Limonoid Compounds Inhibit Sphingomyelin Biosynthesis byPreventing CERT Protein-dependent Extraction of Ceramidesfrom the Endoplasmic Reticulum*□S

Received for publication, January 19, 2012, and in revised form, April 19, 2012 Published, JBC Papers in Press, May 7, 2012, DOI 10.1074/jbc.M112.344432

Françoise Hullin-Matsuda‡§1, Nario Tomishige‡, Shota Sakai‡, Reiko Ishitsuka‡, Kumiko Ishii‡, Asami Makino‡,Peter Greimel‡, Mitsuhiro Abe‡, Elad L. Laviad¶, Michel Lagarde§, Hubert Vidal§, Tamio Saito�, Hiroyuki Osada�,Kentaro Hanada**, Anthony H. Futerman¶, and Toshihide Kobayashi‡§2

From the ‡Lipid Biology Laboratory, RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan, §Inserm U1060-UniversitéLyon1, 69621 Villeurbanne, France, the ¶Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel,the �Chemical Biology Core Facility, RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan, and the **Department ofBiochemistry and Cell Biology, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo 162-8640, Japan

Background: Pharmacological inhibitors of sphingolipid metabolism and transport are useful for both biological andtherapeutic research.Results: High throughput microscopy-based screening identified limonoids as inhibitors of sphingomyelin biosynthesis bypreventing the membrane extraction of ceramide.Conclusion: Some therapeutic properties of limonoids might be related to their effects on ceramide metabolism.Significance: This study provides insights into the role of the ceramide domains in sphingolipid metabolism.

To identify novel inhibitors of sphingomyelin (SM) metabo-lism, a new and selective high throughput microscopy-basedscreening based on the toxicity of the SM-specific toxin, lysenin,was developed. Out of a library of 2011 natural compounds, thelimonoid, 3-chloro-8�-hydroxycarapin-3,8-hemiacetal (CHC),rendered cells resistant to lysenin by decreasing cell surface SM.CHC treatment selectively inhibited the de novo biosynthesisof SM without affecting glycolipid and glycerophospholipidbiosynthesis. Pretreatment with brefeldin A abolished thelimonoid-induced inhibition of SM synthesis suggesting thatthe transport of ceramide (Cer) from the endoplasmic reticulumto the Golgi apparatus is affected. Unlike the Cer transporter(CERT) inhibitor HPA-12, CHC did not change the transport ofa fluorescent short chain Cer analog to the Golgi apparatus orthe formation of fluorescent and short chain SM from the cor-responding Cer. Nevertheless, CHC inhibited the conversion ofde novo synthesized Cer to SM.We show that CHC specificallyinhibited the CERT-mediated extraction of Cer from theendoplasmic reticulum membranes in vitro. Subsequent bio-chemical screening of 21 limonoids revealed that someof them, such as 8�-hydroxycarapin-3,8-hemiacetal andgedunin, which exhibits anti-cancer activity, inhibited SMbiosynthesis and CERT-mediated extraction of Cer frommembranes. Model membrane studies suggest that 8�-hy-

droxycarapin-3,8-hemiacetal reduced the miscibility of Cerwithmembrane lipids and thus induced the formation of Cer-rich membrane domains. Our study shows that certainlimonoids are novel inhibitors of SM biosynthesis and sug-gests that some biological activities of these limonoids arerelated to their effect on the ceramide metabolism.

Recent progress in sphingolipid (SL)3 research highlights therole of these complex lipids in cell growth, differentiation, andapoptosis (1). In addition, SLs have attracted considerableattention because it was shown that they participate in the for-mation of lipid rafts in biomembranes (2). These lipid domains,which are characterized by a tight packing with a relatively highdegree of lateral mobility, are in a permanent associated/disso-ciated equilibrium state in the membrane (3).Two important points have to be considered when studying

the biological activities of SLs as follows: first, the specific sub-cellular localization of the enzymes involved in their metabo-lism, and second, their biophysical properties that require spe-cific transport mechanisms for movement between themembranes as well as translocation across the bilayer (1, 4, 5).Ceramide (Cer) occupies a central position in SL metabolism

* This work was supported by the Lipid Dynamics Program of RIKEN andGrants-in-aid for Scientific Research 22390018 and 24657143 (to T. K.) fromthe Ministry of Education, Culture, Sports, Science, and Technology ofJapan.

□S This article contains supplemental Figs. S1–S5, Materials and Methods, andadditional references.

1 To whom correspondence may be addressed: Lipid Biology Laboratory,RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan. Tel.:81-48-467-9536; Fax: 81-48-462-4981; E-mail: hullin-matsuda@riken.jp.

2 To whom correspondence may be addressed: Lipid Biology Laboratory,RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan. Tel.:81-48-467-9534; Fax: 81-48-467-9535; E-mail: kobayasi@riken.jp.

3 The abbreviations used are: SL, sphingolipid; BFA, brefeldin A; Cer, ceramide;CerS, ceramide synthase; CHC, 3-chloro-8�-hydroxycarapin-3,8-hemiacetal;DHS, dihydrosphingosine; HC, 8�-hydroxycarapin-3,8-hemiacetal; SM, sphin-gomyelin; SPH, sphingosine; C6-NBD-SM, 6-[N-(7-nitrobenzo-2-oxa-1,3-dia-zol-4-yl)-aminohexanoyl]sphingosylphosphoryl choline; C5-DMB-Cer, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl-D-erythro-sphingosine; C5-DMB-PC, 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine; DPH, 1,6-diphenyl-1,3,5-hexatriene; PC, phosphati-dylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; ER,endoplasmic reticulum; GlcCer, glucosylceramide; Ged, gedunin;hCERT, human CERT; HPTLC, high performance TLC; Fiss, fissinolide;DSC, differential scanning calorimetry.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 29, pp. 24397–24411, July 13, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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being generated either by de novo synthesis or by acidic or neu-tral sphingomyelinase activity (6, 7). The de novo synthesis ofCer occurs on the cytosolic side of the endoplasmic reticulum(ER) (8) by a family of ceramide synthases (CerS), eachmembersynthesizing Cer having different acyl chain lengths (9). Next,Cer is specifically transported by the Cer transfer protein(CERT) (10) to the trans-Golgi region where the synthesis ofsphingomyelin (SM) occurs via the action of SM synthase 1 (11)on the luminal side of theGolgi. CERT extracts Cer from the ERmembrane and then transports it to the Golgi in a nonvesicularmanner (12). Cer is also transported to the cis-Golgi for thesynthesis of glucosylceramide (GlcCer), the precursor of com-plex glycosphingolipids. GlcCer is synthesized on the cytosolicside of the Golgi by GlcCer synthase (13, 14).SM plays an essential role in cell proliferation (15), and the

