The 2003 ASBMB-Avanti Award in Lipids Address: applications of novel synthetic lipids to biological problems

Chemistry and Physics of Lipids 129 (2004) 111–131

Review

The 2003 ASBMB-Avanti Award in Lipids Address: applications of novel synthetic lipids to biological problems Robert Bittman∗ Department of Chemistry and Biochemistry, Queens College and The Graduate School of The City University of New York, Flushing, NY 11367, USA Received 24 January 2004; received in revised form 24 January 2004; accepted 28 January 2004

Abstract This paper is an overview of the 2003 Avanti Award in Lipids address that was presented by Robert Bittman at the American Society for Biochemistry and Molecular Biology (ASBMB) Annual Meeting held in San Diego, CA in conjunction with meetings of five other FASEB Societies, April 15, 2003. The theme of the lecture is: “How can the chemical synthesis of unnatural lipids provide insights into problems ranging from cell biology to biophysics?” The following examples are presented: (1) novel ceramide analogs as experimental anticancer agents, (2) photoactivatable sphingosine 1-phosphate analogs as probes of protein targets of this bioactive lipid, (3) a 13 C-enriched cerebroside as a quantitative probe of glycosphingolipid (GSL) transbilayer distribution in bilayers with and without sphingomyelin, (4) cis and trans unsaturated sphingomyelin analogs as modulators of the existence of cholesterol-enriched microdomains (rafts) that may facilitate fusion of alphaviruses with target membranes, (5) ceramide as an indirect enhancer of the permeabilization of membranes induced by cholesterol-specific cytolysins, (6) fluorescent GSL analogs of widely disparate structure as probes of the molecular features responsible for the selective internalization of GSLs in caveolae of living mammalian cells, (7) enantiomeric lysophosphatidic acid (LPA) analogs as probes of receptor subtypes that mediate LPA signaling, and (8) phosphonocholine analogs of the antitumor ether lipid ET-18-OCH3 as tools for discerning the primary targets that are critical for cytotoxic activity in tumor cells. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Alphavirus; Antitumor lipid; Ceramide; Cytolysin; Glycosphingolipids; Lysophosphospholipids

1. Introduction It is a great honor to accept the 2003 ASBMBAvanti Award and to be included in the list of the distinguished biochemists to have received this Award from the Biophysical Society and the ASBMB in past years (Table 1). I am very grateful to the ASBMB for selecting me and to the Avanti Polar Lipids Co. for ∗

Tel.: +1-718-997-3279; fax: +1-718-997-3349. E-mail address: robert [email protected] (R. Bittman).

sponsoring this Award. I am especially indebted to the many talented graduate students and postdoctoral researchers who have worked with me throughout my career. I thank them for their dedication, motivation, and creativity; in fact, I am sharing the Award with them. I also thank my past teachers from whom I have had the privilege to learn. The inspiration and guidance I received in chemistry from my mentor Professor Andrew J. Streitwieser, Jr. of the University of California at Berkeley, where I received the Ph.D. degree in chemistry with an emphasis in physical-organic chem-

0009-3084/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.chemphyslip.2004.01.004

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Table 1 Previous recipients of the Avanti Award in Lipids from the ASBMB and the Biophysical Society

Table 2 Number of “hits” for the keywords “lipid rafts” vs. year of publication

1996 1997 1998 1999 2000 2001 2002 2003

Year

Number of hits

1999 2000 2001 2002 2003

39 107 200 270 352

ASBMB Biophysical ASBMB Biophysical ASBMB Biophysical ASBMB Biophysical

Society Society Society Society

Robert M. Bell Ching-hsien Huang Lewis C. Cantley Richard M. Epand Edward A. Dennis Ronald N. McElhaney Christian R. H. Raetz John F. Nagle and Stephanie Tristram-Nagle

istry, and in biophysics from Dr. Manfred Eigen of the Max Planck Institute for Physical Biochemistry in Göttingen, Germany, where I was introduced to biophysical methods (fast kinetics of enzyme–coenzyme binding), are especially memorable. Finally, I thank the many outstanding collaborators with whom I have had the good fortune to work on a wide range of exciting research projects for their collegiality, dedication, and insightfulness. As a lipid chemist/biochemist, I am gratified that the role of lipids in cell function is now widely recognized. The realization that lipids play a central role in regulation of a myriad of cellular processes, and

are not only the structural backbones of cell membranes and sources of metabolic energy, is evident in the attention given to lipids and “lipid biology” in prominent journals such as Science and Nature and at symposia held at national and international conferences. Table 2 illustrates the increase in the number of “hits” in a PubMed search for the keywords “lipid rafts” versus the year of publication. It is clear that lipids are no longer relegated to the role of being “wallflowers” among biomolecules (Raloff, 1997), taking a backstage place to proteins and nucleic acids as the key players in cell function. They have, at last, gained respect, and can now be considered to be part of a new trifecta—genomics, proteomics, and lipidomics (Fig. 1)—in the race for the

Fig. 1. The new “trifecta”—proteomics, genomics, and lipidomics.

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spotlight among biomolecules. (Actually, there is a “quadrafecta” since one should also include glycomics (the study of the glycans in a biological system).) Lipidomics refers to the study of all of an organism’s repertoire of lipids. The entire lipidome is in the process of being mapped by using some of the powerful recent advances in analytical technology (particularly mass spectrometric methods). That lipids are now in the spotlight is evidenced in a new large-scale 5-year National Institutes of Health (National Institute of General Medical Sciences) “glue” grant for the Lipid MAPS Consortium that brings together diverse research groups to identify and quantify all of the lipids within cells in the hope of improving our understanding of the roles of lipids in health and disease (http://www.lipidmaps.org). Additional examples of such grants are the establishment of a center in lipidomics and pathobiology focused on the characterization and roles of sphingolipids (http://hcc.musc.edu/research/shared resources/lipido mics.cfm) and plant lipids (http://www.kclifesciences. org). Since lipids are recognized as important and powerful participants in a huge number of diverse bioactivities, it will be exciting to see how the nascent science of lipidomics develops to meet the challenges associated with such an ambitious undertaking. This lecture will present an overview of several examples of how my research group has sought to unleash the power of synthetic organic chemistry to gain new insights into the molecular features required for the bioactivity of selected lipids. Our focus has been on the principal components of the cell membrane— glycerophospholipids, sphingolipids, and sterols. To improve our understanding of the interactions among these components, we have synthesized unnatural analogs of each of these components and studied their physical and biological behavior in model membranes and in cells. In this lecture, recent examples of the applications of novel glycero- and sphingolipids to problems of interest in cell biology, virology, and physical biochemistry will be described.

