Inhibitors of Glycosphingolipid Biosynthesis Thomas Kolter and Konrad Sandhoff lnstitut fur Organische Chemie und Biochemie der Universitat Gerhard-Domagk-Str. 7 53727 Bonn Germany Dedicated to Prof. Hans Paulsen on the occasion of his 75th birthday. 1 Structure and Function of Glycosphingolipids Glycosphingolipids (GSLs) are characteristic components of the outer leaflet of the plasma membrane of eukaryotic cells.‘ Each GSL contains a hydrophobic ceramide moiety that acts as membrane anchor and a hydrophilic extracellular oligosaccharide chain. Ceramide itself consists of a long chain amino alcohol D-ervthro-sphingosine which is acylated with a fatty acid. It is also a structural component of a plasma membrane phospholipid sphingomyelin. GSLs are heterogeneous with respect to both their carbohydrate and ceramide portion. Sphingolipids with unusual ceramide structures are found in the skin where they contribute to the epidermal water permeability barrier. Variations in the type number and linkage of sugar residues within the oligosaccharide chain give rise to the wide range of naturally occurring GSLs. More than 300 different struc- tures have been characterised from natural sources. GSL structures depend on the species and can be classified into series which are characteristic for a group of evolutionary related organisms. Beside the species dependence GSLs form cell-type specific patterns on the cell surface. In particular sialic acid containing GSLs of the ganglio-series the gangliosides are abundant on neuronal cells. Moreover these patterns change with cell growth differentiation viral transformation ontogenesis and oncogenesis. Together with glycoproteins and glycosaminogl ycans the GSLs contribute to the glycocalix which covers the cell surface with a carbohydrate wall. At the cell surface GSLs can interact with toxins viruses and bacteria.* These pathogens take advantage of the close spatial neighbourhood between specific carbohydrate recognition sites and the plasma membrane. E.g.the cholera toxin B subunit has to bind to ganglioside GM 1 before the A subunit of the toxin can enter the cell. GSLs can also interact with membrane bound receptors and enzymes and are involved in cell type specific adhesion processes. Various physiological events can be influenced by GSLs eg. embryogenesis neuronal and leukocyte differentiation cell adhe- sion and signal transduction.3 Lipophilic products of GSL metabo- lism such as sphingosine ceramide and their phosphorylated derivatives play a role in signal transduction event^.^ Finally GSLs form a protective layer on biological membranes protecting them from inappropriate degradation and uncontrolled membrane fusion. Limited knowledge about the precise in vivo function of GSLs is available today. A variety of observations indicate that they can participate in different biological events but in most cases def- Thomas Kolter was born in 1963 and studied chemistry at the University of Bonn.He received his Ph.D. with A. Ciannis about the chemistry of chiral aminoaldehydes and the development of pep- tidomimetics . His major research interests are preparative and bioorganic aspects related to glycoconjugate metabolism. inite proofs for their importance are missing. In general the conservation of the overall GSL structure during evolution and the absence of inherited diseases affecting GSL biosynthesis indicate their functional importance for the living organism. 2 Sphingolipid Biosynthesis GSLs and sphingomyelin occur predominantly on the plasma mem- brane of eukaryotic cells. Since their biosynthesis and degradation proceed within cellular organelles GSLs and their precursors are also found on intracellular membranes communicating with the plasma membrane by processes of membrane fusion and fission. The enzymes involved in sphingolipid biosynthesis are membrane bound proteins. Little is known about their structure catalytic mechanism biosynthesis and regulation. De novo biosynthesis of GSLs6 takes place in the same intracellular compartments as gly- coprotein biosynthesis and is coupled to intracellular vesicular transport of the growing molecules through the cisternae of the Golgi apparatus and to the plasma membrane. It starts with the formation of ceramide at the membranes of the endoplasmic retic- ulum (ER).The condensation of the amino acid L-serine with a fatty acyl coenzyme A usually palmitoyl coenzyme A to 3-ketosphin-ganine is catalysed by the enzyme serine palmitoyl transferase (SIT). The SPT is a pyridoxalphosphate dependent enzyme. Two mechanistic pathways for the enzyme-catalysed reaction can be considered which are distinguished by the order of the loss of carbon dioxide and the acylation.8 The finding that tritium labelling of the 2-position in serine is retained during sphingolipid biosyn- thesis in rats supports the first mechanism (Figure 2 pathway A). Another study using [ 2,3,3-*H,)serine as substrate and H. cijerrii as enzyme source led to the observation that the deuterium at the 2-position of serine is eliminated during condensation. This finding supports the second mechanism (Figure 2 pathway B).8 A related reaction which occurs in haem biosynthesis the condensation of succinyl coenzyme A and glycine is catalysed by the enzyme aminolevulinate synthase according to the second mechanism. Sequence homologies on the cDNA level between this two enzymes suggest a common three-dimensional structure and mechanism. The SPT has a lower activity than the