enzymes regulating SL metabolism have been reported as tar-gets in cancer therapy (16, 17). However, the effective use oftherapeutic molecules has been hampered by their toxicity.Therefore, to find new types of inhibitors that affect Cermetab-olism and transport as well as SMmetabolism, we used an orig-inal microscopy-based automated assay to screen a chemicallibrary of natural compounds. This type of lipid-specific probe-based cell screening appears to be a very efficient technique forhigh throughput analysis of small compounds that affect lipidmetabolism. We recently developed this visual technique cou-pled to biochemical analysis to successfully identify small mol-ecules that interfere with cholesterol metabolism and transport(18) using the nontoxic cholesterol-binding protein � toxindomain 4 (19). In the present screening, lysenin, a SM-specificpore-forming toxin (20, 21), was used in the presence of dihy-drosphingosine (DHS or sphinganine) to exclude the inhibitors ofthe serine palmitoyltransferase, which disrupt all SL metabolism(22).Thus,we focusedon thebiosynthetic steps afterDHSsynthe-sis. Screening of a library of 2011 natural small compounds andderivatives revealed that 3-chloro-8�-hydroxycarapin-3,8-hemi-acetal (CHC), a limonoid, selectively inhibited de novo biosynthe-sisofSM.Subsequent screeningof21 limonoids showedthat someof them, such as 8�-hydroxycarapin-3,8-hemiacetal (HC) andgedunin, a palm tree-derived limonoidwith reported anti-malariaand anti-cancer activities (23, 24), inhibited SM biosynthesis. Theresults thus indicate that limonoid compounds are novel inhibi-tors of SL metabolism and suggest that some of their biologicalactivities are partially explained by their inhibition of Cer metab-olism and transport.

EXPERIMENTAL PROCEDURES

Materials—L-[U-14C]Serine (164 mCi/mmol), [methyl-14C]-choline chloride (40–60 mCi/mmol), and D-erythro-[3-3H]-sphingosine (SPH) (15–30Ci/mmol)were fromPerkinElmer LifeSciences. DHS (60 Ci/mmol), N-[1-14C]palmitoyl-D-erythro-sphingosine (C16-Cer) (50mCi/mmol) andN-[1-14C]hexanoyl-D-erythro-sphingosine (C6-Cer) (55 mCi/mmol) were fromAmerican Radiolabeled Chemicals Inc. (St. Louis, MO). Brefel-din A (BFA) was from Biomol International Corp. L-Serine wasfrom Nacalai, Japan. ISP-1/myriocin (serine palmitoyltrans-ferase inhibitor (25)) and fumonisin B1 (ceramide synthaseinhibitor (26)) were purchased from Sigma. The CERT inhibi-tor (27), (1R,3R)-N-(3-hydroxy-1-hydroxymethyl-3-phenyl-

propyl)dodecanamide (HPA-12) was prepared as described(28). 6-[N-(7-Nitrobenzo-2-oxa-1,3-diazol-4-yl)-amino-hexanoyl]sphingosylphosphorylcholine (C6-NBD-SM), 6-[N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)-aminohexanoyl]-D-erythro-sphingosine (C6-NBD-Cer), N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl-D-erythro-sphingosine or Bodipy FL-C5-ceramide (C5-DMB-Cer), and 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine or Bodipy FL-C5-phosphatidylcholine (C5-DMB-PC), 4�,6-diamidino-2-phenylindole, dihydrochloride(DAPI) and 1,6-diphenyl-1,3,5-hexatriene (DPH) were fromMolecular Probes. HPTLC plates were from Merck. 1,2-Dipalmitoyl-sn-phosphatidylcholine (DPPC), egg phosphati-dylcholine (PC), egg phosphatidylethanolamine (PE), DHS,SPH, N-[12-[(7-nitro-2–1,3-benzoxadiazol-4-yl)amino]dode-canoyl]-D-erythro-sphingosine (C12-NBD-Cer), N-hexanoyl-D-erythro-sphingosine (C6-Cer), and N-palmitoyl-D-erythro-sphingosine (C16-Cer) were from Avanti Polar Lipids. TheSpectrum Collection Chemical Library containing 2011 com-pounds was purchased from MicroSource Discovery SystemsInc. (Gaylordville, CT). In some of the experiments, geduninwas also provided by Tocris Bioscience (Ellisville, MO). Li-monin (Microsource ID 10008733) was supplied by ChemicalBiology Core Facility, RIKEN-ASI.Cell Culture—CHO-K1 and HeLa cells were routinely cul-

tured as described previously (18) in medium supplementedwith 10% fetal bovine serum (FBS) (referred to as completemedium).Mediumwith 1%Nutridoma-SP (RocheApplied Sci-ence) was used as a serum-freemedium to study the drug effecton SL metabolism in CHO or HeLa cells.Fluorescence Image Analysis by IN Cell Analyzer—CHO-K1

cells in 96-well plates were treated for 48 h with 50 �M of drugsin complete medium without or with 1 �M DHS. Cells werethen treatedwith lysenin (200ng/ml) (29) for 30min.At the endof the incubation, cells were washed with phosphate-bufferedsaline (PBS) and fixed with 3% paraformaldehyde. They werestained with the fluorescent lipid marker C5-DMB-PC to stainthe membrane and with DAPI to stain nuclei, to follow cellshape and viability. Fluorescence of each well was observed byIN Cell Analyzer 1000 (GE Healthcare) with a �20 objective.Three to five images per well were acquired and analyzed usingIN Cell Analyzer analysis software developer. The fluorescencepattern was also controlled with a Zeiss LSM 510 confocalmicroscope.Fluorescence Confocal Microscopy—For live cell experi-

ments, cells were grown on glass-bottom dishes (Iwaki, Japan),incubated in DMEM/Ham’s F-12 nutrient mixture (1:1) with-out phenol red containing 15mMHepes (pH 7.0), and observedusing a Zeiss LSM 510 confocal microscope equipped withC-Apochromat 63� WKorr (1.2 NA) objective.Assay for Intracellular Transport of C5-DMB-Cer and Recy-

cling of C6-NBD-SM—Cells were incubated with fluorescentlipids as described previously (27, 30–32). See supplementalmaterial for detailed method.Metabolic Labeling of Cell Lipids with [14C]Serine and

[14C]Choline—Subconfluent CHO cells were incubated at37 °C in Nutridoma-supplemented F-12 medium (serum-free)

