Review

Inhibitors of sphingolipid metabolism enzymes

Antonio Delgado a,b, Josefina Casas a, Amadeu Llebaria a, José Luís Abad a, Gemma Fabrias a,⁎

a Research Unit on Bioactive Molecules (RUBAM); Department of Biological Organic Chemistry, Chemical and Environmental Research Institute of Barcelona,(IIQAB-C.S.I.C), Jordi Girona 18-26, 08034 Barcelona, Spain

b University of Barcelona, Faculty of Pharmacy, Unit of Pharmaceutical Chemistry (Associated Unit to CSIC), Avda. Joan XXIII, s/n, 08028 Barcelona, Spain

Abstract

Sphingolipids are a family of lipids that play essential roles both as structural cell membrane components and in cell signalling. The cellularcontents of the various sphingolipid species are controlled by enzymes involved in their metabolic pathways. In this context, the discovery ofsmall chemical entities able to modify these enzyme activities in a potent and selective way should offer new pharmacological tools andtherapeutic agents.© 2006 Elsevier B.V. All rights reserved.

Keywords: Biosynthesis; Sphingolipids; Metabolism; Inhibitor; Enzyme

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19582. Biosynthesis of ceramide: inhibitors of the de novo pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1958

2.1. Serine palmitoyltransferase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19582.2. Ceramide synthase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19592.3. Dihydroceramide desaturase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1960

3. Biosynthesis and catabolism of sphingolipids: the pivotal role of ceramide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19613.1. Inhibitors of the sphingomyelin cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19613.2. Ceramidase inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19643.3. Inhibitors of glucosylceramide biosynthesis and catabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1965

3.3.1. Inhibitors of glucosylceramide synthase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19673.3.2. Inhibitors of glucosylceramide catabolic enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1968

3.4. Ceramide kinase inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19704. Biosynthesis and metabolism of sphingosine-1-phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1970

4.1. Inhibitors of sphingosine kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1970

Abbreviations: CBA, Conduritol B aziridine; CBE, Conduritol B epoxide; CDases, Ceramidases; Cer, Ceramide(s); CerK, Ceramide kinase; CerP, Ceramide-1-phosphate; CerS, Ceramide synthase; DAG, Diacylglycerol; DES, Dihydroceramide desaturase; DHS, D,L-threo-Dihydrosphingosine; DMS, N,N-Dimethyl-sphingosine; DNJ, Deoxynojirimycin; ER, Endoplasmic reticulum; ERT, Enzyme replacement therapy; FB1, Fumonisin B1; GlcCer, Glucosylceramide; GlcCerase,Glucocerebrosidase; GlcCerS, Glucosylceramide synthase; GSH, Glutathione; GSLs, Glycosphingolipids; LacCer, Lactosylceramide; LacCerS, Lactosylceramidesynthase; NBDGJ, N-Butyldeoxygalactonojirimycin; NBDNJ, N-Butyldeoxynojirimycin; NNDNJ, N-Nonyldeoxynojirimycin; NOE, N-Oleoylethanolamine; NOV,N-Octylvalienamine; PKC, Phosphokinase C; SAP, Sphingolipid-activator protein; SLs, Sphingolipids; SM, Sphingomyelin; SMase, Sphingomyelinase; SMS,Sphingomyelin synthase; Sph, Sphingosine; SphK, Sphingosine kinase; SphP, Sphingosine-1-phosphate; SphPL, Sphingosine-1-phosphate lyase; SPT, Serinepalmitoyltransferase; SRT, Substrate reduction therapy⁎ Corresponding author. Fax: +34 932045904.E-mail address: gfdqob@cid.csic.es (G. Fabrias).

4.2. Inhibitors of sphingosine-1-phosphate lyase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1972Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1972References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1972

1. Introduction

Sphingolipids (SLs) are a family of lipids that play essentialroles as structural cell membrane components and also in cellsignalling. From a structural standpoint, SLs derive fromsphingosine (Sph), a long chain amino alcohol, which isacylated with a long chain fatty acid to give ceramides (Cer), thecentral core of glycosphingolipids (GSLs) and sphingomyelin(SM). Living cells contain several Cer and Cer metabolitespecies differing in the length and degree of unsaturation oftheir acyl chain, which appears to be relevant to their biologicalactivities. Although SLs have been classically related to cellpermeability, their roles in cell signalling and intercellularcommunication are well recognized. Moreover, abnormal SLsmetabolism is nowadays known to occur in some diseases, suchas, inter alia, certain sphingolipidoses [1], cancer [2], diabetes[3], and atherosclerosis [4]. As it will be discussed next, thecellular contents of the various SLs species are controlled byenzymes involved in their metabolic pathways. In this context,the search for potent and selective inhibitors of these enzymesoffer new insights for the discovery of therapeutic agents.

2. Biosynthesis of ceramide: inhibitors of the de novo pathway

Cer is the central molecule in SLs and GSLs biosynthesis.There are twometabolic routes leading to Cer: the anabolic or thede novo pathway and the catabolic one. The first one comprises aseries of enzymatic processes leading ultimately to Cer fromsimple components, such as palmitoyl CoA and serine (Sections

2.1 Sections 2.2 Sections 2.3), whereas the catabolic route leadsto Cer from hydrolysis of complex SLs, mainly SM (Section 3.1)and GSLs (Section 3.3.2) (Fig. 1).

2.1. Serine palmitoyltransferase

Serine palmitoyltransferase (SPT) catalyzes the first step inthe biosynthesis of sphingolipids, which is the condensation ofserine and palmitoyl CoA to produce 3-ketodihydrosphingosine[5]. SPT is a member of a pyridoxal phosphate-dependent α-oxoamine synthase family. The mammalian enzyme is aheterodimer of two subunits, LCB1 and LCB2, both of whichare located in the endoplasmic reticulum. Specific missensemutations in the human subunit LCB1 gene cause hereditarysensory neuropathy type I. Inhibitors of SPT include sphingo-fungins, lipoxamycin and myriocin (Fig. 2), which are naturalproducts with potent and highly selective activity. It has beensuggested that inhibition occurs by reaction of these compoundswith SPT to form adducts that mimic the natural intermediatesof the catalytic reaction [5].

The relevance of sphingofungin B stereochemistry to SPTinhibitory activity was demonstrated by examining the effect ofseveral stereoisomers (Fig. 3) on sphingolipid biosynthesis bothin cell free extracts and in intact cells. They inhibitedsphingolipid biosynthesis in the order 2S,3R,4R,5S,14R=2S,3R,4R,5S,14S≫2R,3R,4R,5S,14R>2S,3S,4R,5S,14R ≈2S,3R,4R,5S > 2R,3S,4R,5S,14R≈ 2R,3S,4S,5R,14R≈2S,3R,4S,5R,14R [6]. These results indicate that although theC-14 hydroxyl group of sphingofungin B confers potent SPT

Fig. 1. Biosynthesis of Cer: the de novo pathway.

inhibition, its configuration is not crucial for activity. In contrast,the configurations of the stereogenic centers at positions α, β, γand ∂with respect to the carboxyl group are important to activity.