other enzymes of ceramide biosynthesis and catalyses the rate-limiting step of this pathway It preferentially utilises fatty acid CoA esters with a chain length of Konrad SundliofS was born in 1939 and Jtudied chemistry ut the University of Munich. He received his Ph.D. with Horst Jatzkewitz and Feodor Lynen in 1965. He joined the Max-Planck-lnstitut fur Psychiatrie Miinchen the Johns Hopkins Universitv Baltimore and the Weizmann Institute Rehovot. In I979 he became~full professor for hiochemistrv and director of the institute jbr Organic Cliemistrv and Bio-chemistrv in Bonn. Among the honours he received is the use of the eponvm ‘Sundhofl diseaJe ’ for a certain inherited disorder and the award of the Richard- Kuhn-medal from the Gesell-schaft Deutscher Chemiker. His research interests include the analvsis of lysosomal storage diseases arid the biochemis try of glycolipid metuboliJm. CHEMICAL SOCIETY REVIEWS 1996 Sphingomyelin IGM2 4 0 OH HO OH OH cH bH Figure 1 Structures of sphingomyelin and ganglioside GDl a the most abundant glycosphingolipid (GSL) in adult human brain Abbreviations refer to partial structures Cer -ceramide GlcCer -glucosylceramide GM2 -ganglioside GM2 16 carbon atoms resulting in long chain bases with a C chain In the following NADPH-dependent reaction 3-ketosphinganine is reduced to D-erythro-sphinganine by the enzyme 3-ketosphinganine reductase Sphinganine is acylated to dihydroceramide by the enzyme sphinganine N-acyltransferase Dihydroceramide is sub-sequently desaturated to ceramide in the dihydroceramide desat- urase reaction The order of introduction of the double bond and aclyation was controversial for some time but it can be regarded as accepted that dihydroceramide is desaturated and not sphinga- nine Therefore sphingosine the parent compound of the sphin- golipids,is not an intermediate in sphingolipid biosynthesis Instead of this it is formed during sphingolipid degradation Besides sphin- ganine and sphingosine another long chain base phytosphingosine [ C homologue (2S,3S,4R)-2-amino- 1,3,4-octadecanetriol] is the structural constituent of many plant yeast and mammalian epi- dermis sphingolipids Ceramide is the common precursor of GSLs and sphingomyelin In the case of GSLs in vertebrates a glucose or a galactose moiety is P-glycosidically linked to the 1-position of ceramide through the action of glycosyltransferases The transferases utilise nucleotide activated sugars Galactosylation of ceramide takes place pre- dominantly in oligodendrocyte cells of the brain and in the kidneys Galactosylceramide (GalCer) and sulfatide (GalCer-3-sulfate) occur in high concentrations in the multilamellar layer of the myelin sheet which covers the axons of neuronal cells On the other hand the biosynthesis of most GSLs of vertebrates requires the glucosyla- tion of ceramide The GlcCer synthase transfers a glucose residue from UDP-glucose to ceramide LacCer the common precursor for the five GSL series found in vertebrates is formed by the addition of a galactose moiety from UDP-Gal to GlcCer catalysed by galac- tosyltransferaseI Ceramide is also a precursor for sphingomyelin a structural com- ponent of the plasma membrane It is a 1-ceramide phosphocholine and occurs largely on the extracellular leaflet of the plasma mem- brane I The sphingomyelin content of plasma membranes depends on the cell type and can reach 25% Sphingomyelin is functionally distinguished from glycerophospholipids like phosphatidyl choline by the higher melting temperature of sphingomyelin bilayers due to the prevalence of saturated alkyl chains and the occurrence of inter- molecular H-bonds between the 3-OH group and the amide-NH Sphingomyelin biosynthesis requires the transfer of phosphoryl- choline headgroup from phosphatidyl choline to ceramide Diacylglycerol is liberated in this step which suggests a tight cou- pling between sphingolipid and glycerolipid metabolism Indeed an inverse correlation between the amounts of sphingomyelin and phosphatidyl choline is observed in many membranes 2.1 Topology The first three steps of sphingolipid biosynthesis leading to dihy- droceramide are catal ysed by membrane-bound enzymes at the cytosolic face of the endoplasmic reticulum (ER) Since formation of glucosyl ceramide occurs on the cytosolic face of the Golgi appa- ratus or a pre-Golgi compartment dihydroceramide has to be trans- ported from the ER to the Golgi apparatus either by vesicle flow or by a protein-mediated process Introduction of the next sugar residue leading to lactosyl ceramide appears to be restricted to the lumenal site of the Golgi apparatus This implicates a membrane translocation of glucosylceramide which is thought to be facilitated by a protein a yet uncharacterised flippase Biosynthesis of higher GSL proceeds on the lumenal site of the Golgi apparatus Therefore the oligosaccharide chain of the membrane-bound GSLs is orien-tated anticytosolic This orientation is topologically equivalent to the situation in the plasma membrane where the GSLs face the ex tracell ular space Sphingomyelin synthesis takes place on the lumenal site of an early Golgi or pre-Golgi compartment This requires an additional membrane translocation on the stage of ceramide It is not clear whether this process is facilitated by a protein 2.2 Biosynthesis of complex glycosphingolipids Since the majority of cellular functions of complex GSLs can be attributed to sialic acid containing gangliosides their biosynthesis is briefly discussed here