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in the absence or the presence of limonoids or inhibitors forindicated periods. Then [14C]serine or [14C]choline (1 �Ci/ml)was added, and the incubationwas extended for 2 or 4 h, respec-tively, in the absence or presence of the drugs. At the end of theincubation time, lipids were extracted and analyzed by HPTLCas described below. See supplemental material for detailedinformation. To study the degradation of [14C]SM, subconflu-ent CHOcells were labeled for 2 hwith [14C]serine. After wash-ing, cells were incubated with Nutridoma-supplementedmedium containing 10 mM L-serine at 37 °C in the presence orabsence of CHC. Cells were harvested at the indicated times.Metabolism of Fluorescent C5-DMB-Cer in Cells—Subcon-

fluent CHO cells were pretreated for 1 h with limonoids inserum-free F-12 medium at 37 °C. The C5-DMB-Cer com-plexed with BSA (1.25 �M final concentration) was then added.Cells were incubated at 37 °C for 2–4 h. Lipids were extractedand analyzed by HPTLC as described below. Fluorescent spotswere quantified with Typhoon 9140 (GE Healthcare).Study of the Conversion of [3H]Cer to [3H]SM in Cells—CHO

cells were analyzed for the conversion of [3H]Cer to [3H]SM asdescribed previously (27) with somemodifications as indicatedin the supplemental material.Lipid Analysis and Determination of Lipid Content—At the

end of the incubation time, cells were collected and lipidsextracted by the method of Bligh and Dyer (33). Lipids wereseparated onHPTLC plates (27, 34), and radioactive spots werequantified with a BAS 5000 image analyzer (Fuji Film Inc.,Tokyo, Japan). For determination of the effect of limonoids onphospholipid and SM content, total phospholipid and SM con-tent after HPTLC separation were evaluated by phosphorusquantification (35). See supplemental material for detailedinformation.Purification of Recombinant hCERT—His6-tagged recombi-

nant human CERT (hCERT) was purified as described (10, 36).InVitroAssay of Ceramide Extraction from Isolated ERMem-

branes and from Artificial Liposomes—ER membranes wereprepared fromCHOcells as described (37) and analyzed for theabsence of SM synthase activity usingC6-NBD-Cer as substrate(10). The extraction of Cer from ER membranes was then per-formed as described (10, 36). Various concentrations oflimonoids were preincubated with ER membranes containing3H-labeled Cer (200 �g of proteins) or with hCERT (200 pmol)for 20min on ice in transport bufferHNE (50mMHepes-NaOH(pH 7.5), 100 mM NaCl, 0.5 mM EDTA). The extraction wasstarted by mixing hCERT and labeled ER membranes in HNE(150 �l final volume), and the incubation lasted for 30 min at37 °C. Background of extraction was evaluated by addition ofBSA (200 pmol) instead of hCERT. Incubation was stopped onice, and tubes were centrifuged immediately at 100,000 � g for1 h at 4 °C. Lipids were extracted from supernatants and pellets(33) and separated by HPTLCwith a solvent mixture of chloro-form/methanol/acetic acid (94:5:5, v/v). Radioactive spots werequantified with a BAS 5000 image analyzer.Extraction of 14C-labeled long chain Cer from artificial lipo-

somes was performed as described (10). Limonoids or DMSO(control, 0.1% final concentration) were preincubated withlipid vesicles composed of egg yolk PC, egg yolk PE, and N-[1-14C]palmitoyl-D-erythro-sphingosine ([14C]C16-Cer) (32:8:0.2,

mol/mol) (40 �g) (referred as “liposome preincubation”) orwith hCERT (100 pmol) (referred as “CERTpreincubation”) for10 min on ice in HNE. Then the extraction was initiated bymixing hCERT and lipid vesicles in HNE (100 �l final volume).Themixture was incubated for 30 min at 37 °C. Incubation wasstopped on ice, and tubes were centrifuged immediately at100,000 � g for 30 min at 4 °C. The radioactivity of the super-natant and pellet was counted with a scintillation counter, andthe radioactivity in the supernatant indicated the amount ofCer extracted from the vesicles.Measurement of Fluorescence Anisotropy of DPH—DPPC

vesicles were incubated with increasing concentrations of HCfrom 100:1 to 5:1 molar ratio for 15 min at 37 °C. Egg PC/eggPE/C16-Cer (32:8:2mol/mol) vesicles were incubatedwith 8�M

HC for 15 min at 37 °C. After addition of 0.5 mol % DPH, thefluorescence was monitored as described previously (38). Seesupplemental material for additional information.Differential Scanning Calorimetry (DSC)—Lipid films of

DPPC, C16-Cer, and limonoid HC were formed and thenhydrated in 50mMHepes-NaOHbuffer (pH 7.5) containing 0.5mM EDTA. The final lipid concentration in the vesicles was1 mM. DSC thermograms were recorded at a scan rate of60 °C/h for all samples, and each scan was performed at least 15times. The obtained data were analyzed as described previously(39). See supplemental material for additional information.

RESULTS

Fluorescence Image Screening of aChemical Library Identifiesa Limonoid Compound as an Inhibitor of SL Metabolism—Inthis study, we screened a commercial chemical library of natu-ral compounds for inhibitors of SLmetabolism using an INCellAnalyzer 1000 automated fluorescence imaging system (18).For this purpose, we employed the SM-specific toxin lysenin(20, 40). After binding to SM-enriched domains in the plasmamembrane, lysenin induces pore formation and cell lysis (21).Compounds that reduce the cell surface SM content make thecells resistant to lysenin. Cell number, shape, and fluorescenceintensity were analyzed using the IN Cell Analyzer 1000 fol-lowed by LSM510 confocalmicroscopy (Fig. 1,A–H). DHSwasadded to the medium to exclude serine palmitoyltransferaseinhibitors and to focus the assay on the steps that take placeafter DHS synthesis. Lysenin treatment caused cell shrinkage asrevealed by Bodipy labeling (Fig. 1E) and a smaller condensednucleus as monitored by DAPI (Fig. 1F). The nucleus is clearlyobservable in the differential interference contrast image afterlysenin treatment (Fig. 1H). From the initial screen of 2011compounds, only CHC, a limonoid derivative, significantlyinhibited lysenin-induced cell death under these experimentalconditions. Bodipy staining showed that cells were well spread(Fig. 1A), and the nucleus shape was normal (Fig. 1B) as con-firmed by the differential interference contrast image (Fig. 1D),which was distinct from that in Fig. 1H.Specific Inhibitory Effect of 3-Chloro-8�-hydroxycarapin-3,8-

hemiacetal on de Novo SM Biosynthesis—These results suggestthat CHC affects the biosynthesis, degradation, or recycling ofSM. To study the effect of CHC on de novo SL synthesis, CHOcells were preincubated with various concentrations of CHCfor 22 h and then labeled with [14C]serine in the presence or