2. Unnatural ceramides as experimental antitumor agents The first example involves the synthesis of unnatural analogs of ceramide that have higher antiprolif-

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erative activity than natural ceramide against tumor cells. It is well established that either endogenous or exogenous ceramide can initiate apoptotic cell death in physiological (Kolesnick and Kronke, 1998; Pettus et al., 2002) and pharmacological (Jarvis and Grant, 1998; Ogretman and Hannun, 2001) settings. A thorough review of ceramide-driven lethal stress signals is beyond the scope of the present discussion. Let it suffice to note that numerous signaling systems are affected by ceramide and a great deal of research efforts have been focused on the role of ceramide as a messenger for induction of apopotosis. Thus it appears that ceramide coordinately modulates cytotoxic and cytoprotective signaling systems, leading to cell death by activating enzymes in stress-signaling cascades and suppressing the activity of enzymes in various growth/survival pathways (Ruvolo, 2003). Diminished activity through a given stress pathway can upset the normal balance between cell death and cell survival. The failure to regulate apoptosis ultimately favors proliferation with catastrophic consequences, which may culminate in the initiation of tumor formation. As a possible new approach to experimental anticancer chemotherapy, we have sought to employ unnatural analogs of ceramide that escape recognition as substrates of sphingolipid-utilizing enzymes present in most mammalian cells and thus evade metabolic conversion to mitogenic sphingolipids (such as ceramide 1-phosphate, glycosylceramide, and sphingosine 1-phosphate). Furthermore, some unnatural ceramide analogs may elevate the level of endogenous ceramide levels by inhibiting enzymes involved in ceramide turnover. To investigate this approach, we have modified the structure of ceramide at a variety of sites in the molecule, as depicted in Fig. 2. We have altered the length of the N-acyl chain, modified the amido functionality by replacing it with an ester or sulfonamide or by reducing the carbonyl group, altered the stereochemistry at C2 and C3, modified the 1-hydroxy group by converting it to a methoxy group or incorporating it into a five-membered ring, modified the 3-hydroxy group (by replacing it with hydrogen, methoxy, or ethoxy), incorporated the C4–C5 double bond into a ring (aromatic, heteroaromatic, or cyclopropyl), moved the trans double bond along the sphingoid chain, introduced additional sites of unsaturation, added hydroxyl groups, and modified the

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Fig. 2. Sites of manipulation of the ceramide molecule.

length of the sphingoid chain (He et al., 2000; Chun et al., 2000, 2003a,b). Since many studies have shown that 4,5-dihydroceramide fails to initiate apoptosis of many cells (Bielawska et al., 1993; Karasavvas et al., 1996), we probed the role of the trans C4–C5 double bond by altering its location within the sphingoid backbone (He et al., 2000; Chun et al., 2002, 2003a). Fig. 3 shows the structures of some of these analogs. Short-chain (i.e., N-octanoyl) ceramide analogs were used because they are membrane permeable. Fig. 4 outlines the synthetic route used to introduce an additional trans double bond at C6–C7 of the sphingoid backbone (Chun et al., 2002). N-Boc-l-serine methyl ester acetonide was used as the starting material, which was prepared from l-serine. Reaction of the ester with 2 equivalents of the carbanion of methyl

phenyl sulfoxide gave a ␤-ketosulfoxide. The anion of the latter was formed by using potassium carbonate in DMF, which reacted with (E)-1-bromo-2-tetradecene to afford the ␣-ketosulfoxide intermediate shown in Fig. 4. This intermediate underwent elimination of PhS(O)H at room temperature to give a dienone. Diastereoselective reduction of the ketone with sodium borohydride/ceric chloride in methanol or DIBAL-H in tetrahydrofuran (THF) gave the desired erythro isomer together with the undesired threo isomer in a ratio of ∼5:1. The diastereomers were separated by column chromatography, and the product was obtained after acid hydrolysis of the acetonide and N-Boc groups and N-acylation with p-nitrophenyl octanoate. Can the efficacy of ceramide to induce cell death be improved upon by altering the position of the double bond? In collaboration with Professor Barbara S.

Fig. 3. Structures of ceramide analogs bearing a modified sphingenine backbone.

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Fig. 4. Synthesis of
4,6 -diene-C8-ceramide using sulfoxide chemistry.

Beckman, we found that (4E,6E,2S,3R)-2-N-octanoyl4,6-sphingadienine (
4,6 -diene-C8-ceramide), which differs from natural ceramide in having an additional trans double bond at C6–C7, was more efficient than ceramide in blocking MCF-7 cell growth (Struckhoff et al., 2004). (Although we considered
4,6 -dieneceramide to be an unnatural analog of ceramide at the time of synthesis (Chun et al., 2002), it should be noted that longer-chain amides of this 4,6-sphingadienine were recently isolated and identified from silk moth larvae, and were found to promote neurite outgrowth in a rat pheochromocytoma (PC12) cell line (Kwon et al., 2003).)
4,6 -Diene-C8-ceramide was markedly more potent than N-octanoylceramide (IC50 11.3 ␮M versus 32.9 ␮M in TNF-␣-resistant MCF-7 cells, and IC50 13.7 ␮M in versus 37.7 ␮M in TNF-␣-sensitive MCF-7 cells) (Struckhoff et al., 2004). Moreover,
4,6 -diene-C8-ceramide was more effective than N-octanoylceramide in inducing cell death in MDA-MD-231 and NCI/ADR-RES breast cancer cell lines (IC50 3.7 ␮M versus 11.3 ␮M, and 24.1 ␮M versus 86.9 ␮M, respectively) (Struckhoff et al., 2004). Both
4,6 -diene-C8-ceramide and N-octanoylceramide exhibited a selective cytotoxicity against tumor cells, indicating that
4,6 -diene-ceramide or its analogs may have potential as a therapeutic agent. As shown in Fig. 5, there was little loss of viability of normal breast epithelial cells at concentrations of
4,6 -diene-C8-ceramide and N-octanoylceramide