Viridiofungins are also SPT inhibitors of natural origin [7].They are less potent and specific that sphingofungins andmyriocin, since they also inhibit other enzymes sensitive to di-and tricarboxylic acids, such as squalene synthase [8]. Further-more, viridiofungins do not inhibit Saccharomyces SPT, but theydo inhibit this enzyme in Candida albicans [7]. Two othercompounds that have been extensively used as SPT inhibitors areL-cycloserine andβ-chloro-L-alanine (Fig. 2). Nevertheless, thesetwo chemicals inhibit several other pyridoxal phosphate-dependent enzymes [9,10].

2.2. Ceramide synthase

Ceramide synthase (CerS) catalyzes the acylation of theamino group of Sph, sphinganine and other sphingoid basesusing acyl CoA esters of varying chain lengths. CerS activity isfound both inmicrosomes [11] andmitochondria [12]. Inhibitionof CerS by several fungi metabolites has been reported, whichinclude Fumonisins [13], the fumonisin-relative AAL-toxin [14](Fig. 4) and australifungins [15] (Fig. 5). The fumonisins familyare produced by Fusarium verticillioides and Fumonisin B1(FB1) is the most prevalent member of this class of compounds.FB1 contains an aminoeicosapentol backbone with twohydroxyl groups esterified with 3-carboxy-1,5-pentanedioicacid. A similar structure is present in AAL-toxin, produced bythe fungus Alternaria alternata var. lycopersici. Several studies[16] suggest that inhibition occurs because CerS recognizes theaminopentol moiety (Fig. 4), which competes for binding to thesphingoid base substrate, as well as the dicarboxylic acid sidechains, which act as analogs of fatty acyl-CoA phosphate groups

and interfere the binding of the acyl donor with the catalyst. TheO-deacylated form of FB1 is a weak CerS inhibitor. However, itis also a substrate of CerS and its N-acylated forms are potentCerS inhibitors [16]. This sequence of metabolic transforma-tions may occur in vivo and play a role in the diseases caused byfumonisins. In this regard, these N-acylated forms conform anew category of ceramide synthase inhibitors. Structure-function relationship studies showed that both erythro- andthreo-2-amino-3-hydroxy- and all (2S) stereoisomers of 2-amino-3,5-dihydroxyoctadecanes (Fig. 4) are CerS inhibitorsand are acylated by CerS with the highest apparent Vmax/Km forthe 2S,3R analogues.

FB1 inhibits fungal CerS in vitro, but its activity in wholecells is very weak, probably due to deficient internalization. Incontrast, australifungin (Fig. 5), a micotoxin isolated fromSporormiella australis, is a very potent inhibitor of fungal CerSfrom several species. However, both the alpha-diketone andbeta-ketoaldehyde functional groups present in this compoundconfer australifungin a high chemical reactivity, which limits itsuse [15].

Fig. 3. Stereoisomers of sphingofungin B.

Fig. 2. Inhibitors of SPT.

2.3. Dihydroceramide desaturase

Dihydroceramide desaturase (DES) is the last enzyme in thede novo biosynthesis of ceramide [17]. Two different dihy-droceramide desaturases, DES1 [18] and DES2 [19], have beenso far reported. Although their amino acid sequences have highidentity, they differ in their enzymatic characteristics, sincewhile DES1 exhibits high dihydroceramide desaturase activityand very low C-4 hydroxylase activity, DES2 is similarly activeas both a dihydroceramide desaturase and a C-4 hydroxylase(Fig. 6).

The cyclopropene-containing sphingolipid GT11 (Fig. 7) is acompetitive inhibitor of DES [20,21]. Structure–activity correla-tion studies [21,22] showed that the natural 2S,3R stereochem-istry, the amide function, the presence of a cyclopropene ring inplace of the ceramide double bond and a free hydroxyl group atC1 are required for inhibition, as evidenced from comparison ofGT11 with C1-modified analogues GT34, GT37, and GT30 (Fig.7) [21]. Furthermore, the α-ketoamide (GT85, GT98, and GT99),urea (GT55) and thiourea (GT77) analogues (Fig. 7) retaininhibitory activity, whereas the respective carbamate (GT45) wasinactive [22]. Likewise, N-methyl substitution of GT11 (com-pound GT54) results in the loss of inhibitory activity [22]. Theineffectiveness of carbamate andmethylamido derivativesmay bedue to interference with hydrogen bonding events required for

inhibition, (i.e. enzyme–inhibitor interactions or arrangement ofthe inhibitor into the active conformation).

In primary cultured neurons, GT11 inhibits dihydroceramidedesaturase in a concentration dependent manner (IC50 of 23 nM)with a potency by three orders of magnitude higher than thatfound in vitro [23]. However, although this inhibitory effect isspecific at concentrations up to 1 μM, higher concentrations(from 5 μMupwards) of GT11 not only abolishes desaturation ofdihydroceramide, but also impairs de novo sphingolipid biosyn-thesis by an indirect suppression of SPT activity. Metabolicstudies with radioactively labeled GT11 analogs indicated that theinhibitor is subjected to catabolism by ceramidases (CDases) toyield the cyclopropene-bearing long chain base [23]. Althoughthe free cyclopropene base does not inhibit dihydroceramidedesaturase [23], it cannot be completely disregarded thatdeacylated GT11 or its phosphate are involved in the effect ofhigh concentrations of GT11. Further studies are needed to clarifythe mechanism underlying the effects of high concentrations ofGT11 in cultured cells.

Two dihydroceramide analogues with different alkyl chainlengths have been prepared, in which the 3-hydroxy group hasbeen replaced by a fluorine atom (Fig. 8). Theywere investigated

Fig. 4. Fumonisin and related compounds as CerS inhibitors.

Fig. 5. Australifungin. Fig. 6. Activities of dihydroceramide desaturases DES1 and DES2.

as potential DES inhibitors, but they showed a slight inhibition[24].

A dihydroceramide analogue with a cyclopropane ring at C-5and C-6 of the sphinganine backbone has been reported as aputative suicide inhibitor of the dihydroceramide desaturase (Fig.8) [25]. The authors do not specify the configuration of thecyclopropane stereogenic centers. They postulate that the C4reactive intermediate generated in the first step of the desatura-tion reaction [26] binds covalently to an amino acid side chain ofthe desaturase, thus inhibiting the enzyme irreversibly, andleading to the covalent modification of the protein.