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absence of the compound. CHC induced a selective dose-de-pendent inhibition of [14C]SM synthesis displaying a 50% inhi-bition of SM synthesis at �5 �M (Fig. 2A). A small decrease inthe glycolipids GlcCer and GM3 (N-acetylneuranimyl-lactosyl-ceramide) was observed at high concentrations. CHC did notaffect cell viability up to 100�M (data not shown). In contrast tothe effect on SM, CHCdid not significantly influence the incor-poration of [14C]serine into phosphatidylserine (PS) and PE(Fig. 2B). One hour of preincubation with CHCwas as effectiveas 22 h of preincubation to specifically inhibit SM synthesis(Fig. 2C). This is similar to the effect observed with the CERTinhibitorHPA-12 (Fig. 2C) (27) and suggests thatCHC itself butnot itsmetabolites plays a role in the inhibition of SM synthesis.We also examined the biosynthesis of phosphatidylcholine

(PC) because SM is synthesized by the transfer of phosphocho-line from PC to Cer. Similar to HPA-12 incubation, CHC treat-ment did not significantly affect the incorporation of [14C]cho-line into PC (Fig. 2D). Quantification of the phosphoruscontent after lipid extraction and TLC analysis indicated thatSMcontent displayed a 40–50%decreasewhenCHOandHeLacells were grown for 2 days in the presence of 5 �M CHC (Fig.2E) suggesting that the compound did not display cell specific-ity. SMcomposed�5%of total phospholipids both inCHOandHeLa cells. The total phospholipid contentwas not significantlyaltered by a 2-day treatment with 5 �M CHC (Fig. 2F).These results indicate that CHC specifically inhibits SM bio-

synthesis and thus, as a result, cells became resistant to lysenin.However, this does not rule out the possibility that CHC accel-erates the degradation of SM. Determination of the turnover ofradiolabeled de novo synthesized SM (Fig. 2G) clearly indicatedthat CHC did not affect SM turnover, thus excluding the pos-sibility that CHC could stimulate SM degradation by the acti-vation of sphingomyelinase.Fluorescent SM analogs recycle between the plasma mem-

brane and the recycling endosomes (31, 32, 41, 42). Slow recy-cling back to the plasmamembranemay decrease plasmamem-brane SM. Thus, the influence of CHC on SM recycling wasdetermined (Fig. 3, A–F). CHO cells preincubated in the pres-ence and absence of CHCwere first labeledwith the fluorescent

SM analog, C6-NBD-SM, and the fluorescent lipids were theninternalized in the recycling endosomes. Cell surface fluores-cence was then quenched with sodium dithionite (30, 43), andcells were further incubated in the absence of dithionite. There-emerging cell surface labeling indicates the recycling ofC6-NBD-SM to the plasma membrane. The results show thatCHC did not significantly affect the recycling of a fluorescentSM analog.We alsomeasured the effect of CHCon bulk vesicular flow to

the plasma membrane via the Golgi apparatus with a GFP-la-beled tsO45 mutant of vesicular stomatitis virus G protein (44,45). GFP-vesicular stomatitis virusG accumulates in the ER at anonpermissive temperature and is subsequently transported tothe Golgi and the plasma membrane at a permissive tempera-ture (supplemental Fig. S1). No notable differences weredetected between the control (supplemental Fig. S1, A–C) andCHC-treated cells (supplemental Fig. S1, D–F), indicating thatCHC did not inhibit the intracellular transport of GFP-vesicu-lar stomatitis virus G. Altogether, these results suggest thatCHC specifically inhibits the de novo biosynthesis of SM.CHC Inhibits the Intracellular Transport of de Novo Synthe-

sized Cer to the Site of SM Synthesis—We then examinedwhether the formation of Cer was the target of CHC by meas-uring the CerS activity in vitro. Both CerS-2 and CerS-5 activi-ties were unaffected by the presence of CHC (supplemental Fig.S2, A and B). Furthermore, CHC did not inhibit the activity ofthe SM synthase in vitro using as substrates the fluorescentshort chain Cer analogs C5-DMB-Cer and C12-NBD-Cer andthe radioactive short chain Cer 14C-C6:0 Cer (supplementalFig. S2C).Next, we examined the possibility that CHC inhibits the

transport of Cer from the ER, where it is synthesized, to theGolgi apparatus, where it is converted to SM by SM synthase.BFA induces fusion of the Golgi apparatus with the ER (46) andthus abolishes the inhibitory effect of the CERT inhibitorHPA-12 (36). BFA treatment of CHO cells induced a 2–3-foldincrease in SM synthesis (Fig. 4) as reported previously (47). SMsynthesis in the presence of BFA was comparable in CHC-treated and control cells. The BFA treatment similarly sup-

FIGURE 1. Screening of inhibitors of SM metabolism by fluorescence imaging. CHO cells were grown in the presence of 1 �M DHS with (A–D) or without 50�M CHC (E–H) for 2 days in complete medium. Cells were stained with the fluorescent lipid marker Bodipy-C5-PC and the nuclear DNA marker DAPI. Images wereacquired by confocal microscope. A and E, bodipy image; B and F, DAPI image; C and G, merge of DAPI and Bodipy; D and H, differential interference contrastimage. Bar, 20 �m.

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pressed the inhibition of SM biosynthesis induced by HPA-12(Fig. 4) (27). In contrast, SM synthesis was inhibited both in thepresence and absence of BFA in cells treated with the ceramidesynthase inhibitor fumonisin B1 (data not shown). Theseresults indicate that CHC does not inhibit SM synthase andhenceCHC, similar toHPA-12, appears to exert an effect on theintracellular transport of endogenously formed Cer from theER to the Golgi.De novo synthesized Cer is transported to the Golgi appara-

tus by CERT (10, 36). The transport of an exogenously addedfluorescent short chain Cer analog, C5-DMB-Cer, to the Golgi

apparatus is also dependent on CERT activity (27, 48).We thenexamined the effect of CHC on the intracellular distribution ofC5-DMB-Cer (49). First, CHOcells were labeledwithC5-DMB-Cer at 4 °C in the absence and the presence of the drugs (Fig. 5,A–C). After washing, cells were chased at 37 °C with and with-out the drugs. Before the chase, the fluorescence was distrib-uted at the plasma membrane in the control as well as drug-treated cells. After the chase, the fluorescence accumulated inthe perinuclear region in the control and CHC-treated cells(Fig. 5, D and E, respectively). In contrast, in HPA-12-treatedcells, the fluorescence did not accumulate at the perinuclear