that were cytotoxic to MCF-7 breast carcinoma cells. Some of the mechanisms that contribute to the cytotoxic activity of
4,6 -diene-ceramide were investigated (Struckhoff et al., 2004). A prolonged elevation of the intracellular ceramide level was observed in MCF-7 cells treated with
4,6 -diene-C8-ceramide, which may contribute to its enhanced cytotoxicity. When MCF-7 cells were treated with
4,6 -dieneC8-ceramide or with natural ceramide, induction of apoptosis via the mitochondrial pathway was noted by 8 h, as demonstrated by cytochrome c release from mitochondria, loss of transbilayer phospholipid asymmetry (measured by Annexin V staining), and a decrease in the mitochondrial membrane potential. Thus ceramide analogs may represent new experimental therapeutic agents in breast cancer treatment by triggering tumor cell apoptosis. The most effective analogs would be metabolically stable, raise the level of intracellular ceramide, appropriately activate stressinduced signaling pathways, and increase the vulnerability of cancer cells to oxidative stress—perhaps by depleting cellular glutathione (GSH) levels and elevating levels of reactive oxygen species. It has been proposed that the unsaturation in ceramide at C4–C5 is required for proapoptotic activity because oxidation of the 3-hydroxy group of ceramide affords an ␣,␤-conjugated ketone, which may undergo a condensation reaction with mitochondrial

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Fig. 5. Comparison of the antiproliferative activities of
4,6 -diene-C8-ceramide (open symbols) and N-octanoylceramide (closed symbols) against MCF-7 cells (dashed curves) vs. normal human breast epithelial cells (hTERT-HME) (solid curves). Cells were treated with the ceramides for 24 h. Cell viability was estimated using the MTT assay. Permission to reproduce this figure from Struckhoff et al. (2004) has been obtained from the American Society for Pharmacology and Experimental Therapeutics.

GSH (Radin, 2003). A more extensively conjugated ketone would be formed from a ceramide having a 4,6-sphingadienine backbone; therefore, reaction with GSH may be favored with the oxidation product obtained from
4,6 -diene-C8-ceramide. On the other hand, oxidation of 4,5-dihydroceramide would form a nonconjugated ketone, which would not undergo a condensation reaction with GSH. Similarly,
6 -eneC8-ceramide would form a nonconjugated ketone on oxidation of the C3-hydroxy group. This is consistent with the finding that the antiproliferative activity of
6 -ene-C8-ceramide is much lower than that of
4,6 -diene-C8-ceramide (Struckhoff et al., 2004).

3. Photoactivatable probes of sphingosine1-phosphate (S1P): binding of S1P to the S1P1 receptor and to rat plasma proteins The lysosphingolipid S1P elicits many cellular responses through both extra- and intracellular targets. Some activities of S1P result from its behavior as a ligand for three families of nine G-protein coupled

receptors (GPCRs, designated as S1P1 –S1P5 , GPR3, 6, 12, and GPR63) in higher eukaryotes that regulate many intracellular cell signaling pathways involving cell growth, survival, and motility (Spiegel and Milstien, 2003a; Hla, 2003). In addition, S1P also has second messenger functions arising from its binding to as yet unidentified intracellular targets (Spiegel and Milstien, 2003b). In an initial approach to identify some of the protein targets that regulate S1P biology, we collaborated with Professor Gabor Tigyi in a study of the binding of photoaffinity probes of S1P to one of the five known S1P receptors (S1P1 ) and to plasma proteins. Photoaffinity ligands employ a precursor of a reactive intermediate (such as a carbene or nitrene, or a benzophenone) linked to the ligand that binds to a site in the target. We synthesized two photoactivatable probes of [32 P]S1P (Lu et al., 2003). The radioactivity was incorporated using recombinant sphingosine kinase and [␥-32 P]ATP. As shown in Fig. 6, a benzophenone moiety (which generates a triplet state on photolysis) was inserted at the end of the sphingoid chain of compound 1 by a Mitsunobu reaction.

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Fig. 6. Structures of two photoactivatable probes of [32 P]S1P: compounds 1 and 2.

In compound 2, a trifluoromethylphenoxydiazirine moiety, which is a carbene precursor, was inserted at the end of the sphingoid chain by using a similar coupling reaction. Diazirine and benzophenone probes have been widely used for covalent modification of hydrophobic proteins (Brunner, 1989; Dormán and Prestwich, 1994). To determine the affinity and selectivity of the probes for the S1P1 receptor we carried out radiolabeled binding assays in the dark (Lu et al., 2003). Scatchard analysis gave Kd values of 84 ± 10 nM and 36 ± 2 nM for the binding of compound 1 and S1P, respectively, to the S1P1 receptor expressed in RH7777 cells. Thus compound 1 closely mimics the binding properties of the endogenous ligand for S1P1 . No equilibrium constant could be estimated for compound 2 because the binding of this probe to the S1P1 receptor in the same membranes was not saturable up to 200 nM, which is indicative of nonspecific binding. The binding pocket of the S1P1 receptor appears to accommodate the S1P-benzophenone photoprobe better than the S1P-diazirine probe. In order to identify proteins in rat plasma that bind to S1P and thus regulate its activity, each of these radiolabeled photoprobes was incubated with plasma from normal or analbuminemic rats, photolyzed, and the labeled proteins were analyzed by SDS–PAGE. Specific binding of compounds 1 and 2 to an unidentified 25kDa protein in plasma from analbuminemic rats was found, as reflected by the ability of cold S1P to block the photolabeling and by the lack of competition in the presence of an excess of other cold lysophospholipids. The analbuminemic rats still have some albumin in their plasma, which is produced by the skeletal muscle rather than by the liver; therefore, a band was labeled at 67-kDa in addition to that at 25-kDa (Lu et al., 2003). The method used to synthesize these probes can be modified to prepare compounds with different linkers between the sphingoid base and the photoreactive moiety. Such probes may provide information about

the binding sites of the biological targets of S1P at the molecular level.

