leishmaniasis is caused by parasitic protozoans of the genus leishmania spread by the bite of infected sand flies [ 14 ] . it is endemic in subtropical and tropical countries and approximately 2 million cases are estimated every year . 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 [ 68 ] . 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 ) , and found promising activity in the lipophilic extract of neospongia sp . bioassay - guided isolation furnished renieramycin a ( 1 ) as an active constituent . here , since this sponge was known to contain highly cytotoxic renieramycin p ( 2 : ic50 0.53 nm against p388 cells)[1012 ] , 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   to yield hexane , chcl3 , and 60 % meoh layers . the chcl3 layer , which showed the most potent leishmanicidal and cytotoxic activity ( ic50 3 and 18 ng / ml , respectively ) , was separated by ods flash chromatography using meoh / h2o ( 5:5 and 7:3 ) , ch3cn / h2o ( 7:3 and 85:5 ) , meoh , and chcl3/meoh / h2o ( 70:30:5 ) . the fraction eluted with mecn / h2o ( 7:3 ) which showed less cytotoxicity ( ic50 values : 450 ng / ml against p388 and 70 ng / ml against la / egfp ) was purified by reversed phase hplc using mecn / h2o ( 38:62 ) with 0.2 m nacl , and the final purification by reversed phase hplc using mecn / h2o ( 35:65 ) containing 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 suggested this pseudomolecular ion peak coincided with that of renieramycin a . analysis of 2d nmr data including the hohaha   and hmbc   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 cd3od was not consistent with those of the literature . comparison of h - nmr data in the same solvent ( cdcl3 ) 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 ic50 value of 0.2 g / ml . on the other hand , it showed cytotoxicity against p388 murine leukemia cells at the ten times higher concentration ( ic50 2.2 g / ml ) . several antileishmanial compounds including cyclic peroxides , pyridoacridine alkaloids , and manzamine alkaloids   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 h and 150 mhz for c. chemical shifts were referenced to the cd3od signals ( h 3.3 and c 49 , respectively ) . fabms spectra were measured on a jeol jms700 tandem mass spectrometer using nba as a matrix . the animal specimens were collected by hand using scuba off kuchinoerabu - jima island in the satsunan islands ( 302831n ; 1301173e ) in july 2001 and identified as neopetrosia sp . by dr . rob van soest , university of amsterdam . 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 ic50 ( 0.42 g / ml ) of amphotericin b ( icn , ohio , usa ) , la / egfp were cultured at 5  10 cells / ml with various concentrations of the drug , and their fluorescence signals frozen animals ( 1.5 kg ) were exhaustively extracted with meoh ( 2l ) and etoh ( 2l  2 ) , and the combined extracts were concentrated and partitioned between h2o and chcl3 . the organic layer was subjected to the modified kupchan procedure : first partitioned between n - hexane and meoh / h2o ( 90:10 ) , then the meoh / h2o ( 90:10 ) layer was diluted with h2o to make meoh / h2o ( 60:40 ) which was extracted with chcl3 . the chcl3 layer was separated by ods flash chromatography using meoh / h2o ( 5:5 and 7:3 ) , ch3cn / h2o ( 7:3 and 85:5 ) , meoh , and chcl3/meoh / h2o ( 70:30:5 ) . fractions eluted with mecn / h2o ( 7:3 ) was concentrated and separated by reversed phase hplc [ phenomenex luna phenyl - hexyl , 20  250 mm ] using mecn / h2o ( 38:62 ) with 0.2 m nacl . the active fraction was further purified by reversed phase hplc [ cosmosil 5c18-arii , 10  250 mm ] using mecn / h2o ( 35:65 ) with 0.2 m nacl to afford renieramycin a ( 1 , 0.5 mg ) ; [ ]d 30 ( c 0.02 , meoh ) ; h- and c - nmr see table 1 ; fabms m / z 571 [ m+4h+h ] ; esims m / z 567 [ m+h ] . nmr spectra were recorded on a jeol a600 nmr spectrometer operating at 600 mhz for h and 150 mhz for c. chemical shifts were referenced to the cd3od signals ( h 3.3 and c 49 , respectively ) . fabms spectra were measured on a jeol jms700 tandem mass spectrometer using nba as a matrix . the animal specimens were collected by hand using scuba off kuchinoerabu - jima island in the satsunan islands ( 302831n ; 1301173e ) in july 2001 and identified as neopetrosia sp . by dr . 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 ic50 ( 0.42 g / ml ) of amphotericin b ( icn , ohio , usa ) , la / egfp were cultured at 5  10 cells / ml with various concentrations of the drug , and their fluorescence signals frozen animals ( 1.5 kg ) were exhaustively extracted with meoh ( 2l ) and etoh ( 2l  2 ) , and the combined extracts were concentrated and partitioned between h2o and chcl3 . the organic layer was subjected to the modified kupchan procedure : first partitioned between n - hexane and meoh / h2o ( 90:10 ) , then the meoh / h2o ( 90:10 ) layer was diluted with h2o to make meoh / h2o ( 60:40 ) which was extracted with chcl3 . the chcl3 layer was separated by ods flash chromatography using meoh / h2o ( 5:5 and 7:3 ) , ch3cn / h2o ( 7:3 and 85:5 ) , meoh , and chcl3/meoh / h2o ( 70:30:5 ) . fractions eluted with mecn / h2o ( 7:3 ) was concentrated and separated by reversed phase hplc [ phenomenex luna phenyl - hexyl , 20  250 mm ] using mecn / h2o ( 38:62 ) with 0.2 m nacl . the active fraction was further purified by reversed phase hplc [ cosmosil 5c18-arii , 10  250 mm ] using mecn / h2o ( 35:65 ) with 0.2 m nacl to afford renieramycin a ( 1 , 0.5 mg ) ; [ ]d 30 ( c 0.02 , meoh ) ; h- and c - nmr see table 1 ; fabms m / z 571 [ m+4h+h ] ; esims m / z 567 [ m+h ] .
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 g / ml .