Finally, in a recent article Schulz et al. have suggested that 4-hydroxyfenretinide is an inhibitor of dihydroceramide desatur-ase [27].

3. Biosynthesis and catabolism of sphingolipids: the pivotalrole of ceramide

Cer plays a central role in SLs biosynthesis. As depicted in Fig.9, Cer can arise either from simple components in the so called denovo biosynthesis (Section 2), from reacylation of Sph producedby catabolic processes (“salvage pathway”), degradation of SMthrough the “sphingomyelin cycle” or GlcCer hydrolysis. In allcases, the reverse anabolic pathways converting Cer to the aboveprecursors also occur. Thus, the overall metabolic processesaround Cer compose an intricate scenario in which severalenzymes are implicated in the forward and reverse steps, offeringexcellent targets for therapeutic intervention.

3.1. Inhibitors of the sphingomyelin cycle

SM is the primary sphingophospholipid in mammalian cells.As much as two thirds of the total cellular SM resides at theplasma membrane, where it plays important structural andfunctional roles. The major pathway for SM synthesis iscatalyzed by the phosphatidylcholine:ceramide phosphocholinetransferase (sphingomyelin synthase; SMS), which transfers thephosphocholine group from phosphatidylcholine to ceramide togenerate sphingomyelin and diacylglycerol (DAG). Thisenzyme has the important ability to directly regulate, inopposite directions, ceramide and DAG levels within thecells, potentially controlling opposite cellular processes such as

cell proliferation, growth arrest, and apoptosis [28]. SMS is anintegral membrane enzyme located in the Golgi apparatus.Despite its importance in sphingolipid metabolism, SMSs-encoding cDNAs have only been recently identified [29–31].

Compound MS-209 (Fig. 10), a quinolone-derivative thatblocks P-glycoprotein and multidrug resistance-associatedprotein-1, has been reported as a SMS inhibitor [32].Tricyclodecan-9-xanthogenate (D609, Fig. 10) was firstreported as an inhibitor of phosphatidylcholine-specific phos-pholipase C from Bacillus cereus [33,34]. Moreover, it has beenreported that D609 also inhibits SMS in different cell types [35–37]. However, the xanthate group is chemically unstable insolution and is readily oxidized. Recently, synthesis and activityof chemically stable S-(alcyloxymethyl) analogues of D609have been reported. Among them, the pivaloyloxymethylanalogue (D609 prodrug in Fig. 10) inhibited SMS moreefficiently than D609 in cells [38].

SM hydrolysis can be carried out by isoforms of sphingo-myelinase (SMase), of which at least five isotypes have been sofar described. Most mammalian cells are capable of signallingthrough the SM pathway, which can be activated both byreceptor ligands (TNF, interleukines) and stress (UV, oxidants,radiation) [39]. SM hydrolysis by SMases produces phosphor-ylcholine and the intracellular effector Cer. The former isreleased into the aqueous environment, while Cer diffuseswithin membranes, acting as a second messenger. Generation ofCer in various cellular systems is currently recognized as criticalto the initiation of vital cellular processes such as differentia-tion, cell proliferation and apoptosis.

SMases differ in their catalytic properties, subcellularlocation and probably in their mode of regulation. AcidSMase catalyzes the lysosomal degradation of SM. Inheriteddeficiencies of acid SMase activity result in various clinicalforms of the Niemann–Pick disease, which are characterised bymassive lysosomal accumulation of SM [40]. Most of the acidSMase is lysosomal, but an alternately spliced Zn-dependentsecretory form exists [41]. This second form is differentiallyglycosylated and processed, and is believed to function ininflammatory processes, including atherogenesis [42]. It issecreted by macrophages, human skin fibroblasts, and humanvascular endothelial cells and is the only SMase responsible foran extracellular hydrolysis of SM [43,44]. Recently, it has beensuggested that secreted SMase may play a critical role in thedevelopment of apoptosis and organ failure in sepsis [45]. Onthe other hand, neutral SMase is a Mg-dependent enzymelocated in plasma membranes [46]. In addition, a Mg-independent neutral SMase is also found in cytosol [47], and

Fig. 7. GT11 and analogues as DES inhibitors.

Fig. 8. Dihydroceramide analogues as DES inhibitors.

an alkaline SMase has been identified in the gastrointestinaltract [48].

Several physiological inhibitors of acid SMase have beendescribed. Thus, Kolzer et al. reported L-α-phosphatidyl-D-myo-inositol-3,5-bisphosphate (PtdIns3,5P2) as a specific acid SMaseinhibitor (Ki 0.53 μM.) [49], and Testai et al. showed that L-α-phosphatidyl-D-myo-inositol-3,4,5-triphosphate (PtdIns3,4,5P3)is a non-competitive inhibitor of acid SMase from humanoligodendroglioma cells, with an IC50 value of 3 μM [50]. Theseresults provide some insights into the structural features requiredfor the selective, efficient inhibition of acid SMase (i.e. the 3-phosphoinositide moiety may be critical) and may also be used asstarting point for the development of new potent acid SMaseinhibitors [50]. Recently, ceramide-1-phosphate (CerP) [51] andsphingosine-1-phosphate (SphP) [52] have also been describedas physiological inhibitors of acid SMase in intact macrophages.However, different mechanisms of action have been unveiled forboth inhibitors [53]. CerP also inhibited acid SMase in cell

homogenates, whereas SphP was only active in intact cells [52].On the other side, glutathione (GSH) has been shown to be aninhibitor of neutral SMase in a dose-dependent manner atphysiological concentrations with a greater than 95% inhibitionobserved at 5 mMGSH [54]. Since GSH depletion is observed ina variety of cells in the process of cellular injury and apoptosis,these studies suggest that this may be an important mechanismfor activation of neutral SMase. It is worth noting that thismechanism links oxidative stress and signalling through productsof SM hydrolysis [55].

There are a number of compounds, structurally unrelated toSM, that have found application as SMase inhibitors. This is thecase of desipramine, SR33557, NB6, C11AG and GW4869(Fig. 11).

Desipramine has been frequently used as a selective acidSMase inhibitor. This compound, and possibly also othersimilarly acting tricyclic antidepressants, induce proteolyticdegradation of acid SMase [56] by interfering with the bindingof the enzyme to the lipid bilayers and thereby rendering itsusceptible to proteolytic cleavage by lysosomal proteases [57].Compound SR33557 is also an specific acid SMase inhibitor(72% inhibition at 30 μM) [58]. This compound avoids apoptosisinduced by TNF inML-1a cells. Recently, NB6 has been reportedas an inhibitor of the SMase gene transcription [59] and it hasbeen used in in vivo experiments to propose that activation of theplasmatic isoform of acid SMase may play a critical role in thedevelopment of apoptosis and organ failure in sepsis [45].Amtmann et al. described a series of new guanidinium derivativesas inhibitors of neutral SMase. Lipophilicity was found to becorrelated with the inhibitory potential of the compounds and theundecylidene aminoguanidine C11AG (Fig. 11) was the most

Fig. 9. Biosynthesis of SLs from Cer.