4. A 13 C NMR approach for quantitative estimation of the effects of sphingomyelin on the transbilayer distribution of GalCer in SUVs ␤-Galactosylceramide (GalCer) is an important component of the outer leaflet of the plasma membrane of many cells as well as a key component of the myelin sheath. Since glycosphingolipids (GSLs) are attachment sites for certain viruses and bacteria, it is obvious that GSL membrane topology has important consequences. In collaboration with Dr. Rhoderick E. Brown, we were interested in determining whether sphingolipid-sphingolipid interactions influence the transmembrane distribution of GalCer in sonicated vesicles. NMR methods are often used to estimate the transbilayer distribution of phospholipids. In this method, a paramagnetic ion is added to the external medium of vesicles to quench the 31 P NMR signal arising from a specific phospholipid in the outer leaflet of the bilayer. To estimate the transbilayer distribution of GalCer in vesicles, we could not, of course, use 31 P NMR since no phosphate group is present in the headgroup of this cerebroside. The following novel 13 C NMR approach was used to quantitate the sidedness of GalCer in SUVs. We enriched the C6 atom of the galactosyl moiety of N-palmitoyl-GalCer with 13 C (to 99.8% purity) by chemical synthesis, incorporated [6-13 C]GalCer (1 or 2 mol%) into sonicated vesicles prepared with 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC) in the presence or absence of sphingomyelin, and then quenched the outer leaflet signal arising exclusively from the C6 nucleus of the galactosyl moiety of GalCer by adding 5 mM Mn2+ to the pre-formed vesicles (Mattjus et al., 2002). The synthesis of GalCer specifically enriched with 13 C at C6 is outlined in Fig. 7 (Mattjus et al., 2002, see supplemental information). Briefly, the activated

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Fig. 7. Synthesis of [6-13 C]-N-palmitoyl-GalCer. (A) Preparation of the trichloroacetimidate of [6-13 C]galactosyltetraacetate (the galactosyl donor). (B) Preparation of d-erythro-2-azido-3-(O-benzoyl)sphingosine (the galactosyl acceptor). (C) Coupling of donor and acceptor, hydrolysis of protecting groups, azide reduction, and N-acylation.

[13 C]galactosyl donor was prepared by peracetylation of commercially available [6-13 C]galactose, followed by conversion of the anomeric position to the trichloroacetimidate. Then, the diazo transfer reaction was used to convert sphingosine to (2S,3R)-2-azidosphingosine, which was silylated regioselectively in the presence of 2 equivalents of imidazole (Im) to afford 1-O-(tert-butyldimethylsilyl)2-azidosphingosine. After benzoylation, the O-silyl

ether was deprotected with tetra-n-butylammonium fluoride in the presence of imidazole to give 2azido-3-(O-benzoyl)sphingosine, which was used as the galactosyl acceptor. The reaction between the galactosyl donor and acceptor was carried in the presence of boron trifluoride etherate and molecular sieves. Methanolytic degradation of the benzoyl and acetate esters provided [6-13 C]-2-azido-␤galactosylsphingosine. The synthesis of N-palmitoyl-

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[6-13 C]-␤-GalCer was completed by azide reduction (Staudinger reaction using triphenylphosphine in wet THF) and in situ N-acylation with p-nitrophenyl palmitate. Small unilamellar vesicles (SUVs) of 250-nm diameter were prepared from POPC and 1 or 2 mol% of [6-13 C]GalCer. When 5 mM Mn2+ (a concentration that does not induce leakage of vesicle contents or induce aggregation) was added to the SUVs, the 13 C-NMR signal arising from [6-13 C]GalCer in the outer leaflet (at δ 61.4 ppm, shaded resonance in Fig. 8) was quenched, but the signal arising from [6-13 C]GalCer in the inner leaflet was unaffected. We compared the integrated signal intensity at δ 61.4 ppm in the absence (Fig. 8, bottom spectrum) and presence (top spectrum) of Mn2+ and found that GalCer was preferentially localized (70%) in the inner leaflet of SUVs prepared from POPC. The galactosyl headgroup of GalCer is smaller and less hydrated than the phosphocholine headgroup of POPC, which may account for the tendency of GalCer to be enriched in the inner leaflet and allow the more sterically demanding component (POPC) to be accommodated in the more loosely packed outer leaflet of the SUVs.

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Incorporation of egg sphingomyelin in increasing concentrations into the POPC vesicles (POPC/SPM molar ratios of 1:1 and 1:2; [GalCer], 1 mol%) caused a marked change in the transbilayer distribution of [613 C]GalCer. The right-hand panel of Fig. 8 shows that 54, 48, and 40 mol% of GalCer was localized in the inner leaflet of SUVs prepared with POPC/egg sphingomyelin at 2:1, 1:1, and 1:2 molar ratios, respectively, compared with the 70 mol% of GalCer localized in the inner leaflet in the absence of egg sphingomyelin. 31 P and 13 C NMR spectra recorded in the presence of Pr3+ or Mn2+ (to shift the resonances arising from phospholipids in the outer leaflet without affecting those from phospholipids in the inner leaflet) revealed that ∼72% of the sphingomyelin molecules were localized in the outer leaflet of these SUVs. To test our hypothesis that sphingolipid-sphingolipid interactions are important in regulating the transbilayer distribution of GalCer in SUVs, we recently started to examine whether other phospholipids can mimic the effect of egg sphingomyelin. The transbilayer distribution of GalCer was not altered when dipalmitoylphosphatidylcholine, 3deoxy-N-palmitoylsphingomyelin, or 4,5-dihydroN-palmitoylsphingomyelin was incorporated into

Fig. 8. 13 C NMR spectra of [6-13 C]GalCer in POPC SUVs in the presence and absence of Mn2+ , and the transbilayer distribution of [6-13 C]GalCer in POPC SUVs in the absence and presence of egg sphingomyelin. Permission to reproduce this figure from Mattjus et al. (2002) has been obtained from ASBMB.

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POPC vesicles. Subtle differences in the interfacial region of bilayers formed from dipalmitoylphosphatidylcholine and 3-deoxy- or 4,5-dihydro-Npalmitoylsphingomyelin relative to sphingomyelin result in differences in hydrogen bonding and hydration. For example, the headgroup of sphingomyelin is smaller and less hydrated than that of phosphatidylcholine, and the 4,5-trans double bond affects the orientation of the 3-hydroxy group of sphingomyelin and the extent of hydrogen bonding (Epand, 2003; Steinbauer et al., 2003). The inability of dipalmitoylphosphatidylcholine, 3-deoxy-N-palmitoylsphingomyelin, and 4,5-dihydro-N-palmitoylsphingomyelin to duplicate the effect of egg sphingomyelin on the transbilayer distribution of GalCer in SUVs may be a result of the diminished capacity of these phospholipids to interact with GalCer. Therefore, structural features in the interfacial region of the sphingomyelin molecule seem to modulate the GSL transbilayer distributions in SUVs.