Most GSL found in vertebrates share lacto- sylceramide as a common precursor and structural element The sequential addition of further sugar residues including sialic acid requires the action of membrane-bound glycosyl transferases in the INHIBITORS OF GLY COSPHINGOLIPID BIOSYNTHESIS -T KOLTER AND K SANDHOFF PLP pathwayA "-K H0+f1310 PalmitoylCoA H H IY II -"Oy%ooQ n0 PalmitoylCoA Figure 2 Mechanism of serine palmitoyltransferase (SPT) See text for details lumen of the Golgi apparatus LacCer and its sialylated derivatives GM3 GD3 and GT3 serve as precursors for more complex ganglio- sides of the 0,a b and c series C series gangliosides have been found only in trace amounts in human tissues Sequential glycosylation of these precursors is performed by less specific glycosyltransferases which transfer a respective sugar residue to glycosyl acceptors which differ only in the number of sialic acids bound tq the inner galactose In vitro data indicate that the sialyltransferases I and I1 are much more specific for their glycolipid substrates than sialyltransferases 1V and V as well as the Gal and GalNAc transferase The distribu- tion of glycosyltransferases within the stacks of the Golgi apparatus has been investigated with the aid of inhibitors of vesicular mem- brane flow Monensin a cationic ionophore impedes vesicular mem- brane flow between proximal and distal Golgi cisternae and causes an increased biosynthetic galactose labelling of GlcCer LacCer GM3 GD3 and GM2 while labelling of more complex gangliosides was reduced Brefeldin A which causes fusion between the ER and largely cis-and medial Golgi causes label reduction in gangliosides GMla GDla GDlb GTlb GQlb and to some extent sphin- gomyelin in neuronal cells Although glycosyltransferase activities are not exclusively found in one Golgi subcompartment these data suggest that GM3 and GD3 are formed in early Golgi compartments whereas more complex GSL like GM la are formed in a late compart- ment Besides de novo biosynthesis GSLs can also be formed in salvage pathways utilizing monosaccharides sphingosine and possi- bly also ceramide released in glycoconjugate catabolism (part 4) 23 Regulation The maintenance of balanced GSL patterns on individual cell sur- faces requires a stringent control of GSL biosynthesis degradation and intracellular traffic The regulation of GSL metabolism and transport is not well understood and only a few indications about it are available6 SPT seems to be the first control point for sphin- golipid formation The enzyme activity correlates with the relative amounts of sphingolipids found in different tissues Sphingosine reduces the SPT activity in cultured neurons and removal of lipids from the skin leads to increased SPT activity During ontogenesis and cell transformation a correlation between GSL expression and the activity of the glycosyltransferases leading to its synthesis has been observed Therefore transcriptional control of glycosyltransferases seems to be a major point of regulation Since most glycosyltransferases have been cloned within the last few years information required for the understanding of transcrip- tional control is expected to be available in the near future Besides regulation on the genomic level some findings hint on epigenetic regulation mechanisms Feedback control of several glycosyl- transferases either by its respective reaction product or final prod- ucts within the corresponding series has been observed in vitro Also the phosphorylation status of the gl ycosyltransferases can influence their activity Lowering the pH of murine cerebellar cell culture media from 7 4to 6 2 resulted in a reversible shift of ganglroside biosynthesis from the a- to the b- series This observation can be explained by the complementary pH profiles of the key regulatory glycosyltransferases sialyltransferase I1 and GalNAc transferase 3 Glycosphingolipid Degradationg The final degradation of GSLs occurs in the lysosomes of the cells The plasma membrane containing GSLs destined for degradation are endocytosed and traffic through the endosomal compartments to reach the lysosome A detailed model of the topology of this process CHEMICAL SOCIETY REVIEWS 1996 LSenne PalmitoylCoA Senne palmrtoyltransferase (PLP) “2 0 3-Dehydrosphingan1ne 3-Dehydrosphingan~ne Redudase (NADPH)i y2 D-erythroSphinganine + RCO-SCoA Sphinganlne N-acyltransferase i HNPCH3 D-erythro-Dih ydroceramlde Dihydroceramde Desaturase Hr\l OH Ceramide + UDP-Glc Glucosytlransferasei 9cH3 CIS OH Glucosylceramide Lactosylceramlde Figure 3 Biosynthesis of lactosylceramide Heterogeneity within the lipid portion is not indicated has been suggested Within the lysosome hydrolysing enzymes Sphingolipid degradation is not necessarily restricted to occur in sequentially cleave off the sugar residues to produce ceramide which the lysosome Sphingomyelin and ceramide can be cleaved by is deacylated to sphingosine This can leave the lysosome reenter the sphingomyelinases and ceramidases of various subcellular localisa- biosynthetic pathway or be further degraded More than ten different tion Prior to degradation sphingosines with the natural erythro exohydrolases are involved in GSL degradation If any of these configuration are phosphorylated by a sphingosine kinase with enzymes is deficient the corresponding lipid substrate accumulates cytosolic localisation The sphingosine- 1 -phosphate generated in and is stored in the lysosomal compartment This leads to inherited this reaction can