FIGURE 2. CHC selectively inhibits de novo biosynthesis of SM. A and B, CHO cells were treated with increasing concentrations of CHC for 22 h followed by2 h of labeling with [14C]serine in the presence of CHC. Lipids were analyzed by HPTLC. A, SM (closed diamond), Cer (open circle), GlcCer (closed triangle),ganglioside GM3 (open square). B, PS (closed square), PE (open triangle). Results are expressed as percentage of the control after normalization of the radioactivityby the protein content and are mean and average deviation of two independent experiments. C, shorter preincubation time with CHC significantly decreasesSM biosynthesis; CHO cells were preincubated for 1 h with 10 �M CHC (black bar) or with 1 �M CERT inhibitor, HPA-12 (gray bar). Then cells were labeled with[14C]serine for 2 h in the presence of the drugs. Lipids were analyzed by HPTLC. Results are expressed in percentage of the control after normalization of theradioactivity by the protein content. They are the mean and S.E. of three independent experiments for CHC (**, p � 0.01 for SM values compared with control)and mean and average deviation of two independent experiments for HPA-12. D, CHC does not modify PC synthesis; CHO cells were preincubated for 1 hwithout (control) or with CHC (10 �M, black bar) or 1 �M HPA-12 (gray bar) and then pulse-labeled with [14C]choline for 4 h. Lipids were analyzed by HPTLC.Results are expressed in percentage of the control after normalization of the radioactivity by the protein content and are the mean and average deviation oftwo independent experiments. E and F, CHC treatment decreases selectively SM content without significant effect on total PL; CHO and HeLa cells wereincubated without (gray bars) or with CHC (5 �M, black bars) for 2 days. Medium was replaced after 24 h. SM content (E) and total phospholipid content (F) wereanalyzed as described under “Experimental Procedures.” Data are expressed in nanomoles of phosphorus/mg of proteins. G, CHC does not stimulate SMdegradation; CHO cells were labeled 2 h with [14C]serine and then chased with medium containing 10 mM serine and 1 �M (closed circle) and 10 �M (closedtriangle) CHC or DMSO solvent (0.1% final concentration) as the control (open circle) for the indicated times. Lipids were extracted and analyzed as describedunder “Experimental Procedures.”

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area (Fig. 5F) (27). These results suggest that CHC did notinhibit the transport of the exogenously added C5-DMB-Cer tothe Golgi apparatus. This is further supported by the observa-tion that CHC did not modify the conversion of C5-DMB-Certo C5-DMB-SM (Fig. 5G).

The naturally occurring Cer are highly hydrophobic due totheir long acyl chains. Consequently, their physical propertiesdiffer from those of their fluorescent or short chain counter-parts. Indeed, model membrane studies showed that fluores-cent short chain Cer analogs underwent spontaneous mem-brane transfer much faster (50, 51) than natural C16-Cer (52).Thus, it is speculated that CHC did not affect the transport ofC5-DMB-Cer due to the spontaneously rapid transfer of thelipid.

Next, we studied the effect of CHC on the conversion of theendogenously formed Cer to SM by metabolically labeling thede novo synthesized Cer pool. For this purpose, CHO cells werepulse-labeled with [3H]DHS at 15 °C to allow the formation of[3H]Cer without further conversion to [3H]SM (53). Cells werethen incubated with and without drugs at 4 °C and chased at37 °C in the presence of the CerS inhibitor fumonisin B1. Incontrast to the fluorescent short chain Cer, CHC inhibited theformation of SM from endogenous Cer to a similar extent asHPA-12 treatment (Fig. 5H) (27). This result indicates thatCHC inhibited the transport and, as a consequence, the conver-sion of endogenously formed Cer to SM.CHC Inhibits the CERT-mediated Cer Extraction from Iso-

lated ER Membranes—The initial step of CERT-mediatedtransport ofCer from the ER to theGolgi apparatus requires theextraction of Cer from the ER. To determine the influence ofCHC on Cer extraction from the ER membrane, we used an invitro assay utilizing CHOERmembranes containing long chain[3H]Cer formed from SPH and palmitoyl-CoA (10). First, weconfirmed that recombinant hCERT extracted [3H]Cer but not[3H]SPH from the ER membrane (Fig. 6). Interestingly, prein-cubation of the ER membrane with CHC inhibited Cer extrac-tion similar to the CERT inhibitor HPA-12 (Fig. 6). This indi-cates that CHC interferes with the CERT-mediated extractionof long acyl chain Cer from ER membrane.Screening of 21 Additional Limonoids Identified as New

Inhibitors of SLMetabolism—The results indicate that CHC is anew type of inhibitor of SL biosynthesis. Because CHC belongsto a large family of natural compounds, called limonoids, weexamined the effect of other natural and derivatized limonoidson SL biosynthesis (see Fig. 7 for limonoid structure). Fig. 8Aindicates that gedunin (Ged, compound 6), khayanthone (com-pound 7), xylocarpus A (compound 16), ethandrophragmin(compound 19), and HC (compound 20) inhibited more than

FIGURE 3. CHC does not modify the recycling of C6-NBD-SM. CHO cells were preincubated with 0.1% DMSO (control, A–C) or 5 �M CHC (D–F) for 30 min andthen incubated with C6-NBD-SM for 30 min (A and D) at 37 °C. Cell surface NBD fluorescence was quenched by dithionite (B and E) and chased for 1 h at 37 °C(C and F). Bar, 10 �m.

FIGURE 4. BFA treatment overcomes the CHC-induced inhibition of SMsynthesis. CHO cells were treated for 1 h with CHC (10 �M) or HPA-12 (1 �M).Cells were then incubated in the absence (gray bars) or the presence (blackbars) of BFA for 30 min, followed by 2 h of labeling with [14C]serine in thepresence of BFA. Inhibitors were present throughout incubation. Radioactiv-ity in SM is expressed in percentage of that of the control without BFA treat-ment after normalization of the radioactivity by the protein content. Data arethe mean and average deviation of two independent experiments.

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half of thede novo synthesis of SM.CHCandHPA-12were usedas positive controls. As observed previously with CHC, theselimonoids did not affect the biosynthesis of other phospholip-ids, e.g. PS, PE (supplemental Fig. S3A), and PC (supplementalFig. S3B). Furthermore, the SM content was significantlydecreased when CHO cells were grown for 2 days in the pres-ence of 5 and 10 �M HC (Fig. 8B), whereas the total phospho-lipid content was unchanged (Fig. 8C) as observed previouslywith CHC (see Fig. 2, E and F).In contrast, anthothecol (compound 1) and cedrelone (com-

pound 4) inhibited SM biosynthesis (Fig. 8A) in a nonspecificway because they decreased the biosynthesis of PS and PE,respectively (supplemental Fig. S3, A and B). Both compounds

were cytotoxic during longer incubation times (22 h, data notshown). Preliminary results indicated that long time treatment(22 h)with 10�Mmethylangolensate (compound9),mexicano-lide (compound 10), or humilinA (compound 12) did not affectSM biosynthesis in contrast to the CerS inhibitor fumonisin B1(supplemental Fig. S3C). Odoratone (compound 13) was toxicat concentration below 1 �M (data not shown).