5. Are lipid rafts involved in the fusion of Semliki Forest virus (SFV) and Sindbis (SIN) virus with large unilamellar vesicles (LUVs)? Semliki Forest virus (SFV) and Sindbis virus (SIN) are the prototypic alphaviruses, all of which have a lipid envelope and a RNA genome, and use anthropods as vectors to infect their host organisms (Strauss and Strauss, 1994). These viruses, like many other viruses, infect cells by binding to receptors on the plasma membrane, followed by receptor-mediated endocytosis, transport to endosomes, and fusion of the viral membrane with the endosomal membrane. Fusion is mediated by the heterodimeric glycoprotein E1/E2 in the viral envelope. The transmembrane glycoprotein E1 and a water-soluble ectodomain of E1 interact with cholesterol in LUVs (Ahn et al., 2002). During fusion, E1 undergoes a conformational change triggered by low pH. A homotrimer of E1 is required for fusion of SFV (Kielian et al., 1996) and SIN (Smit et al., 1999). The low pH induced fusion of SFV and SIN with cells or model membranes requires the presence of both cholesterol and sphingolipids in the target membrane (Moesby et al., 1995; Wilschut et al., 1995; Smit et al., 1999) and may involve a discrete hemifu-

sion intermediate (Smit et al., 2002). Since transient formation of lipid rafts in membranes also requires the presence of both cholesterol and sphingolipids with predominantly saturated N-acyl chains, and since rafts have been shown to be involved in the cell entry of other viruses (Bavari et al., 2002; Triantafilou and Triantafilou, 2003), we were interested in determining whether fusion of SFV and SIN requires the presence of rafts in the target membranes (LUVs). We synthesized unnatural analogs of sphingomyelin with different degrees of N-acyl chain unsaturation, as well as with different unsaturation geometry (cis versus trans). In collaboration with Professor Jan Wilschut, these sphingomyelin analogs were incorporated into LUVs (together with phosphatidylcholine, phosphatidylethanolamine, and cholesterol), and the abilities of the LUVs to form lipid rafts and to support viral fusion were analyzed (Waarts et al., 2002). A pyrene-excimer assay of fusion that reports both the kinetics and extent of fusion of pyrene-labeled viruses with LUVs was used (Smit et al., 2003). The extent of lipid raft formation in the LUVs was estimated by using a gradient flotation analysis (Waarts et al., 2002). Fig. 9 shows that sphingomyelin from brain or with a N-stearoyl or N-elaidoyl chain (lanes A–C) gave LUVs that formed rafts, as measured by flotation of [3 H]cholesterol, and also supported extensive fusion. In contrast, rafts were not formed with LUVs prepared with sphingomyelins containing cis unsaturation (N-oleoyl- or N-linoleoylsphingomyelin), yet these LUVs did support fusion (lanes D and E); LUVs containing N-oleoylceramide also underwent extensive fusion with SFV but did not form rafts (lane F). (Lane G represents the control without sphingolipid.) We also used plant sterols and synthetic analogs of cholesterol that varied in the length or degree of unsaturation of the side chain. There was a lack of correlation between the ability of these sterols to support lipid raft formation and their ability to support efficient fusion of SFV and SIN with LUVs. Viral fusion was rapid and extensive with LUVs containing sphingomyelins and sterols that differed in their abilities to form lipid rafts. Thus lipid rafts are not required for fusion of these alphaviruses viruses with LUVs. It remains to be shown whether SFV and SIN utilize raft domains as sites for cell entry or for fusion with the endosomal membrane.

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6. How does ceramide enhance the efficiency of membrane permeabilization induced by cholesterol-specific cytolysins?

Fig. 9. Lack of correlation between the extent of SFV fusion with LUVs containing unnatural sphingomyelins and the ability of the LUVs to form rafts. LUVs were prepared from phosphatidylcholine, phosphatidylethanolamine, cholesterol, and sphingolipid (molar ratio, 1:1:1.5:1). Lane A, brain SM; lane B, N-stearoyl-SM; lane C, N-elaidoyl-SM; lane D, N-oleoyl-SM; lane E, N-linoleoyl-SM; lane F, N-oleoylceramide; lane G, no sphingolipid. Fusion was measured at pH 5.5 and 37 ◦ C. Detergent (Triton X-100)-insoluble complexes floating to the top of an Optiprep density gradient at 4 ◦ C were detected by scintillation counting of the [3 H]cholesterol initially incorporated into the LUVs. Permission to reproduce this figure from Waarts et al. (2002) has been obtained from ASBMB.

The bacterial toxin Vibrio cholerae cytolysin (VCC) forms pores in membranes containing cholesterol (Zitzer et al., 1997). Membrane permeabilization is inefficient in SUVs composed of phosphatidylcholine and cholesterol, but is greatly enhanced by incorporation of ceramide into the target bilayers (Zitzer et al., 1999). In collaboration with Professor Michael Palmer, we found that a broad variety of unnatural ceramide analogs were as effective as derythro-ceramide in enhancing membrane permeabilization induced by VCC and streptolysin O (SLO), another cholesterol-specific but structurally unrelated cytolysin (Zitzer et al., 2001). We concluded that ceramide did not interact directly with the toxin. Instead, a lipid molecule having a conical shape (a property shared by all of the synthetic ceramide analogs and by diacylglycerol (DAG)) brings about a change in the energetic state of membrane cholesterol that results in an enhancement in the interaction between cholesterol and the cytolysin. Fig. 10 is a cartoon of a possible mechanism that accounts for how ceramide and DAG may modulate

Fig. 10. Model for membrane permeabilization by cholesterol-specific cytolysins in the presence of ceramide or DAG.