be cleaved by an enzyme localised on the cytoso- lipid storage diseases with broad clinical and biochemical hetero- lic face of the endoplasmic reticulum the sphingosine- 1-phosphate geneity For GSLs with long carbohydrate chains of more than four lyase The enzyme is PLP-dependent and the reaction corresponds sugar residues the presence of an enzymatically active exohydrolase to a retro aldol cleavage The products are ethanolamine phosphate is sufficient for degradation in VIVU However degradation of mem and an unsaturated aldehyde (Fig 5) brane bound GSLs with short oligosaccharide chains requires the cooperation of an exohydrolase and a protein cofactor a so-called sphingolipid activator protein Several sphingolipid activator pro- 4 Sphingolipids in Signal Transduction teins are now known including the GM2 activator and the saposins To clarify the role of cell surface GSLs is not the only motivation to SAP-A -B -C and -D Inherited deficiencies of either lysosomal modulate sphingolipid metabolism Lipophilic intermediates of hydrolases or activator proteins give rise to GSL storage diseases GSL catabolism have been identified as putative signalling mole- INHIBITORS OF GLYCOSPHINGOLIPID BIOSYNTHESIS -T KOLTER AND K SANDHOFF PH &*q:*7an on LacCer Sialyltrans-I ferase I1 Sialyltrans-ferase I IV GalNAc-transferase 01 Galactosyl-transterase II Ho' Figure 4 Biosynthesis of a and b series gangliosides the predominant GSLs in adult human brain The biosynthesis of 0 series and c series gangliosidesh IS not shown Biosynthesis of 0 series gangliosides starts from LacCer by the action of GalNAc transferase to yield GA2 that of c series gangliosides from GD3 by the action of an a2,8 sialyltransferase to yield GT3 Heterogeneity within the lipid portion is not indicated cules involved in the transmission of extracellular signals to intra- cellular regulatory systems 45 In the case of the structurally related glycerolipids it has been well established for several years that extracellular agents are able to cause the formation or the release of lipid-derived second messengers like diacylglycerol ,inositol-1,4,5-trisphosphate and others Therefore the function of phospholipids is not restricted to being structural constituents of the lipid bilayer of biological membranes Also sphingosine ceramide and their 1-phosphorylated derivatives are currently discussed as signalling molecules Increasing evidence suggests that ceramide plays a role compar able to its structural and functional glycerolipid counterpart diacylglycerol(DAG) DAG together with inositol- 1,4,5 trisphos phate is released from phosphatidylinositol-4,5-b1sphosphateby phospholipaseC in response to an extracellular signal The observa tion that sphingomyelin hydrolysis can also be induced by extra cellular agents in various cell types like lymphocytes myelocytes or fibroblasts led to the discovery of the so called sphingomyelin cycle Tumour necrosis factor a,y-interferon or interleukin-1 which act on receptors in the plasma membrane but also calcitriol 376 CHEMICAL SOCIETY REVIEWS 1996 Sphrngosine-I -phosphate Figure 5 Sphingosine 1 phosphate lyase reaction which acts on intracellular receptors cause the formation of ceramide The cellular and molecular effects of these extracellular agents inhibition of cell growth induction of differentiation modulation of protein phosphorylation or regulation of gene transcription are mimicked by application of a membrane perme- able ceramide derivative C2-ceramide which is the abbreviation used for N-acetyl-sphingosine Importantly the effects of C2- ceramide are generally not observed with the corresponding satu- rated derivative C2-dihydroceramide This finding suggests a specific interaction between ceramide and an intracellular target molecule The identity of the cellular targets of ceramide and other molecules downstream within the signal flow is not known unambiguously A ceramide-dependent kinase a phosphatase and a protein kinase C subtype are currently under investigation In general ceramide appears to mediate antimitogenic effects like cell differentiation cell cycle arrest and cell senescence The most spectacular among the various cellular roles of ceramide is that of a physiological mediator of programmed cell death Programmed cell death or apoptosis is a well-defined process regulated by biochemical pathways which are only partially clar- ified to date It is controlled by receptor-mediated mechanisms which in turn activate intracellular signal cascades which influence the phosphorylation status of target proteins and finally gene expression Beside the sphingomyelin cycle several signal trans- duction pathways to apoptosis seem to be involved in this process which is necessary for normal development of organs tissues and the immune system A large number of events are reported to be influenced by sphin- gosine or sphingosine- 1-phosphate In these cases a clear coupling between extracellular receptor activation intracellular elevation of sphingosine or sphingosine- 1-phosphate and corresponding cellular responses is less evident compared to ceramide Other effects of sphingolipids on signal transduction5 are the inhibition of protein kinase C by sphingosine and IysoGSLs lacking the amide-bonded fatty acid Sphingosine-1-phosphate mediates mitogenic effects in contrast to ceramide It induces pro- liferation of Swiss 3T3 cells and stimulates the liberation of calcium ions from internal sources Since ceramide sphingosine and