Interestingly, as observed previously with CHC and HPA-12(Fig. 4), BFA treatment abolished the inhibitory activity of thefour newly identified limonoids that were active in SM biosyn-thesis (Fig. 9A) suggesting that they might also affect Cer avail-ability. Next, we selected three structurally distinct limonoids(see Fig. 7 and under “Discussion”), the active HC and Ged as

FIGURE 5. A–G, CHC does not inhibit the intracellular transport of fluorescent short chain Cer to the Golgi apparatus and its metabolism; CHO cells were labeledwith C5-DMB-Cer for 30 min at 4 °C and then preincubated with 0.1% DMSO (A and D), 5 �M CHC (B and E), or 2.5 �M HPA-12 (C and F) for 30 min at 4 °C (upperpanels, A–C). Cells were then incubated for 30 min at 37 °C (lower panels, D–F). Bar, 10 �m. G, CHC does not inhibit the conversion of fluorescent short chain Certo SM; CHO cells were preincubated for 22 h with increasing concentrations of CHC in serum-free F-12 medium at 37 °C. Then C5-DMB-Cer was added foradditional 2 h. Lipids were extracted and analyzed by HPTLC. Fluorescent spots corresponding to the fluorescent SM were quantified with Typhoon 9140, andvalues are expressed in percentage of the control after normalization of the fluorescent unit intensity by the protein content; H, CHC inhibits the conversion ofendogenous [3H]Cer to SM; CHO cells were pulse-labeled for 30 min at 15 °C with [3H]dihydrosphingosine and then treated with 0.1% DMSO (control, closeddiamond), CHC (5 �M, closed square), or HPA-12 (1 �M, open triangle) for 30 min at 4 °C. Cells were then incubated at 37 °C for various times in the presence offumonisin B1 (40 �M final). Lipids were extracted and analyzed by HPTLC. SM content is expressed as arbitrary unit (AU) per �g of proteins.

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well as the nonactive fissinolide (Fiss, compound 5), to deter-mine their influence on the recombinant hCERT-mediatedextraction of Cer from isolated ER membranes. HC as well asGed reduced Cer extraction in a concentration-dependentmanner (Fig. 9B). In contrast, even at concentrations of up to 20�M, Fiss did not affect CERT activity. The presence of HC alsointerfered with the recombinant hCERT-mediated extractionof Cer from artificial liposomes composed of PC, PE, and[14C]C16-Cer (10). HPA-12 was used as positive control,whereas neither DMSO (control, 0.1% final concentration) norFiss affected the Cer extraction (Fig. 9B).Hemiacetals are usually considered unstable. Nevertheless,

cyclic hemiacetals, unlike their acyclic counterparts, tend toexhibit a remarkable stability (54), especially if pyranoid orfuranoid rings form, such as in the case of glucose and fructosethat predominantly exist in their cyclic form in solution. Toprobe the stability of HC, a cyclic hemiacetal, we subjected it toacidic (pH 5.0) and neutral (pH 7.0) conditions for 24 h at 37 °C.Mass spectroscopic (MS) analysis revealed no significantchange in either sample (supplemental Fig. S4A). A major peakatm/z 485 [M � H]� was detected corresponding to the intacthemiacetal and a minor peak at m/z 467 corresponding to thefragment without the hydroxyl. This indicates that the cyclichemiacetal HC and its derivatives can tolerate the pH range of5–7, which is a range that would be encountered after cellularuptake.We also showed that the HC content in the cell extracts after

1 and 24 h of incubation was stable (supplemental Fig. S4B), as

revealed by MS-MS quantification (precursor ion m/z 485.5,[M � H]�) with multiple reaction monitoring mode (supple-mental Fig. S5). The steady cellular content of HC after 1 and24 h of incubation is in good agreement with the similar inhi-bition of SM biosynthesis at short and long time incubationperiods (see Fig. 2C). These results suggest that the activelimonoids decreased the de novo SM biosynthesis by inhibitingtheCERT-dependent extraction of long acyl chainCer from theER membrane.Limonoid HC Induces the Formation of Cer-rich Membrane

Domains—The intermembrane Cer transport catalyzed byCERTwas recently demonstrated to bemarkedly reducedwhenCer is in a tightly packed environment. Conversely, Cer in fluidmembranes was shown to be available for CERT-mediatedtransfer (55). These results indicate that the membrane matrixsurrounding Cer, i.e. Cer miscibility, crucially affects CERTactivity. To examine the effect of limonoids on the fluidity ofbulk membrane, we measured the fluorescence anisotropy ofDPH (38) incorporated into limonoid-containing liposomes(Fig. 10,A and B). DPH localizes to the hydrophobic core of themembrane bilayer, thus providing information on the packingproperties of the bulk membrane. DPH anisotropy in DPPCvesicles with or without HC (the range of the molar ratioDPPC/HC was from 100:1 to 5:1) exhibits identical trendsbetween 20 and 60 °C with an apparent transition temperatureof �41 °C (Fig. 10A). Similarly, HC presence in the PC/PE/Cerliposomes (PC/PE/Cer/HC, 32:8:2:0.4) did not modify DPHanisotropy between 20 and 60 °C (Fig. 10B).We further examined the effect of limonoids on the physical

properties of model membranes using DSC (Fig. 10, C–F). Theheating and cooling scans of pure DPPC liposomes (Fig. 10C)displayed a characteristic pretransition peak at 35 °C followedby a typical gel (L�) to liquid crystalline (L�) phase transitionpeak at 41 °C, as described (56). It is known that Cer exhibits amain endothermic transition at 90 °C (57). The addition of HC(1:8 drug/lipid molar ratio) to pure DPPC liposomes abolishedthe pretransition temperature, but it did not significantly mod-ify the main phase transition peak (Fig 10D). This is in goodagreementwith theDPH result. In a 1:8 Cer/DPPCmixture, theendothermic peak became broadened (Fig 10E). The coolingthermogram showed the coexistence of a large 41 °C peak and asmall 52 °C peak, indicating the presence of phase-separatedDPPC- and Cer-enriched domains (58). The presence of HC inthis Cer/DPPC mixture sharpened the transition peak arisingfrom the Cer-enriched domains. In addition, according to thecooling scans, the higher temperature transition peak of theCer-enriched domains shifted to 55 °C in the presence of HC(Fig 10F). These results suggest that limonoids, like HC, reducethe miscibility of Cer in DPPC liposomes by promoting theformation of Cer-rich domains.