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the interaction of the toxin with cholesterol. In the absence of ceramide or DAG (left-hand side of Fig. 10), cholesterol (which is also a cone-shaped lipid) is well accommodated in the bilayer. The phosphocholine headgroup of phosphatidylcholine (PC) and sphingomyelin (SM) provides headgroup coverage to cholesterol so that the sterol is not exposed to an energetically unfavorable state. In fact, there is a notion that PC makes an umbrella for cholesterol (Huang and Feigenson, 1999). The right-hand side of Fig. 10 shows how a cholesterol-specific toxin may be activated in an indirect manner by the cone-shaped lipids, ceramide and DAG. When either of the latter lipids occupies the sites between adjacent phospholipids, cholesterol will dissociate from the bilayer (especially in membranes with a high cholesterol content). To avoid unfavorable contacts with water, cholesterol molecules occupy binding sites in the cholesterolspecific cytolysins. Thus ceramide and DAG activate VCC and SLO in an indirect manner, since they do not act on the toxins specifically yet they augment membrane permeabilization by several hundred fold. In a sense, cholesterol may function as a second messenger to ceramide and/or DAG. It is conceivable that the mechanism depicted in Fig. 10 also applies to other membrane-associated proteins that have specific binding sites for cholesterol.

7. Use of synthetic glycosphingolipid (GSL) analogs to examine the mechanism of endocytosis in different cell types by fluorescence microscopy Fluorescent analogs of glycosphingolipids such as lactosylceramide (LacCer) allow one to use fluorescence microscopy to monitor how these lipids undergo endocytosis and sorting within living mammalian cells. Many lipids containing a boron dipyrromethene difluoride (BODIPY) are available from Molecular Probes (Eugene, OR). They are widely used in lipid, nucleic acid, and protein research because of their photostability, attractive spectral characteristics (high extinction coefficient and high fluorescence quantum yield), and ability to form excimers on increasing concentration in membranes (which changes the emission from green to red wavelengths) (Pagano et al., 2000). In the fluorescent analog of LacCer, the BODIPY moiety is linked to the nitrogen of the ceramide

moiety of LacCer; the linker is typically a short fatty acid chain in place of the normal N-acyl chain. When a complex of the BODIPY-lipid with defatted albumin is added to cells at low temperature, one observes immediate labeling of the plasma membrane. On warming, BODIPY-LacCer is internalized almost exclusively via caveolae. In normal cells, LacCer is subsequently targeted to the Golgi apparatus in a process that involves microtubules, phosphatidylinositol 3-kinase, rab 7, and rab 9. However, in sphingolipid storage diseased cells, BODIPY-LacCer as well as other BODIPY-GSL analogs accumulate in endosomes and lysosomes (Choudhury et al., 2002). The factors that determine why BODIPY-LacCer is taken up from the cell membrane by a clathrin-independent or caveolar-like mechanism are presently unknown. In a recent collaborative study with Dr. Richard E. Pagano, we sought to alter some molecular features in the GSL molecule and then analyze the pathway of initial entry of these GSL analogs into human skin fibroblasts and other cell lines. We synthesized a new BODIPY-GSL analog, BODIPY-maltosylceramide (MalCer), which differs from LacCer in carbohydrate structure. In MalCer, the headgroup is Glc(␣1 → 4)Glc whereas in LacCer the headgroup is Gal(␤1 → 4)Glc (Fig. 11). In addition, analogs of BODIPYLacCer were prepared in which the length of the sphingoid base as well as the length of the linker to the fluorophore was varied. Fig. 12 outlines the synthesis of BODIPY-MalCer with a five-carbon linker in the N-acyl chain. The glycosyl donor was per-O-acetylmaltosyl trichloroacetimidate, which was prepared in an analogous manner as the [6-13 C]glactosyl donor (see Fig. 7). 2-Azido3-O-benzoylsphingosine was used as the glycosyl acceptor. After glycosylation and deprotection of the benzoyl and acetate ester functionalities, the azido group was reduced to an amino group with triphenylphosphine in wet THF, and N-acylation was carried out by using the N-hydroxysuccinimidoyl (NHS) ester of C5 -BODIPY, affording the product. The internalization of these analogs and of BODIPY-MalCer was compared in several mammalian cell types pretreated with selective endocytic inhibitors such as chlorpromazine (CPZ), which blocks clathrin-mediated uptake, and nystatin, which blocks caveolar-mediate uptake. A comparison of the internalization of the two fluorescent-labeled GSLs

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Fig. 11. Structures of BODIPY-LacCer (top) and BODIPY-MalCer.

shows identical behavior in control fibroblasts and in nystatin- and CPZ-treated fibroblasts (Fig. 13). We concluded that BODIPY-LacCer and BODIPYMalCer are taken up exclusively by the caveolarmediated pathway of endocytosis, not by the clathrindependent route (Singh et al., 2003). Despite marked differences in the conformation in the vicinity of the carbohydrate/sphingolipid interface and in sphingoid chain length of the GSLs, the mechanism of uptake was not altered.

These results suggest that the ceramide backbone portion of the GSL molecule is the major determinant of caveolar endocytosis. The hydrophobic core of the sphingosine backbone is not a critical region for directing LacCer to utilize the caveolar pathway, since variations in this region did not alter the mechanism of uptake from the cell membrane. It appears that the lipid/water interfacial region of the ceramide portion of the GSL molecule may be critical in directing the lipid to utilize the caveolar-mediated pathway. To test

Fig. 12. Synthesis of BODIPY-MalCer.

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Fig. 13. Comparison of the effects of nystatin and CPZ on the internalization (5 min, 37 ◦ C) of BODIPY-LacCer and BODIPY-MalCer in rat fibroblasts (RFs). Note the nearly identical internalization response of BODIPY-LacCer and BODIPY-MalCer to the pretreatment with nystatin and CPZ.

this hypothesis, we plan to modify the interfacial region and stereochemistry of the sphingoid backbone of BODIPY-LacCer.