Fig. 10. SMS inhibitors.

active [60]. Collagen induced arthritis in mice could be preventedand/or alleviated by C11AG treatment [61]. Compound GW4869(Fig. 11) was discovered during a high throughput screening onMg2+ dependent neutral SMase. This compound acted as anoncompetitive inhibitor with the substrate SM (IC50: 1 μM) andexhibited significant and specific inhibitory activity with no orminor inhibitory activity versus other hydrolytic enzymes, suchas bacterial phosphatidylcholine-PLC and bovine proteinphosphatase 2A. Importantly, GW6948 showed no inhibitionof the human acid SMase [62].

On the other hand, several compounds isolated from naturalsources exhibit interesting inhibitory activities against SMases.

This is the case of scyphostatin (Fig. 12), a competitive inhibitor(IC50: 1 μM) of neutral SMase, isolated from the mycelialextract of Trichopeziza mollissima [63,64] Other SMaseinhibitors of natural origin are Macquarimicin A (IC50

146 μM for neutral SMase and 616 μM for acidic SMase)[65], Alutenusin, a non-competitive inhibitor of neutral SMase[66], Chlorogentisylquinone [67], Manumycin A (Fig. 12), anirreversible specific inhibitor of neutral SMase [68], and α-Mangostin, an acidic SMase inhibitor isolated from themicroorganism Garcinia speciosa [69].

Based on the inhibitory activity of scyphostatin, differentanalogues have been designed. The first one was spiroepoxide 1

Fig. 11. Inhibitors of SMase.

Fig. 12. Natural and synthetic SMase inhibitors.

(Fig. 12), an irreversible and specific neutral SMase inhibitor[70]. The primary hydroxyl group seems crucial for enzymeinhibition, since a drastic decrease on the inhibitory activity wasobserved after its replacement by H or phenyl groups [71]. Lateron, several analogues sharing the epoxy and cyclohexenonestructural features of Scyphostatin and Manumycin A have beensynthesized [72] and tested. Thus, compounds 2 and 3 (Fig. 12)turned out to be selective irreversible inhibitors of neutralSMase. Preliminary structure–activity studies suggested that thepresence of the cyclohexenone moiety was crucial for theactivity [68]. On the other hand, Pitsinos et al. reported thepreparation and inhibitory SMase activity of analogues 4–6 (Fig.12), which lack the epoxy moiety. Compounds 4 and 6 werefound to be irreversible neutral SMase inhibitors, whilecompound 5 was a weak inhibitor. As in the spiroepoxideanalogues, the presence of a primary hydroxyl group increasesenzyme inhibition, whereas an unsaturated side chain con-tributes to a higher affinity for neutral SMase. Furthermore, theepoxy moiety seems not to be a strict requirement for enzymeinhibition [73]. In this context, compound 7 (Fig. 12), lackingthe epoxy function of the natural congener, has been reported asthe first scyphostatin analogue with a reversible neutral SMaseinhibitory activity [74]. Claus et al. also described the neutralSMase inhibition by this new analogue in several in vivo testsystems (monocytes, macrophages, hepatocytes) by monitoringits antiapoptotic effects and the inversion of phorbol ester-induced translocation of green fluorescent protein labeled kinase(protein kinase C-alpha) [74]. Recently, irreversible inhibition ofneutral SMase by a new family of compounds, namedsphingolactones, has been reported. Compounds with asaturated butyrolactone (8–9) were better inhibitors than thecorresponding unsaturated analogues (10–11), and (2E,4E)-hexadienoic acid derivative 8 was the most active [75].

Lister et al. [76] prepared sphingomyelin analogues differing atthe C-1 or C-3 position of the Sph backbone, which were testedagainst neutral and acid SMase. Compounds modified at the C-1position were not active, whereas the 3-O-methyl(compound 12,Fig. 13) and the 3-O-ethylsphingomyelin analogues were selectiveinhibitors of neutral SMase, with IC50 values of 50 μM and140 μM, respectively. On the other hand, Hakogi et al. [77]synthesized the sphingomyelin carbon analogues 13 (Fig. 13),which showed moderate inhibitory activity toward neutral SMase

from B. cereus (IC50=120 μM for n=1 and 78 μM for n=2).Taguchi et al. [78] described the synthesis and activity of simplifiedSM analogues with improved solubility and higher hydrolyticstability. These goals were achieved by shortening the N-acylgroup, replacing the phosphodiester moiety by an ester orcarbamate function and using a dialkylamino or pyridyl group asthe trimethylammonium unit surrogates. Among them, carbamateanalogues of general structure 14 (Fig. 13) were good inhibitors ofneutral SMase, with IC50 values of 2–80μM.Moreover, carbamate15 prevented Cer generation and apoptotic neuronal cell death in amodel of ischemia based on organotypic hippocampal slice cultures[79].

On the other hand, Yokomatsu et al. [80] reported thesynthesis and activity of the CerP difluoromethylene analogues16 (Fig. 13), in which the alkenyl chain of the parent sphingoidbase and the phosphate moiety were replaced by a phenyl groupand an isosteric difuoromethylenephosphonic acid, respectively.The diastereomeric mixture showed a non-competitive neutralSMase inhibition in bovine brain microsomes with an IC50 of400 μM.

In addition, single enantiomers 16, ent-16 and the deoxyana-logue 17 (Fig. 13) were also evaluated. The deoxyderivative 17(IC50=181 μM) was found to be 2-fold more potent than 16,which demonstrated that the 3-OH group is not critical for neutralSMase inhibition. On the other hand, neutral SMase inhibitionsignificantly increases in analogues with unnatural configurationon the sphingoid base. Thus, ent-16 (IC50=99 μM)was ca. 4-foldmore potent than 16. This trend was even more striking withdeoxy derivatives (17 vs. ent-17), since ent-17 (IC50=3.3 μM;Ki=1.6 μM) was found to be ca. 60-fold more potent than 16.These results reveal that the stereochemistry of the sphingoidchain backbone is critical for interaction with the enzyme. Themode of inhibition for ent-17 was determined to be non-competitive. This analogue is also a good acid SMase inhibitorfrom bovine brain lysosomes, showing a 48% inhibition at3.3 μM concentration [81].