DISCUSSION

Limonoids are a large family of natural compounds and havebeen employed in traditional medicine (59). Recent reportshave highlighted the antimalarial and antiproliferative activitiesof limonoids, such as Ged (23, 60–62). However, their molec-ular mechanism of action is not well understood. This study

FIGURE 6. CHC inhibits the CERT-mediated extraction of Cer from isolatedER membrane in vitro. 3H-Labeled Cer were synthesized by CHO ER mem-branes from D-erythro-[3-3H]SPH. CHC (2 and 10 �M) and DMSO (0.1%, con-trol) were preincubated for 20 min on ice with the labeled ER membranes, andthe CERT inhibitor HPA-12 (5 �M) was preincubated with hCERT (200 pmol) for20 min on ice. The extraction was started by mixing hCERT and labeled ERmembranes. Background of extraction was evaluated by adding BSA insteadof hCERT. After 30 min at 37 °C, incubation tubes were centrifuged and lipidsextracted from supernatant and pellet fractions. Lipids were separated byHPTLC as described under “Experimental Procedures.” Cer (black bars) andsphingosine SPH (gray bars) extracted in the supernatant are expressed inpercentage of Cer or SPH present in the ER membranes. Values are themean � average deviation of two independent experiments for BSA, HPA-12,and CHC and mean � S.D. of four independent experiments for control andhCERT.

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FIGURE 7. Chemical structure of the limonoids analyzed during this study. The indicated boldface numbers of the compounds are used in Figs. 8A and 9A,supplemental Fig. S3, A–C, and in the text. The A–D rings (red) of the primarily tetracyclic core are indicated in Ged (compound 6) structure. All compounds canbe found at MicroSource Discovery Systems Inc.

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indicates for the first time that certain limonoids, includingGed, are unique inhibitors of SM biosynthesis.The screening method was based on the cytotoxicity of lys-

enin, a SM-specific pore-forming toxin (20, 21). Thus, in thissystem, each compound capable of decreasing the SM on thecell surface would render the cells resistant to lysenin. Thislipid-specific protein-based cell screening appears to be a veryefficient technique for high throughput analysis of small com-pounds affecting lipid metabolism (18). In the present screenperformed in the presence of DHS, we focused on SM biosyn-thesis and excluded serine palmitoyltransferase inhibitorsbecause a number of them such as ISP-1/myriocin, sphingofun-gins, lipoxamycin, and sulfamisterine have been reported (25,63–66). Out of a library of 2011 natural products andderivatives, we identified 3-chloro-8�-hydroxycarapin, CHC, alimonoid derivative. Biochemical analysis indicated that CHCand other limonoids selectively inhibit de novo SM biosynthe-sis. In the initial steps of SMbiosynthesis, DHS is converted intodihydroceramide by CerS. Mammalian cells contain six mem-bers of the CerS family (9). Each CerS exhibits a different sub-strate fatty acid specificity (67, 68). CHC did not affect the invitro activity of CerS2 and CerS5, responsible for the synthesisof the very long chain Cer, i.e.C22-C24 Cer, andC16 Cer, respec-tively. This suggests that CHC inhibits a step that takes placeafter Cer formation.

It is noteworthy that BFA treatment, which induces fusion ofthe Golgi apparatus and the ER (46), rescued the limonoid-induced inhibition of SM biosynthesis. This demonstrates firstthat the SM synthase 1 activity is not influenced by theselimonoids, and second, it demonstrates that the activelimonoids inhibit SM biosynthesis by interfering with Cer traf-ficking from the ER to theGolgi apparatus, as observedwith thespecific CERT inhibitor, HPA-12 (27). However, in contrast toHPA-12, these limomoids donot appear to inhibit the transportof the short chain fluorescent Cer analog to theGolgi apparatusor its subsequent conversion to corresponding SM. Neverthe-less, the conversion ofde novo synthesizedCer to SMwas inhib-ited indicating that these limonoids selectively inhibit theCERT-regulated transport of endogenous long chain Cer. In aseparate set of in vitro experiments, we confirmed that theselimonoids inhibited the CERT-dependent extraction of endog-enously formed long chain Cer from isolated ERmembranes, aswell as the extraction of long chain Cer from artificial lipo-somes. Such a differential effect based on the acyl chain lengthof Cer may be due to differences in the physical properties oflong and short chain Cer, the latter of which is known to moverelatively more freely between membranes. Model membranestudies showed that a fluorescent short chainCer analog under-went spontaneous membrane transfer much faster (t1⁄2, minuteorder) (50, 51) than natural C16-Cer (t1⁄2, days) (52). CERT effi-

FIGURE 8. Limonoid compounds Ged and HC specifically inhibit SM biosynthesis. A, certain limonoids inhibit SM biosynthesis; CHO cells were treated for1 h with various limonoids (10 �M) in serum-free medium and labeled for 2 h with [14C]serine in the presence of limonoids. See Fig. 7 for names of compounds.Lipids were extracted and analyzed as in Fig. 2A. Results are expressed as percentage of the control after normalization of the radioactivity by the proteincontent. B and C, HC treatment selectively decreases SM content without significant effect on total PL; CHO cells were incubated without (white bars) or withHC (5 �M, gray bars and 10 �M, black bars) for 2 days. SM content (B) and total phospholipid content (C) were analyzed as described under “ExperimentalProcedures.” Data are expressed in nanomoles of phosphorus/mg of proteins. They are the mean and S.E. of three independent experiments (**, p � 0.02; ***,p � 0.01 for SM values compared with control).

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ciently reduces the transfer time of long chain Cer (53), selec-tively transferring Cer with C14 up to a chain length of C20 (36).In contrast, the rapid spontaneous transfer of C6-NBD-Cer

(t1⁄2 � 0.4 min) masks the CERT-mediated transfer (36).Because of its hydrophobicity, natural long chain Cer is embed-ded in the ER membrane. Its transport to the Golgi requires a

FIGURE 9. BFA treatment rescues the limonoid-induced inhibition of SM biosynthesis. A, CHO cells were treated for 1 h with limonoids (10 �M) or HPA-12(1 �M). Then they were incubated in the absence (gray bars) or the presence (black bars) of BFA for 30 min, followed by 2 h of labeling with [14C]serine. Lipidanalysis was performed as in Fig. 2. SM content is expressed in percentage of that of the control without BFA treatment after normalization of the radioactivityby the protein content. B, limonoids HC and Ged inhibit the CERT-mediated extraction of Cer from ER membrane in vitro; limonoids HC (closed diamond), Ged(open circle), Fiss (20 �M closed square), or 0.1% DMSO (control) were preincubated with isolated ER membranes containing 3H-labeled Cer for 20 min on ice asdescribed under “Experimental Procedures.” The extraction was started by the addition of hCERT. After 30 min of incubation at 37 °C, tubes were centrifugedand lipids extracted from supernatant and pellet. Lipids were analyzed as described under “Experimental Procedures.” Cer extracted are expressed in percent-age of the Cer content in the ER membranes. Values are mean and average deviation of two independent experiments for HC and representative of oneexperiment for Ged and Fiss. C, HC inhibits CERT-mediated extraction of Cer from artificial liposomes in vitro; 4 �M limonoids (HC and Fiss) or HPA-12 or 0.1%DMSO (control) were preincubated with liposomes composed of egg yolk PC, egg yolk PE, and ([14C]C16-Cer) (32:8:0.2, mol/mol) ( � liposome preincubation)or with hCERT ( � CERT preincubation) for 10 min on ice, and then hCERT was mixed with lipid vesicles. After 30 min at 37 °C, the mixture was centrifuged. Theradioactivity of the supernatant represented the Cer extracted from the vesicles. Values are expressed in percentage of hCERT activity (100% corresponding tothe Cer extracted by hCERT in the control) and are the mean and average deviation of two independent experiments.