sphingosine- 1-phosphate are metabolically coupled it is not clear which of these molecules is responsible for a distinct effect and why this pathway is mitogenic in some cells and antiprolife- rative in others To date it awaits elucidation how the cell avoids confusion between the function of these molecules either as meta- bolic intermediates or second messengers In other words how the cell regulates normal metabolism as opposed to signal dependent events 5 Inhibition of Sphingolipid Biosynthesis The precise role of cell surface GSLs as well as their metabolic intermediates for cell function IS not defined A strategy to clarify their cellular and molecular roles is the interruption of their biosyn- thetic pathway at a definite step This can be achieved either by inhibitors or by the generation of mutant cells (or animals) which are deficient in a distinct biosynthetic enzyme (see ref 7 for review) The effects of these approaches are twofold the cell is depleted of metabolites downstream of the inhibited or mutated enzyme On the other hand metabolites upstream of the blocked step can accumulate allowing the investigation of their biological func- tion This is of particular importance for sphingolipid biosynthesis since ceramide and the catabolic metabolite sphingosine as well as their phosphorylated derivatives are currently discussed as sig- nalling molecules As we are focusing on low molecular mass inhibitors of GSL bio-synthesis mutant cells as well as inhibitors of sphingolipid degrada- tion are outside the scope of this review However important contributions to the understanding of GSL metabolism and function may arise with the aid of these valuable tools Several inhibitors of GSL biosynthesis have been described They have been isolated from natural sources or have been gener- ated by design and chemical synthesis Most of them act on early steps of the synthetic pathway and have a lipid-like structure The compounds share the advantage of a higher membrane-permeabil- ity compared to carbohydrate-based inhibitors Suitable inhibitors of glycosyltransferases are only available for the steps associated with the addition of the first two sugars leading to glucosyl- and lac- tosyl-ceramide 5.1 Inhibition of Serine Palmitoyltransferase(SPT) 5 I I Cycloserine Fluoroalansne Chloroalanine The SPT IS inhibited by suicide inhibitors of PLP-dependent enzymes which are directed against the serine binding site L-Cycloserine'o leads to a reduction of GalCer levels in mouse brain but has little effect on gangliosides and sphingomyelin SPT is also irreversibly inhibited by P-chloro- and P-fluoro-alanine with IC INHIBITORS OF GLYCOSPHINGOLIPIDBIOSY NTHESIS-T KOLTER AND K SANDHOFF I II I I I I I I I Activation I I 1 II Sphingomyetin Phosphatidylcholine Sphingomyelinase xm 0 II tww OWI HO&OAJkyl Ceramide Diacylglycerol Candidate direct targets CAPP CAPK PKC Downstream effectors Biology Differentiation,Cell-cycle arrest Apoptosis Figure 6 Spingomyelin cycle (modified from ref 4) See text for details 3x values of about 50 p~ II Inhibition can be blocked by high serine cultures The effect of sphingofungincan be reversed by addition of concentrations These compounds seem to be suicide inhibitors of phytosphingosine but not sphingosine This could be expected several PLP-dependent enzymes and therefore of limited use in since yeast sphingolipids contain phytosphingosine instead of clarifying sphingolipid function Especially high SPT activity is sphingosineas major long chain base The sphingofunginsact com-found in human keratinocytes which is inhibited by L-cycloserine petitively with respect to serine for yeast and mammalian SPT I? and ~-chloroalaninewith IC values of 3 0 and 25 p~,respec-tively 5 1 3 Mvriocin (ISP-I) Myr~ocin'~is a structural analogue of the sphingoid backbone and 5 I2 Sphingofingin inhibits biosynthesis of ceramide and the two major GSLs in yeast Two compounds with structural relationship to sphingolipids inositolphosphorylceramide (IPC) and mannosyl-IPC (MIPC) It sphingofungin I3 and C showed a broad spectrum antifungal but no causes a reduction in the rate of transport of GPI-anchoredproteins antibacterial activity They have been isolated from a culture of to the Golgi apparatus and the remodelling of the GPI-anchor to Aspergillus fimigatus and have been investigated towards inhibi-ceramide-containingstructures tion of sphingolipid biosynthesis in yeast (Saccharornyces cere-ISP-1 is a very potent immunosuppressant of fungal origin It viseae) Sphingofungin B caused an inhibition of de novo turned out to be identical with the antibiotics myriocin and thermo-sphingolipid synthesis (IC = 8 nM) measured by incorporation of zymocidin In contrast to the widely used immunosuppressants (3HH]inositolinto yeast sphingolipids l3 Yeast SPT is inhibited by cyclosporineand FK-506,ISP-1 does not interfere with interleukin-sphingofunginB (ICs0 = 20 nM) in vitro and also by the 5-0-acetyl 2 production but suppressed the IL 2 dependent growth of a cyto-derivative sphingofungin C (IC = 20 nM) Inhibition is accom-toxic murine T-lymphocyte cell line CTLL-2 SPT of these cells is panied by growth inhibitionand cell death was observed in growing inhibited in vitro in a noncompetitive manner with an apparent CHEMICAL SOCIETY REVIEWS 1996 @NH OH OH Q-\ cmC 6H 6H L-Cycloserine PHalo-alanine(X = F Ci) Sphingofungin 6 0 N3 OH OH a27 (+)-Myriocin Aziiosphingosine cis4Methylsphingosine 0 Upoxamycin Cd **0 OH Aiternaria toxin 0 Australifungin DttKeo-PDMP N-Bu-DGNJ Epoxy-GlcCer Figure 7Inhibitors of sphingolipid biosynthesis. The absolute configuration of the tricarballylic acid side chains in fumonisin B 1 and alternaria toxin are not indicated. inhibition constant of 0.28 n~.'