3.2. Ceramidase inhibitors

Catabolism of ceramide occurs by the action of CDases,which hydrolyze the amide bond to yield the Sph moiety andfatty acids. According to their pH optima for activity, CDases

Fig. 13. SM analogues as SMase inhibitors.

fall into three groups, acidic, neutral and alkaline. The acidicCDase is localized in the lysosomes and its defect causesFarber's disease [82–84]. Neutral CDases are localized in theendoplasmic reticulum and the mitochondria and they havebeen reported in several species [85–90]. Finally, alkalineCDases have also been found in different organisms frombacteria [91–93] to yeast [94,95] and mammals [96,97].

Several inhibitors of the neutral/alkaline [98,99] and theacidic CDase [100–103] have been reported. The formerinclude the Cer analogue (1S,2R)-D-erythro-2-(N-myristoyla-mino)-1-phenyl-1-propanol (D-MAPP) (Fig. 14) [98], whichhas been extensively used in basic studies. From the chemicalstandpoint, its enantiomer does not inhibit the neutral CDase,but it undergoes a time- and concentration-dependent N-deacylation to an extent similar to that observed with N-hexanoylsphingosine [98].

The structural requirements of Cer and Sph analogues asmitochondrial CDase inhibitors were investigated by Usta et al.[99]. This enzyme was inhibited by all Sph stereoisomers, aswell as by N-methyl-D-erythro-sphingosine, D-erythro-sphinga-nine, D-erythro-4,5-dehydrosphingosine (Fig. 14), (2S)-3-keto-sphinganine, the three diastereoisomers of natural Cer andD-erythro-N-hexadecylcarbamoylsphingosine (Fig. 14). Lesspotent inhibitors included (2S)-N-hexadecanoyl-3-ketosphingo-sine and N-octadecylsphingosine (Fig. 14). The N-octadecyl-sphingosine and the urea analogue are competitive inhibitors. Inthe same study, 1-O-methyl-D-erythro-sphingosine, (cis)-D-erythro-sphingosine, (2S)-3-ketosphingosine, (2S)-3-ketodehy-drosphingosine and N,N-dimethyl-D-erythro-sphingosine werefound to be weak inhibitors. These studies concluded that theprimary and secondary hydroxyl groups, the C4–C5 transdouble bond, and the NH-protons from either the amide or theamine are required for enzyme inhibition. N-oleoylethanola-mine (NOE, Fig. 14) is the acidic CDase inhibitor mostextensively used in cell biology studies, despite having a weakpotency (0.5 M) and poor selectivity [104]. From a chemicalstandpoint, NOE can be regarded as an N-oleoylsphingosineanalogue arising from formal removal of the 1-hydroxy-2-hexadecyl moiety. In contrast to NOE, N-leoylsphingosine is asubstrate of acidic CDase [105], which suggests that chemicalmanipulation of the N-oleoylsphingosine framework might be asuitable approach to the discovery of new acidic CDaseinhibitors. In agreement with this idea, several members of a

family of C2 substituted NOE analogues showed a higher acidicCDase inhibitory potency than NOE [106].

Other acidic CDase inhibitors include (1R,2R)-2-(N-tetrade-cylamino)-1-(4-nitrophenyl)-1,3-propanediol (AD2646) [102]and (1R,2R)-2-(N-tetradecanoylamino)-1-(4-nitrophenyl)-1,3-propanediol (B-13) [100,101,103] (Fig. 15). Both compoundssuppress acid CDase activity with good potencies and the activityof the neutral/alkaline isoform is affected only slightly. Themechanism of inhibition by these compounds is not known. Inrecent articles [106,22], the first family of mechanism-basedinhibitors of the acid CDase with potencies in the low μM rangewas reported (compounds GT – Fig. 15 – and GT85, GT98. andGT99 –Fig. 7) These compounds, which bear an α-ketoamideunit (Fig. 15), have a good selectivity against acidic CDase invitro, since inhibition of neutral CDase requires around 20-foldhigher concentration.

3.3. Inhibitors of glucosylceramide biosynthesis andcatabolism

Glycosylation of Cer at the C1(OH) position providesglycosphingolipids (GSLs), a wide structural family of cellmembrane components that plays important roles in biologicalsystems. They differ in the nature and the number of sugar unitslinked to the Cer scaffold. As depicted in Fig. 16, thebiosynthesis of most GSL in mammals starts in the Golgicomplex with glucosylation of Cer. In this process, catalyzed byglucosylceramide synthase (GlcCerS) (UDP-glucose:N-acyl-sphingosine glucosyltransferase), the α-glucoside residue istransferred from UDP-glucose to Cer with inversion ofconfiguration at the anomeric center of the sugar.

Fig. 14. Inhibitors of the neutral CDases.

Fig. 15. Inhibitors of CDases.

The initially formed glucosylceramide (GlcCer) serves asstarting building block for the synthesis of complex GSLs.Thus, condensation of a galactose unit by means of agalactosyltransferase leads to lactosylceramide (LacCer), astarting point to the biosynthesis of all the hitherto reportedGSLs series [107].

The molecular architecture of GSLs as membrane compo-nents requires an extracytosolic disposition with their hydro-philic carbohydrate head groups oriented towards the outerleaflet of the plasma membrane. In this context, GSLs play aprotective role in the cell against chemical and mechanicaldamage. In addition to these mechanical properties, GSLs areexpressed as complex patterns, which have been accepted to takeactive part in processes such as cell adhesion, cell growthregulation, and differentiation. This “communication role” ofGSLs has emerged since the recognition of lectins ascomplementary carbohydrate binding proteins, and the impli-cation of cell surface glycolipids as physiologically relevantlectin ligands in cell–cell recognition systems [108]. GSLs alsoplay important roles within cellular membranes, where theyform microdomains (“rafts”), which can regulate the activity ofsome receptors in the plasma membrane, and hence signaltransduction [109].

While Cer biosynthesis is catalyzed by membrane-boundenzymes at the cytosolic face of the endoplasmic reticulum(ER), formation of GlcCer takes place at the cytosolic face ofthe Golgi apparatus, and higher gangliosides are biosynthesizedat the luminal face. GSLs are then transported to the cellmembrane by means of exocytosis mechanisms from the Golgi[107]. GSL catabolism takes place in the lysosomes, which arespecialized cell compartments where glycon-specific, pH

optima glycosidases hydrolyze GSLs, arising from endocytosisfrom cell membranes, to the corresponding monosaccharidesand Cer, which are mostly recycled in the cytosol. In the case ofGSLs with short oligosaccharide chains, the lipids to bedegraded are no longer accessible to hydrolases since they arestill embedded in the lipid bilayer structure of the parent cellmembrane. In this case, the participation of the so-called“sphingolipid-activator proteins” (SAP or saposines) is re-quired. It is widely accepted that SAP play an essential role bypresenting the oligosaccharide to the specific glycosidase by“lifting” the glycolipid from the hydrophobic membraneenvironment [110].