8. 1-Acyl-sn-glycero-3-phosphate (lysophosphatidic acid, LPA) analogs—stereopharmacology of ether-linked and amino acid based derivatives of LPA The simple structure of LPA (1-acyl-sn-glycero-3phosphate)—which has a glycerol backbone esterified with a phosphate headgroup and a saturated or unsaturated long acyl chain, and is therefore the simplest glycerophospholipid—belies the diversity of its biological properties. The glycerol C2 atom is a stereogenic center, and naturally occurring LPA has the R (or L) configuration. O-Alkyl or O-alkenyl long chains can replace the fatty acyl chain in some molecular species to give LPAs with distinctly different activities than the predominant acyl-containing LPA. Similarly, analogs of LPA such as cyclic phosphatidic acid

(Fujiwara et al., 2003) and a regioisomer, 1-lyso-2acyl-sn-glycero-3-phosphate (Tokumura et al., 2002), are naturally occurring lipid mediators that evoke different cellular responses than acyl-containing LPAs. This lipid mediator is involved in many physiological and pathophysiological processes. Specific GPCRs present in many cell types mediate the cellular effects of extracellular LPA (Tigyi and Parrill, 2003). Much less information is available about intracellular LPA receptors. However, a new target for LPA was discovered recently; LPA acts on the intracellular peroxisome proliferator-activated receptor-␥ (PPAR␥), a nuclear transcription factor, forming a LPA–PPAR␥ complex (McIntyre et al., 2003). The latter is bound to DNA in a complex with the retinoic acid receptor RXR and regulates nuclear gene transcription. Efforts to elucidate other intracellular roles of LPA are currently underway, especially because of the implication of LPA in atherogenesis (Füller et al., 2003). Specific LPA molecular species containing an unsaturated acyl chain or an ether chain are ligands of PPAR␥ and induce neointimal lesion formation (Zhang et al., 2004).

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Antagonists that selectively block the ability of LPA to act as a PPAR␥ ligand may have novel therapeutic roles; for example, they may be anti-atherogenic and anti-inflammatory drugs. There has been a considerable synthetic effort directed toward the development of pharmacological modulators of LPA signaling pathways. Agonists as well as antagonists that interact selectively with the various G protein-coupled receptor subtypes for LPA (designated as LPA1 –LPA3 and p2y9/GPR23) are being sought. Many structural analogs of LPA have been synthesized for structure–activity relationship studies with cells expressing LPA receptors (Tigyi and Parrill, 2003). We synthesized 1-O-hexadecyl-sn-glycero-3phosphate (1-C16-GP) and its enantiomer 3-Ohexadecyl-sn-glycero-1-phosphate (3-C16-GP) using (S)-glycerol acetonide as the starting material (Yokoyama et al., 2002). The advantage of using the ether analogs of LPA is that the 1-O-alkyl linkage is stable and not subject to intramolecular rearrange-

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ment like their O-acyl counterparts. In collaboration with Professor Gabor Tigyi, we found that both the natural L (R) and unnatural D (S) enantiomers of the 1-ether-2-lyso-PA (see Fig. 14 for the structures) were equally active in several bioassays, such as the activation of chloride ion currents in Xenopus oocytes, the mobilization of intracellular calcium, and the dephosphorylation of these LPA analogs catalyzed by a lipid phosphate phosphohydrolase (LPP-1) in fibroblasts (Yokoyama et al., 2002). These observations are an exception to the commonly found stereoselectivity for ligand recognition by receptors; usually, the naturally occurring enantiomer is preferred over the unnatural one, resulting in large differences in biological activities between the enantiomers. No stereoselectivity was also found for the action of LPA stereoisomers on platelet LPA receptors (Gueguen et al., 1999) and for the action of analogs of LPA in which an ethanolamine backbone replaced the glycerol backbone that links the phosphate group and the long chain (Heise et al., 2001). Replacement of the

Fig. 14. Structures of lysophosphatidic acid analogs with ester, ether, and amino acid linkers.

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glycerol backbone with a serine or tyrosine backbone afforded N-palmitoylserine or N-palmitoyltyrosine phosphoric acid (NP-Ser-PA and NP-Tyr-PA, respectively) that were recognized in a nonstereoselective manner as antagonists of LPA receptors in Xenopus oocytes (Bittman et al., 1996; Liliom et al., 1996). However, enantioselective responses were observed for the stereoisomers of 1-acyl-3-phosphorothioate LPA analogs, in which the 2-hydroxy is blocked as a 2-methoxy group; in fact, an inverted activity relationship was found, with the unnatural S isomer being more effective than the natural R isomer in activating LPA3 (Qian et al., 2003). These structure–activity relationship studies illustrate how the ligand-binding pockets of LPA receptors may be probed by using synthetic LPA analogs. The recent observation that only one stereoisomer of an N-acyl-ethanolamide phosphate, VPC12449, blocked the LPA1 receptor, with the other enantiomer being inactive (Heise et al., 2001), indicates that chirally pure LPA analogs are needed in future drugs. LPA receptor antagonists may have a number of therapeutic applications, such as for the inhibition of aspirin-insensitive platelet aggregation in the prevention of thrombus formation leading to heart attack and stroke (Rother et al., 2003) and for the inhibition of tumor cell metastasis (Yamada et al., 2004).

9. Antitumor ether lipids: use of phosphonocholine analogs of ET-18-OCH3 to gain insights about the targets relevant to tumor cell growth inhibition Antitumor ether lipids (AELs) inhibit the growth of a broad panel of tumor cell lines. The classical example of AELs is 1-O-octadecyl-2-O-methyl-racglycerophosphocholine ET-18-OCH3 or its C16 analog, ET-16-OCH3 (Fig. 15). Unlike many available anticancer compounds, these unnatural, ether analogs

of 2-lysophosphatidylcholine do not interact with cellular DNA and therefore are not mutagenic. It has been difficult to discern the primary targets of action of AELs because they affect an unusually large number of cellular events (Lohmeyer and Bittman, 1994). Therefore, the principal mechanism of action by which AELs generate cytotoxic activity is still being debated. Nevertheless, there is increasing evidence that after AELs are taken up into the cell membrane numerous intracellular signal transduction pathways regulating cell proliferation/cell death are perturbed (Arthur and Bittman, 1998). The finding that racemic ET-18-OCH3 and ET-16OCH3 induce selective tumor cell apoptosis has led to studies of the mechanisms by which these AELs block the survival of tumor cells (Mollinedo et al., 1997). A number of targets of various alkyllysophospholipids have been identified that culminate in (i) the inhibition of the mitogenic extracellular signalregulated kinase (ERK) and the phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B) proliferative and survival pathways, and (ii) the activation of the stress-activated Jun N-terminal kinase (JNK) pathway (Meuillet et al., 2003; Ruiter et al., 2003). The cell death receptor Fas/CD95 appears to be one of the first intracellular targets activated by ET-18-OCH3 , initiating cell death via the mitochondrial apoptotic pathway (Gajate and Mollinedo, 2002). Another direct intracellular target of ET-18-OCH3 is Raf-1, a well-known regulator of the mitogen-activated protein kinase (MAPK) pathway (Samadder et al., 2003). ET18-OCH3 , but not several naturally occurring phospholipids and lysophospholipids, interacts specifically with Raf-1 and blocks the interaction between Raf-1 and activated Ras (Samadder et al., 2003). By inhibiting the activation of Raf-1, ET-18-OCH3 disrupts signaling through the Raf-MEK-ERK signaling cascade. A pair of structurally similar synthetic analogs of AELs with different antiproliferative effects on tumor cells would be useful as tools to distinguish be-

Fig. 15. Structures of AEL analogs.

