MeOH layers. The CHCl 3 layer, which showed the most potent leishmanicidal and cytotoxic activity (IC 50  The FABMS of 1 exhibited an (M+4H+H) + ion at m/z 571, which corresponded to the hydroquinone form; in fact, ESIMS gave an (M+H) + ion at m/z 567. A database search using MarineLit TM suggested this pseudomolecular ion peak coincided with that of renieramycin A [14]. Analysis of 2D NMR data including the HOHAHA [15] and HMBC [16] spectra disclosed three spin systems and two quinone moieties which are the same as renieramycin A ( Figure 2). However, some of the chemical shift values obtained in CD 3 OD was not consistent with those of the literature. Comparison of 1 H-NMR data in the same solvent (CDCl 3 ) with those of the literature enabled us to assign the compound 1 was renieramycin A. Antileishmanial activity of renieramycin A (1) was evaluated using La/egfp. As shown in Figure  3, renieramycin A showed a dose-dependent inhibition against La/egfp with an IC 50 value of 0.2 µg/mL. On the other hand, it showed cytotoxicity against P388 murine leukemia cells at the ten times higher concentration (IC 50 2.2 µg/mL).

Conclusions
Several antileishmanial compounds including cyclic peroxides [17], pyridoacridine alkaloids [18], and manzamine alkaloids [19] have been reported from marine invertebrates. However, the number of antileishmanial compounds isolated from marine source is still limited. We adopted for the first time the newly developed bioassay using recombinant Leishmania amazonensis expressing enhanced green fluorescent protein (La/egfp) to the search of leishmanicidal metabolites from marine organisms, and isolated renieramycin A (1) from a marine sponge Neopetrosia sp. From the less cytotoxic fraction obtained after several steps of chromatographic fractionation, renieramycin A (1) was obtained as an active substance. As expected, 1 showed moderate selectivity for inhibition against La/egfp proliferation over cytotoxicity against P388 cells.
In this study, we have demonstrated the efficacy of the new assay using La/egfp for discovery study of antileishmainal compounds from natural source. NMR spectra were recorded on a JEOL A600 NMR spectrometer operating at 600 MHz for 1 H and 150 MHz for 13 C. Chemical shifts were referenced to the CD 3 OD signals (δ H 3.3 and δ C 49, respectively). FABMS spectra were measured on a JEOL JMS700 tandem mass spectrometer using NBA as a matrix. ESIMS data were obtained using JEOL AccuTOF JMS-T100LC.

Animal material
The animal specimens were collected by hand using SCUBA off Kuchinoerabu-jima Island in the Satsunan Islands (30°28'31"N; 130°11'73"E) in July 2001 and identified as Neopetrosia sp. by Dr. Rob van Soest, University of Amsterdam. They were immediately frozen and kept at -20 ℃ until processed.

Antileishmanial assay
Fluorescence signals of La/egfp promastigotes cultured in 199 medium (NISSUI Pharmaceutical, Tokyo, Japan) in 96-well plates at 25 °C were measured by a fluorescence microplate reader (Fluoro scan Ascent FL., Dainippon Pharmaceutical Co., Osaka, Japan) with excitation at 485 nm and emission at 538 nm. To determine the IC 50 (0.42 µg/mL) of amphotericin B (ICN, Ohio, USA), La/egfp were cultured at 5 x 10 5 cells/mL with various concentrations of the drug, and their fluorescence signals were measured after 72 h incubation.

Isolation
Frozen animals (1.5 kg) were exhaustively extracted with MeOH (2L) and EtOH (2L x 2), and the combined extracts were concentrated and partitioned between H 2 O and CHCl 3 . The organic layer was subjected to the modified Kupchan procedure [7]