Metabolic disorders of GSL metabolism produce glyco-sphingolipidoses, which a rare group of inherited diseaseshaving diverse, often neurodegenerative, and severe phenotypes[1]. They are caused bymutations in catabolic enzyme genes thatreduce the catalyst efficiency giving rise to increased lysosomalstorage products. As it will be discussed below (see Sections3.3.1 and 3.3.2), recent advances in the treatment of some ofthese diseases have been achieved through direct intervention onparticular key enzymes involved in GSL biosynthesis and/ordegradation.

Catabolism of GlcCer (Fig. 16) can take place both atlysosomal and non-lysosomal levels. In the first case, gluco-sylceramide β-glucosidase (GlcCerase or glucocerebrosidase) isresponsible for the hydrolysis of GlcCer into Cer and glucose, aprocess that requires the participation of the sphingolipid-activator protein SAP-C (see above) [111]. On the other side, theexistence of a non-lysosomal glucosylceramidase near the cellsurface has also been documented [112]. This enzyme is anintegral membrane protein that shows different specificity

Fig. 16. Biosynthesis and catabolism of GlcCer.

Fig. 17. Inhibitors of GlcCerS (modified Cer analogues).

towards substrates and inhibitors, as compared with thelysosomal enzyme (Section 3.3.2) [113].

The only known enzyme inhibitors of GSL biosynthesis atthe Cer level are restricted to the glucosylation step (catalyzedby GlcCerS), its catabolic counterparts (GlcCerase and non-lysosomal glucosylceramidase) and the galactosyltransferaseleading to LacCer from the initially formed GlcCer.

3.3.1. Inhibitors of glucosylceramide synthaseThe enzyme glucosylceramide synthase (GlcCerS) is one of

the few glycosyltransferases for which efficient inhibitors areknown [114]. They belong to two different structural classes: (a)those structurally related to Cer, and (b) synthetic analogues ofnaturally occurring iminosugars, such as the α-glycosidaseinhibitor deoxynojirimycin (DNJ).

(a) Ceramide analogues: In the search for GSL biosynthesisinhibitors, Vunnam and Radin [115] described a series ofceramide analogues in which the unsaturated alkyl chain and theamino moiety of the enzyme substrate were replaced by anaromatic ring and an heteocyclic system, respectively (Fig. 17).The most potent inhibitors were 1-phenyl-2-decanoylamino-3-morpholino-propanol (PDMP) and its N-palmitoyl homologue(PPMP). In all cases, the D-threo isomers were the most activeas competitive inhibitors of GlcCerS [116].

These results boosted the synthesis of new analogues arisingfrom modifications of the aromatic and heterocyclic moieties.Some of them, in particular compound hydroxy-P4, exhibited adramatic increase in potency and selectivity for the ceramide-specific glucosyl transferase [117]. Moreover, the ethylene-dioxy derivative (n=1, see Fig. 17) has recently been describedas a specific inhibitor of human GlucCerS in comparison withenzymes from non-human origin [118]. Analogues combiningthe heterocyclic moiety with the Sph backbone have also beenreported (Fig. 18) [119,120]. In all cases, it is worth mentioningthat the stronger inhibitors showed the non-natural (R,R)configuration of D-threo sphingosine. These findings aresomewhat intriguing and pose serious doubts about the role ofthese compounds as real substrate analogues. Nevertheless, it iswidely accepted that the protonated heterocyclic ring is able tomimic the charged transition state of the enzyme/UDP–glucosecomplex and that D-threo-PDMP is a reversible, mixed-typeinhibitor for ceramide but is uncompetitive for the nucleotidesugar donor [116].

(b) Iminosugars and related compounds: Iminosugars(polyhydroxypiperidines) comprise an important group ofinhibitors of glycosylation enzymes [121,122]. In particular,

Fig. 18. Inhibitors of GlcCerS derived from the Sph backbone.

Fig. 19. Iminosugars as GlcCerS inhibitors (in parenthesis, % inhibition of GlcCerS at 200 μM).

Fig. 20. Irreversible inhibitors of GlcCerase.

N-butyldeoxynojirimycin (NBDNJ) and N-butyldeoxygalacto-nojirimycin (NBDGJ) (Fig. 19) have shown inhibitory activityagainst GlcCerS [123,124].

Despite having been described as sugar analogues acting astransition state mimics of the glycosylation step, Butters et al.have shown that inhibition of GlcCerS byNBDNJ is competitivefor Cer and non-competitive for UDP-glucose, which indicates astructural mimicry of NBDNJ with Cer [125]. Structure–activitystudies carried out with iminosugars have evidenced theimportance of the N-alkyl chain for GlcCerS inhibition. Thus,a chain length between 3 and 6 carbon atoms is required, whilelonger chains increase inhibition, but also toxicity (see data inFig. 19). Modeling studies led to the assumption that NBDNJanalogues substituted at the C(2) oxygen atom with a long alkylchain should be potent inhibitors due to their closer similaritywith Cer. However, experimental data did not confirm thishypothesis [126]. Some iminosugars have been approved for the

treatment of certain sphingolipidoses. For example, NBDNJ(miglustat, Zavesca®) [127,128] is currently used as a “substratereduction therapy” (SRT) drug for treating type I Gaucherdisease [129].

3.3.2. Inhibitors of glucosylceramide catabolic enzymesAs mentioned above, catabolism of GlcCer can take place by

the action of two β-glucosidases (glucosylceramide hydrolases)that differ in their location and sensitivity towards selectiveinhibitors. Thus, D-gluconolactone is a competitive inhibitor ofthe non-lysosomal glucosylceramidase [112,113] but not oflysosomal GlcCerase, for which some irreversible inhibitors areknown, such as, for example, conduritol B epoxide (CBE)[130,131,113], cyclophellitol [132], and conduritol B aziridine(CBA) [133] (Fig. 20).

Irreversible GlcCerase inhibitors have been used to gain in-sight into the structure of the enzyme active site [130,131,134],as well as pharmacological tools to induce animal models of

Fig. 21. Polyhydroxypiperidine alkaloids and iminosugars as glucosylceramide hydrolase inhibitors (in parenthesis, non-lysosomal/lysosomal GlcCerase IC50 μMinhibition); ni: no inhibition.

Fig. 22. Analogy of isofagomine with the carbocationic intermediate generatedin the course of the hydrolysis of a β-glycosidic bond.

Fig. 23. Isofagomine analogues with an alkyl chain on C6 position. GlcCeraseinhibition (IC50, nM) is shown in parenthesis. The postulated interaction modelwith GlcCerase active site is depicted at the bottom.