~ SPT inhibition was accompanied by suppression of T cell growth which could be restored by C2 ceramide sphinganine or sphingosine-1-phosphate but not by sphingomyelin GlcCer GalCer or GM3. Later it was assumed that growth suppression of CTTL-2 cells by ISP-I was due to induction of apoptosis in these cells.'6 5.1.4 Lipoxumycin Lipoxamycin has been reported to inhibit SPT from Sacchuromyces cerevisiae (IC50 = 21 nM) in ~itr0.I~ Also the corresponding 13-hydroxy-derivative inhibits (IC50 = 88 nM). Ten-fold lower ICs0- values were obtained against SPT from cultured HeLa cells. The high toxicity of the compound when applied to mice subcuta- neously or topically is remarkable. Also antifungal activity against several human pathogens was found which could be reversed by sphinganine or phytosphingosine. 5.1.5 SPT downregulation Other agents have been reported to reduce SIT activity without inhibiting the enzyme directly. The fact that D-erythro-sphingosines of various chain length can down-regulate SPT activity suggests an autoregulatory mechanism in sphingolipid biosynthesis. The mech- anism of SPT downregulation is not known but interaction of sphingoid bases with a transcriptional factor followed by reduction of SlT biosynthesis would be a possible mechanism. In this way the cell might prevent overproduction of these cytotoxic molecules. INHIBITORSOF GLYCOSPHINGOLIPIDBIOSYNTHESIS-T. KOLTER AND K. SANDHOFF A similar effect is exerted by synthetic analogues of sphingosine D-erythro-azidosphingosine either with trans or cis double bond downregulates SFT activity in primary cultured neurons.I8 These compounds are metabolically stable due to the fact that acylation to ceramide is not possible. In contrast to sphingosine de novo sphin- golipid biosynthesis is strongly inhibited by this compound in concentrations lower than 50 p~. Also the synthetic 4-methyl derivative of cis-sphingosine down- regulates SIT activity (IC50=10 FM) causes a transient increase of the intracellular concentration of calcium ions and behaves as a potent mitogen in quiescent Swiss 3T3 fibroblasts. Thymidine incorporation into DNA is stimulated tenfold at 10 p~ concentra-tion. Moreover the compound initiates drastic morphological altera- tions of the cells and initiates cell death.19 Both the cis-double bond and the 4-methyl group are necessary for the observed effects since the trans- and 5-methyl derivatives are ineffective. Work is in progress to discriminate between the effects -basically biosyn- thesis inhibition mitogenic effect and morphological alterations -with the aid of synthetic analogues of the compound. 5.2 Inhibitors of Sphinganine N-Acyltransferase 5.2.I Fumonisin Fusarium moailiforme is a mould frequently found on corn and grains. Consumption of contaminated agricultural products leads to diseases in animals and correlates with oesophageal cancer in humans. Mycotoxins from F. moniliforme the fumonisins have been shown to cause the diseases associated with F. moniliforrne uptake. Fumonisins B1 and B2 (lacking the 10-hydroxy group of FB 1) have been identified as inhibitors of sphinganine-N-acyl- transferase with IC,,-values of about 0.1 p~.~~.~~They are struc- tural analogues of sphingoid bases with the 1 -OH function missing in fumonisin. This may contribute to persistence of inhibition since long chain bases are cleaved only after phosphorylation in the 1-L-Serine +PalmitoylCoA Cycloserine &Haloalanines Sphingofungins Myriocin (ISP-1) 3-Ketosphinganine Sphinganine Fumonisins Australifungin Dihydroceramide position. The tricarballylic acid moiety and the 5-OH group of fumonisins are important but not critical for inhibition Fumonisin B3 lacking the 5-OH group is still active albeit at I p~ concentra-tion. A similar value is obtained for fumonisin B1H prepared by mild base cleavage of the tricarballylic acid. Inhibition of ceramide formation is accompanied by accumulation of its biosynthetic pre- cursor sphinganine. On the one hand this accounts for some effects of these toxins since long chain bases are known to be toxic and mitogenic at low concentrations. On the other hand the ratio of sphinganine to sphingosine in the serum of animals is a sensitive means to detect fumonisin consumption. 5.2.2 Alternaria toxin Alternaria toxin is a phytotoxin with structural similarity to the sphingolipid backbone. It inhibits sphingolipid biosynthesis (Ks0 =1 p~)on the stage of ceramide formation*l but is of limited use due to cytotoxicity in mammalian cell culture and less potency com- pared to fumonisins. 5.2.3 Australifungin Recently the fungal metabolite australifungin was reported as an inhibitor of sphinganine N-acyltransferase in vitro. The IC value is less or equivalent to fumonisin B I dependent on the cell type.22 Australifungin is a potent antifungal agent and shows no structural similarity to sphingoid bases. 53 Inhibitors of GlcCer Synthase Inhibition of ceramide biosynthesis results in depletion of cell surfaceGSLs and of sphingomyelin. This is a principle disadvantage of such inhibitors as far as they should provide insight into the func- tion of cell surface carbohydrates. The observed phenomena might be obscured by effects due to the inhibition of membrane biosyn- thesis due to depletion of sphingomyelin. Therefore the availability downregulation Sphingosine Azidosphingosine cis-4-Methylsphingosine Spingolipid-degradation Sphingosine Fumonisins (salvage) Australifungin J POMP Cera mide NBDGJ GlcCer Glycosphingolipids Sphingomyelin Figure 8Flow scheme of sphingolipid biosynthesis. 