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tween molecular targets that lead to growth inhibition from those that are not relevant to growth inhibition but are nevertheless affected by the AELs. Although one might predict that stereoisomers of AELs would be useful in this regard, previous studies showed no enantioselective antiproliferative activity. For example, the R and S enantiomers of the well-known antitumor ether lipid ET-18-OCH3 have equal activity (Lohmeyer and Workman, 1992; Bittman et al., 1994; Mollinedo et al., 1997). We synthesized the (R)- and (S)-phosphono analogs of ET-16-OCH3 , which have a carbon-phosphorus bond in place of a carbon–oxygen– phosphorus bond and are therefore not susceptible to hydrolysis at this site (Fig. 15). These compounds are isosteric phosphonocholines, since a methylene group replaces the oxygen atom on the glycerol side of the phosphorus. These saturated phosphonocholines also did not exhibit an enantioselective effect with respect to inhibition of tumor cell growth in vitro or in vivo (Bittman et al., 1994). We recently made additional modifications at the sn3 position, while maintaining the carbon–phosphorus bond. In collaboration with Professor Gilbert Arthur, we discovered that the enantiomers of an unsaturated phosphonocholine ((R)- and (S)-ENE-OCH3 ; see Fig. 15), which have a trans double bond adjacent to the carbon–phosphorus bond, exhibited markedly different antiproliferative effects in some epithelial cancer cell lines (human breast cancer MCF-7 and human neuroblastoma SK-N-SH cells). This is the first example of AELs that exhibit enantioselective activity in vitro in some cell lines. We used this pair of compounds to show the importance of activation of the JNK signaling pathway in the differential cytostatic/cytotoxic effects of these compounds (Samadder et al., 2004). The S enantiomer was used as a “negative control;” if the S isomer affected the activity of a cellular target in cells in which it did not exert an antiproliferative effect (e.g., MCF7 or SK-N-SH cells), then this target was concluded to be not relevant to the mechanism leading to growth inhibition. Two neuroblastoma cell lines, SK-N-MC and SK-N-SH, were used for these comparative studies. While the R and S enantiomers of ENE-OCH3 had a similar inhibitory effect on the growth of SK-N-SH cells, the S isomer was significantly less cytostatic than the R isomer in SK-N-MC cells. Activation of JNK was five- to eight-fold higher in quiescent SK-N-MC

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cells incubated with (R)-ENE-OCH3 than with (S)ENE-OCH3 on the basis of densitometry of the Western blots. In SK-N-MC cells, both enantiomers inhibited PKB activation similarly, and had no effect on ERK activation. These results suggest that the differential effects of the enantiomers of ENE-OCH3 arise primarily from their differential ability to activate JNK and not from the perturbation of the PKB and ERK pathways in these cells. However, in SK-N-SH cells (where there was no enantioselective effect), both enantiomers activated JNK similarly (a 2.5-fold activation) and inhibited MAPK activation to approximately the same degree. Although the enantiomers inhibited PKB to different extents in SK-N-SH cells, there was no significant difference in their antiproliferative effects; therefore, perturbation of PKB activation cannot be the principal site of action of these drugs. The activation of JNK by both enantiomers in SK-N-SH cells can explain the similar effects of the enantiomers on the proliferation and cytotoxicity in these cells. Thus this pair of compounds offers a novel means for relating the postulated cellular effects of AELs to the mechanism of inhibition of cell growth. In this example, we used the enantiomers of ENE-OCH3 to demonstrate a correlation between the activation of JNK and the differential cytostatic/cytotoxic effects of these AELs.

10. Conclusions In this lecture, representative examples have been presented that illustrate how the chemical synthesis of unnatural lipid analogs is a powerful approach for addressing issues involving the properties of selected bioactive lipids. These examples show that the interplay of organic synthesis with physical biochemistry, cell biology, and virology has provided a means for furthering our understanding of the molecular details of the properties of lipids in model membranes and cells. Specifically, the studies described above have indicated that: (1) some novel ceramide analogs are more effective than natural ceramide as anticancer agents; (2) photoactivatable analogs of [32 P]S1P are useful probes for the identification of protein targets of this key lysophospholipid mediator; (3) sphingomyelin but not related phospholipids alters the transbilayer distri-

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bution of GalCer in SUVs; (4) the alphaviruses SFV and SIN do not require the presence of lipid rafts for fusion with model membranes; (5) ceramide has a nonspecific role in augmenting the interaction of cholesterol with the cytolysin VCC; (6) the selective caveolar endocytosis of GSLs does not depend on the conformation around the saccharide/sphingolipid linkage or the length of the sphingoid chain; (7) there is no stereospecific ligand recognition by some LPA receptors but there is a need to develop new chiral LPA analogs that act as LPA receptor antagonists for a number of therapeutic applications; and (8) the enantiomers of an unsaturated phosphonocholine analog of the protypical AEL ET-18-OCH3 are useful probes for discovering which intracellular targets lead to the cytotoxic effects of this drug.

Acknowledgements The continued financial support of this research for the past 30-plus years from the National Institutes of Health (Grant HL 16660) is gratefully acknowledged. I would like to acknowledge my gratitude to the outstanding collaborators in the projects described above

as well as to the many other scientists whom I have been so fortunate to have worked with throughout my career. I also thank two former Ph.D. students for nominating me for this Award—Dr. Sanda Clejan and Dr. Shaukat Ali.

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