Gaucher disease by accumulation of GlcCer [135]. However,most GlcCerase inhibitors are competitive inhibitors and the canbe classified into three different families:

(a) Polyhydroxypiperidine alkaloids and iminosugars: Thepolyhydroxypiperidine alkaloids DNJ [136–141] and castanos-permine [142,143] (Fig. 21) have been described as GlcCeraseinhibitors by their structural similarity with the putative oxocarbe-nium ion that develops in the course of the hydrolysis step.

In a classical work [113], a series of N-alkyl substituted DNJanalogues were reported as GlcCerase inhibitors. Some of them,specially those bearing a N-alkyl spacer and a large hydropho-bic group, showed activities at nanomolar concentrationsagainst non-lysosomal GlcCerase (Fig. 21). In this case, thehydrophobic groups were designed to favour the interactionwith the cell membrane, taking into account the enzymelocation (see above). More recently, some N-alkylated DNJanalogues have been described as chemical chaperones ofGlcCerase, increasing its cellular activity by assisting on theproper folding of the enzyme in the course of its maturationprocess [144]. Among them, N-nonyldeoxynojirimycin(NNDNJ) [145], (Fig. 21) and NBDNJ [146] (Fig. 19) haveshown promising chaperone effects on GlcCerase in differentcell studies. These findings offer a new potential therapeuticalternative for the treatment of Gaucher disease, specially theneuropathic variants, in addition to the substrate reductiontherapy (SRT, see above) or the enzyme replacement therapy(ERT), which consists on the intravenous administration of thedeficient enzyme [147]. Recombinant GlcCerase (Imiglucerase,

Cerezyme®) is currently used in Gaucher disease for thetreatment of non neurological variations [148].

(b) Isofagomine analogues: Isofagomine, a synthetic ana-logue of the alkaloid fagomine (Fig. 22), has been described as ageneral inhibitor of β-glycosidases [149–151]. It was designedtaking into account its close geometric and electronicsimilarities with the carbocationic intermediate that developsin the first step of the β-glycosidic bond hydrolysis.

Very recently, some isofagomine analogues bearing an alkylchain on the C6 position have been described as selectiveGlcCerase inhibitors [152,153]. This finding led the authors tosuggest the presence of a hydrophobic domain close to theGlcCerase active site. According to this hypothesis, bindinginteractions would be reinforced by an ionic bond between theprotonated heterocyclic amine with one of the acidic residues(Glu340) at the enzyme's active site. This is consistent with theobservation that the corresponding N-alkyl derivatives weremarkedly less potent inhibitors (Fig. 23). Some of theseanalogues have also shown interesting chaperone activity onGlcCerase with potential application for the treatment ofGaucher disease (see above).

(c) Aminocyclitols: an important group of GlcCeraseinhibitors belong to this family of compounds. First reportsdealt with GlcCer imino linked analogues in which theglucoside moiety was replaced with the aminocyclitol frame-work present in the natural product β-valienamine and itssaturated analogue β-validamine (Fig. 24) [154,155]. Interest-ingly, both Z and E isomers behaved as potent and selectiveGlcCerase inhibitors, whereas β-validamine analogues wereless potent [154].

Simpler N-alkyl derivatives of β-valienamine [155] werealso strong GlcCerase inhibitors, the N-octyl derivative (N-octyl-β-valienamine: NOV) being the most potent of the series.In a later work [156], N-alkyl and N-acyl derivatives of NOVwere also synthesized and tested as GlcCerase inhibitors inorder to elucidate the role of the hydrophobic portion around thenitrogen atom (Fig. 25). Although the latter lacked inhibitoryactivity, the former analogues were strong GlcCerase inhibitors.

The same authors also tested a series of PDMP analoguescontaining the β-valienamine and β-validamine moieties [157].Some of them showed a strong inhibitory activity, as shown inFig. 26. More recently, a moderate GlcCerase activity has been

Fig. 24. GlcCer imino linked analogues of valienamine and validamine.

Fig. 25. NOV derivatives as GlcCerase inhibitors (in parenthesis, IC50 GlcCerase nM inhibition). ni, not inhibitor.

described for the 4-O-(β-D-galactopyranosyl) derivative of β-valienamine [158] (Fig. 26).

Finally, a series of new inosamine derivatives have recentlybeen described as selective GlcCerase inhibitors (Fig. 27).Interestingly, among the two diastereomeric series of analoguesdescribed in that work, only those of the C1 series showed amarked inhibitory activity against recombinant GlcCerase [159].

As already mentioned for the above families of GlcCeraseinhibitors, some N-alkyl β-valienamine derivatives are able toincrease the enzyme activity through a chaperone effect. This isthe case of NOV (see structure in Fig. 25), which gives rise to anincrease on the enzyme activity when applied to culturedfibroblast cells bearing certain mutated enzymes with lowresidual activities [160].

Finally, biosynthesis of complex gangliosides starts withformation of lactosyl ceramide (LacCer) by galactosydation ofGlcCer by means of a galactosyltransferase (see Fig. 16). AGlcCer analogue bearing an epoxide group on the C4 positionof the Glu moiety has been described as an irreversible inhibitorof this enzyme in chick embryos cultured neurons [161].

3.4. Ceramide kinase inhibitors

Although some sphingosine kinase (SphK) inhibitors areknown to modestly inhibit ceramide kinase (CerK) at highconcentrations in vitro [162], the first specific and effectiveCerK inhibitor has been reported only recently [163]. Thiscompound (K1) is an olefin isomer of the SphK inhibitor F-12509A (see Fig. 29). In vitro kinase assays demonstrated thatK1 effectively inhibits CerK without inhibiting either SphK orDAG kinase. The compound is also active in RBL-2H3 cells, in

which it reduces cellular CerP levels and suppresses IgE/antigen-induced mast cell degranulation.

4. Biosynthesis and metabolism of sphingosine-1-phosphate

Sphingosine-1-phosphate (SphP) is involved in the regula-tion of many cellular functions [164,165]. There is evidence thatSphP acts as an intracellular second messenger involved theregulation of calcium homeostasis and suppression of apoptosis.Interest in SphP has increased enormously after the discovery ofits role as extracellular ligand for specific G protein-coupledreceptors known as the EDG-1 or SphP receptors family [166].

SphP is synthesized from Sph and ATP by the action of SphK[167], for which two different isoforms are known: SphK1[168,169], and SphK2 [170]. They differ in their tissue distributionand cellular location. Interestingly, the SphP generated by theaction of each kinase gives rise to opposite biological effects[171]. Compared with SphK1, SphK2 contains two mostlyunrelated aminoacid sequences, one at the amino terminus andthe other one in the middle of the protein chain.