380 of potent and specific inhibitors of glucosylceramide biosynthesis would be highly desirable Two classes of synthetic compounds are reported to date as inhibitors of GlcCer synthase 23 5 3 1 D-threo-PDMP D-threo-PDMP [D-threo-(1R,2R)-1-phenyl-2-decanoylamino-3-morpholinopropan-1-01] is the most thoroughly investigated member of a series of ceramide analogous inhibitors of GlcCer syn- thase 24 D-threo-PDMP inhibits formation of glucosylceramide in concentrations of 2 5 to 10 p~ and has already been used in func-tional studies 25 D-threo-PDMP shows a mixed type inhibition mode with respect to ceramide and is uncompetitive for the gluco- syl donor The apparent K is 0 7 p~ In concentrations of more than 25 p~ also sphingomyelin biosynthesis and protein transport along the secretory pathway are inhibited 26 PDMP stereoisomers and analogues have been synthesised and investigated among them D- threo-1-morpholino-1 -deoxyceramide (73% inhibition of GlcCer synthase in Madin-Darby canine kidney (MDCK) cells at 5 p~ concentration compared with 20% inhibition through D-rhreo- PDMP) 27 Concentrations of more than 100 p~ D-threo-PDMP or 10 JLM of its palmitoyl derivative PPMP are toxic for HL-60 cells D-threo-PDMP exhibits multiple cellular effects like cell growth inhibition eventually mediated by ceramide accumulation or inhibition of sphingomyelin synthesis and is metabolised by cytochrome P450 Various other effects of PDMP its isomers and analogues are summarized in Ref 25 5 3 2 DGNJ Recently it has been shown that a synthetic derivative of the natu- rally occurring glycosidase inhibitor deoxynojirimycin N-butyldeoxynojinmycin (NB-DNJ) inhibits GlcCer formation with an IC value of 20 JLM NB-DNJ was known to inhibit HIV replica- tion in vitro obviously via inhibition of viral glycoprotein pro- cessing Butyldeoxygalactonojirimycin(N-Bu-DGNJ) is a related compound with improved selectivity (IC = 40 p~)28 GI ycosidases such as /3-gluco- and P-galacto cerebrosidase a-glu- cosidase I and I1 are either not or weakly inhibited by this com- pound Structure-activity relationships revealed that the alkyl chain length requires three carbons for inhibition with C and C being optimal Longer chain length leads to improved inhibition in vitro but also to cytotoxicity in vzvo The corresponding derivatives of mannose fucose and GlcNAc are inactive A major advantage is their metabolic stability and their low toxicity up to 2-5 mM are tolerated The mechanism of inhibition by alkylated iminosugars is not known but it might be argued that they mimic the transition state of the transferase reaction This is of particular importance since with rare exceptions attempts for the development of potent and selective glycosyl transferase inhibitors have not been successful to date 5.4 Inhibition of LacCer Synthase A synthetic truncated derivative of glucosylceramide bearing an additional epoxide function in the 4-position of the glucose residue caused an irreversible and concentration dependent decrease of the specific activity of LacCer synthase 29 In primary cultured neurons of chick embryos the biosynthetic GSL patterns changed in such a way that labelling of GSL downstream from GlcCer was reduced and label accumulated in GlcCer The gluco derivative was active while the derivative with galacto configuration showed no effect Inhibition of LacCer synthase in vitro by epoxy-GlcCer was much less pronounced 250 p~ concentration was required to cause only 30% inhibition of enzyme activity Therefore it cannot be excluded that the observed effect in vivo is due to inhibition of a GlcCer trans- locator or a transcriptional factor 55 Miscellaneous A recent study3 shows that azidothymidine (AZT) which is used as chemotherapeutic agent in the treatment of HIV infection CHEMICAL SOCIETY REVIEWS 1996 inhibits cellular glycosylation of gl ycolipids and glycoproteins in clinical relevant concentrations of 1-5 JLM The primary intra- cellular metabolite of AZT the monophosphate possibly inhibits the uptake of nucleotide sugars by the Golgi apparatus thereby reducing the content of complex acidic GSLs Toxic side effects of AZT especially on maturation of blood stem cells seems to be due to modified glycosylation patterns on these cells and not on inhibi- tion of DNA replication Antisense oligodeoxynucleotides to GM2 synthase and GD3 syn- thase led to downregulation of more complex GSLs downstream of GM3 in the biosynthetic pathway (Figure 4) The human leukaemia cell line HL-60 treated with these antisense-DNAs underwent monocytic differentiation under these conditions and accumulated GM3 31 Since suitable low molecular mass inhibitors for glycosyl transferases are not available today this approach constitutes a promising tool for the investigation of GSL function 6 Perspective Therapeutic Potential of GSL Biosynthesis Inhibitors There are several potential fields for the application of inhibitors of GSL biosynthesis Only three of them are briefly mentioned here 6.1 Chemotherapy of Parasite Infections Various observations indicate that inhibition of sphingolipid bio- synthesis can become helpful in the treatment of diseases caused by lower eukaryotes e g fungal and protozoan