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two-step process catalyzed by CERT as follows: first, extractionfrom the membrane, and second, transfer between the ER andGolgi apparatus membrane (12, 69). The C-terminal steroido-genic acute regulatory protein-related lipid transfer domain ofCERT contains the amphiphilic cavity for Cer binding (in a 1:1binding ratio) and the pleckstrin homology domain for phos-phatidylinositol 4-phosphate binding at the trans-Golgi (36,70). The crystal structure of the CERT-steroidogenic acute reg-ulatory protein-related lipid transfer domain in complex withCer demonstrates that the size and shape of its cavity controlsthe chain length limit and stereo-specificity of Cer recognitionby CERT. For example, the binding of C16- and C18-Cer com-

pletely fills the hydrophobic part of the cavity, whereas in thecase of C6-Cer some empty space remains (70). This is in goodagreement with the fact that CERT efficiently transfers Cerwith a chain length of C14 up to C20.

Synthetic HPA-12 is structurally similar to D-erythro-cer-amide and thus acts as a competitive inhibitor of CERT (27, 36,71). In contrast, the structure of limonoids does not resembleCer or other sphingolipids (Fig. 7). Recently, itwasdemonstratedthat the miscibility of Cer in the membrane affects its CERT-me-diated extraction (55). Cer was less efficiently extracted by CERTfrom tightly packed membranes compared with more fluid ones.This is in line with the DSC analysis, which indicated that the

FIGURE 10. Limonoid HC does not modify the fluorescence anisotropy of DPPC (A) and PC/PE/Cer (B) liposomes. A, anisotropy of DPH (0. 5 mol %) in DPPCvesicles (1 mM) was measured in the presence of increasing concentrations of HC (DPPC/drug molar ratio indicated in the legend). B, anisotropy of DPH (0.5mol %) in 0.8 mM PC/PE/Cer vesicles (32:8:2, mol/mol) was measured in the presence of 8 �M HC. DPH excitation was at 360 nm and emission at 428 nm. C–F,limonoid HC induces the formation of Cer-rich domains; DSC thermograms of DPPC (C), HC/DPPC (1:8 mol/mol) (D), C16-Cer/DPPC (1:8 mol/mol) (E), andHC/C16-Cer/DPPC (1:1:8 mol/mol/mol) (F) were recorded at a scan rate of 60 °C/h, and representative data are shown. In panels, upper and lower graphs indicatethermograms of heating and cooling, respectively. The vertical scale bars in C and D and E and F correspond to 2 and 0.2 kcal/mol/°C, respectively.

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presence ofHC reduced themiscibility ofCer andDPPC inducingthe formation of Cer-rich domains in themembrane.Limonoids include a structurally heterogenous group of nat-

ural products with a prototypical structure of a furan attachedto a tetracyclic core (A–D rings) (59, 72). Synthetic access tolimonoids represents a tremendous challenge due to the com-plexity of their molecular structure, as recently highlighted bythe completion of the total synthesis of azadirachtin, a naturalinsecticide extracted from the neem tree (73). The limonoidsand derivatives of this study can be roughly divided into threegroups based on their structural features (Fig. 7). The firstgroup is composed of the gedunin-like limonoids possessing apredominantly flat and fused core ring system. This group iscomposed of compounds 1, 4, 7, 13, 14, 15, 21, and Ged (com-pound 6) as well as the loosely related 9, 17, and 18. The 3-folddecrease of the inhibitory activity of compound 14 comparedwith 7 suggests a slightly adverse effect of the lactone in ring D.However, a variation in the A ring substitution pattern exertsonly a minor influence on the inhibitory activity, as demon-strated by the comparable activity of Ged, compounds 14 and15. In contrast, the presence of an �-hydroxy-�,�-unsaturatedketone in ring B was accompanied by reduced specificity of theinhibitory activity. The second group, the carapin-like limonoids,composed of compounds 3, 8, 10, 12, and Fiss (compound 5) fea-ture a bridged ketone between theA andB rings and the lack of anepoxide at ringD. This completely abolished any inhibition of SMbiosynthesis. In contrast, in the third group, limonoids featuringmultiple bridged ring systems, such as compounds 2, 11, 16, 19,HC, and CHC, tend to inhibit SM biosynthesis. Nevertheless, ahigh levelofbulky substituents incombinationwithanorthoester,as in compounds 2, 11, and 19, resulted in only moderate inhibi-tory activity. Interestingly, potent inhibitors, such as compounds16, HC, and CHC feature a hemiacetal bridge in their pentacycliccore ring system. Unlike acyclic hemiacetals, their cyclic counter-parts tend to exhibit a remarkable stability (54, 74, 75). This isfurther supported by theMS analysis indicating a sufficient stabil-ity of HC in the pH range of 5–7. In addition, the quite constantcellular level ofHC after 1 and 24 h of incubation suggests that thelimonoid itself, and not a metabolite, plays an active role inthe inhibition of SM biosynthesis. This is further supported bythe direct inhibitory effect of HC on the CERT-dependentextraction of Cer in vitro.

It is worth pointing out that SLmetabolismhas been associatedwith cancer cell proliferation and Plasmodium development (76–79). The limonoid-induced inhibition of SM biosynthesis repre-sents a plausible explanation of the anti-cancer and anti-malariaproperties of these compounds. We hope that these results willhelp spur the search for novel natural products with a high degreeof selectivity to avoid the problem of multidrug resistance oftenencountered in cancer andmalaria therapy.

Acknowledgments—We thank Dr. T. Hayakawa for comments on thephysical properties of limonoids. We are grateful to Drs. A. Yamaji-Hasegawa, M. Murate, and T. Kishimoto for helpful discussion andall themembers of Kobayashi laboratory for their support and criticalreadings of the manuscript.

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and Toshihide KobayashiHubert Vidal, Tamio Saito, Hiroyuki Osada, Kentaro Hanada, Anthony H. FutermanIshii, Asami Makino, Peter Greimel, Mitsuhiro Abe, Elad L. Laviad, Michel Lagarde, Françoise Hullin-Matsuda, Nario Tomishige, Shota Sakai, Reiko Ishitsuka, KumikoProtein-dependent Extraction of Ceramides from the Endoplasmic Reticulum

Limonoid Compounds Inhibit Sphingomyelin Biosynthesis by Preventing CERT

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