Dephosphorylation of SphP to Sph is promoted by differentlipid phosphatases [172], including some specific SphPphosphohydrolases [173,174]. The exact role played by thesephosphohydrolases in sphingolipid signalling is not well known[175] and inhibitors for these enzymes have not been reported.

4.1. Inhibitors of sphingosine kinase

The first inhibitors described for SphK were the threo diaster-omers of Sph as well its saturated counterpart, D,L-threo-dihydrosphingosine (DHS or safingol) (Fig. 28) [176]. SinceDHS was slightly more potent than threo-sphingosines, it wassubsequently used in different studies involving SphP signal-ling. Interestingly, DHS is a competitive inhibitor for SphK1,being a substrate for SphK2 [170]. N,N-Dimethylsphingosine(DMS) (Fig. 28) is an even more potent inhibitor, which hasbeen widely used in studies involving SphK and has become the

Fig. 26. Valienamine and validamine analogues as GlcCerase inhibitors (in parenthesis, IC50 GlcCerase μM inhibition).

Fig. 27. Inosamine derivatives as GlcCerase inhibitors (in parenthesis, IC50GlcCerase μM inhibition). Fig. 28. Inhibitors of SphK.

reference inhibitor for these enzymes. DMS is present in severalcancer cell lines and, although it was first described as aphosphokinase C (PKC) inhibitor [177,178], this compoundwas also found to inhibit SphK [179–181]. DMS is a non-specific SphK inhibitor, competitive for SphK1 and uncompet-itive for SphK2 with Ki's in the low micromolar range for bothenzymes. Some specific SphK inhibitors have been recentlydescribed. Thus, Kim et al. [182] reported on some potent invitro SphK2 inhibitors among a family of amino alcohols with amodified sphingoid core structure. Pyrrolidine analogue SG-14was one of the most interesting, as it was nearly equipotent withDMS on SphK2 at 50 μM, while almost inactive against SphK1isoform.

A number of natural products with SphK inhibitory activityhave been isolated from different sources. For example, theisochroman compounds of fungal origin S-15183a and S-15183b(Fig. 29) inhibited SphK from rat liver with IC50 values of 2.5 and1.6 μM, respectively [183]. In a screening for SphK inhibitors, 4-amino-3-hydroxybenzoic acid esters B-5354a, B-5354b, and B-5354c (Fig. 29) were isolated from a marine bacterium and foundto be SphK inhibitors with IC50 values of 21, 58 and 38 μM,respectively [184]. In addition, B-5354c exhibits a non-competitive SphK inhibition (Ki=12 μM) in human platelets.In contrast, B-5354c was an uncompetitive inhibitor of bothSphK1 (Ki=3.7 μM) and SphK2 (Ki=2.2 μM) [185]. Experi-ments using synthetic derivatives of B-5354c indicated that allthree functional groups, i.e., the long unsaturated aliphatic chain,

and the 4-amino and 3-hydroxyl groups, are required for SphKinhibition [186].

Compound F-12509A (Fig. 29), a new sesquiterpenequinone isolated from a discomycete consisting of a drimanemoiety and a dihydroxybenzoquinone, inhibits SphK in acompetitive manner (Ki=18 μM) [187]. Latter studies [185]revealed that F-12509A is a competitive inhibitor of bothSphK1 (Ki=4 μM ) and SphK2 (Ki=5.5 μM) enzymes.

Finally, a screening of chemical libraries has led to theidentification of some potent human SphK1 inhibitors,structurally unrelated to the sphingolipid structure. Some ofthese compounds exhibit SphK inhibitory activities in thenanomolar range and high selectivity towards SphK in front ofother human lipid and protein kinases (Fig. 30) [188]. Kineticstudies revealed that the compounds were non-competitiveinhibitors of the ATP-binding site of SphK1. No data areavailable on the isoform selectivity of these compounds. Veryrecently [189], compound SKi (Fig. 30) has been used todemonstrate that sphingosine kinase modulates voltage-operat-ed calcium channels activity in GH(4)C(1) cells by removinginhibitory sphingosine. Likewise, Gamble et al. [190] havereported that phenoxodiol (PXD), a synthetic analogue ofgenistein, inhibits sphingosine kinase, and suggests that thisactivity may account in part for the pro-apoptotic andantiangiogenic properties exhibited by this compound in mostcancer cells. Finally, a recent article by the group of Cuvillier[191] has showed that docetaxel and camptothecin induced

Fig. 29. Some SphK inhibitors of natural origin.

Fig. 30. Synthetic SphK inhibitors.

strong inhibition of SphK1 in vitro, with the subsequentelevation of the ceramide/S1P ratio.

4.2. Inhibitors of sphingosine-1-phosphate lyase

The last step in the catabolic route of ceramide is theretroaldolic cleavage of SphP to give ethanolamine phosphateand 2-hexadecenal (Fig. 9). This reaction is catalyzed bysphingosine-1-phosphate lyase (SphPL), a microsomal enzymewhich, like some other aldolases, requires pyridoxal phosphateas coenzyme [192]. The first reported inhibitor of this enzymewas the SphP analogue 1-desoxysphinganine-1-phosphonate(Fig. 31), which is a competitive inhibitor of SphPL [193].Using rat liver microsomes as enzyme source, Boumendjel andMiller [194] reported that 2-vinylsphinganine-1-phosphate (Fig.31) inhibited SphPL in vitro with an IC50 of 2.4 μM. Thestereochemistry of this compound is not defined.

Compound FTY720 (Fig. 31) is a synthetic Sph analogue thatwas rationally designed by modification of myriocin (Fig. 2), andis being investigated as immunosuppressive agent. This com-pound is phosphorylated in vivo to its active form [195,196] bySphK2 [197] and exerts its effects by targeting SphP receptors[198]. In a recent article [199], Bandhuvula et al. havedemonstrated that FTY720 also inhibits SphPL activity in vitroand show that treatment of mice with FTY720 inhibits tissuelyase activity, without affecting either lyase gene transcription orprotein expression. The studies by Bandhuvula et al. raised thepossibility that inhibition of SphP cleavagemay account for someeffects of FTY720 on immune function and that SphPL may be apotential target for immunomodulatory therapy.

Acknowledgments

We thank the dedication and experimental contribution of theRUBAM members to the laboratory data presented in thisreview. Partial financial support from Ministerio de Ciencia yTecnología (Spain) (Project MCYT CTQ2005-00175/BQU),Fondos Feder (EU), Ministerio de Sanidad y Consumo, Spain(Project PIO40767), Fundació “La Marató de TV3” (Projects040730 and 040731) and CSIC (Projects PIFF 2004-80F026and 2005-80511) is acknowledged.

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