infections The great number of infections (one million children die of malaria each year) and the occurrence of drug resistance led to an urgent requirement for new drugs in this field Recently it has been shown that D&-threo-PDMP which is usually used as an inhibitor of glucosylceramide formation and a chain homologue of it effectively inhibited sphingomyelin forma- tion in the human malaria parasite Plasmodium falciparurn Inhibition was achieved with concentrations of less than 1 p~ and accompanied by inhibition of parasite proliferation in culture 32 Plasmodia1 sphingomyelin synthase appears to be a rational target for the development of antimalarial drugs Many observations indicate that inhibition of GSL biosynthesis might become advantageous in the treatment of parasitic or fungal infections In contrast to vertebrates lower eukaryotes like yeast (Saccharomyces cerevisiae) have only a simple set of GSLs which are also predominantly found in the plasma membrane Studies with mutant cells (reviewed in ref 7) indicate that sphingolipids appear to be essential for the viability of yeasts Also inhibition studies with the fungal metabolite myriocin showed that ceramide stores are rapidly depleted in these fast proliferating cells if ceramide is not regenerated by biosynthesis Often natural compounds with sphingolipid-like structure of fungal or marine origin33 have anti- fungal properties Both in MDCK cells and yeast the intracellular transport of glyosyl-phosphatidylinositol-anchoredproteins and of sphingolipids seems to be tightly coupled and commonly regu- lated 34 Since the content of GPI-anchored proteins of the cell surface is particularly high in lower eukaryotes these organisms should be sensitive towards inhibition of this process Furthermore the H +-ATPase of such organisms is dependent on inositolphos- phoryl ceramide which is not found in higher eukaryotes 35 6.2 Antiproliferative Agents Signal transduction is an attractive target for the discovery of anti- proliferative agents A pharmacological approach of this type has the potential advantage that the action of a drug is not necessarily accompanied by toxic side effects associated with the action of traditional chemotherapy based on the inhibition of DNA synthesis Efforts in this direction are rare within this very new area of research The generic synthesis of aryl-fused sphingosine derivatives designed as agents for the topical treatment of inflammatory skin disorders like psoriasis has been reported 36 Compounds of this type inhibit protein kinase C in micromolar concentrations in vitro INHIBITORS OF GLYCOSPHINGOLIPID BIOSYNTHESIS-T KOLTER AND K SANDHOFF 38 1 63 Treatment of Sphingolipidoses Sphingolipidoses are a group of inherited disorders due to impaired proteins responsible for sphingolipid catabolism within the lyso- somes of the cell With rare exceptions a treatment of these often lethal diseases is not possible to date Several factors influence the pathogenesis of the sphingolipidoses Accumulation of lipids occurs mainly in those cell types and organs in which the lipids are predominantly synthesized or taken up by endocytosis In Tay-Sachs disease for example P-hexosaminidase A is deficient This causes accumulation of the ganglioside GM2 in neuronal cells the main site for synthesis of gangliosides (sialic acid-containing GSLs) According to a kinetic severity and onset of these diseases depend on the residual enzyme activities Their decrease beyond a critical threshold value leads to the accumulation of the substrate of the deficient enzyme since substrate influx into the lyso- somes exceeds the degradation rate Substrate influx into the lyso- some due to biosynthesis can be reduced by inhibiting this process 23 24 From kinetic considerations it should be possible to influence the severity as well as the onset of these diseases with the aid of synthetic inhibitors 7 Outlook Many questions about the details of sphingolipid biosynthesis and function remain open and might in part be answered with the aid of enzyme inhibitors or receptor ligands Inhibitors of dihydroce- ramide desaturase are not available They would permit purification of the enzyme and eventually dissect structural and signalling sphingolipid pools Also no suitable inhibitors of sphingomyelin synthase are known They would be interesting candidates for the treatment of malaria infections as discussed above More potent and selective inhibitors are required for the treatment of sphingolipi- doses Ligands of sphingolipid binding proteins might be helpful in con-firming the current hypotheses about details of sphingolipid sig- nalling function The development of antiproliferative drugs based on this approach would yield pharmacological application Acknowledgement Work done in the author’s laboratory was sup- ported by the Deutsche Forschungsgemeinschaft (SFB 284) 8 References 1 C C Sweeley in ‘Biochemistry of Lipids Lipoproteins and Membranes’ ed D E Vance and J Vance Elsevier Amsterdam 1991 pp 327-361 2 K -A Karlsson Annu Rev Biochem 1989,58,309 3 C B ZellerandR B Marchase,Am J Physiol 1992,262,C1341 4 Y A Hannun J Biol Chem 1994,269,3125 5 S Spiegel D Foster and R Kolesnick Curr Opin Cell Biol 1996,8 159 6 G van Echten and K Sandhoff J Biol Chem 1993,268,5341 and references therein 7 A H Futerman Trends Glycosci Glycotechnol 1994,6,143 8 K Krisnangkura and C C Sweeley J Biol Chem ,1976,251,1597 9 K Sandhoff and T Kolter 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