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• W2107082487 abstract "We report the synthesis and characterization of a novel thiourea derivative of sphingomyelin (AD2765). In vitro assays using pure enzyme and/or cell extracts revealed that this compound inhibited the hydrolysis of BODIPY-conjugated or 14C-labeled sphingomyelin by acid sphingomyelinase and Mg2+-dependent neutral sphingomyelinase. Studies in normal human skin fibroblasts further revealed that AD2765 was taken up by cells and inhibited the hydrolysis of BODIPY-conjugated sphingomyelin in situ. In situ and in vitro studies also showed that this compound inhibited the synthesis of sphingomyelin from BODIPY-conjugated ceramide. The specificity of AD2765 for enzymes involved in sphingomyelin metabolism was demonstrated by the fact that it had no effect on the hydrolysis of BODIPY-conjugated ceramide by acid ceramidase or on the synthesis of BODIPY-conjugated glucosylceramide from BODIPY-conjugated ceramide. The overall effect of AD2765 on sphingomyelin metabolism was concentration-dependent, and treatment of normal human skin fibroblasts or cancer cells with this compound at concentrations > 10 μM led to an increase in cellular ceramide and cell death.Thus, AD2765 might be used to manipulate sphingomyelin metabolism in various ways, potentially to reduce substrate accumulation in cells from types A and B Niemann-Pick disease patients, and/or to affect the growth of human cancer cells. We report the synthesis and characterization of a novel thiourea derivative of sphingomyelin (AD2765). In vitro assays using pure enzyme and/or cell extracts revealed that this compound inhibited the hydrolysis of BODIPY-conjugated or 14C-labeled sphingomyelin by acid sphingomyelinase and Mg2+-dependent neutral sphingomyelinase. Studies in normal human skin fibroblasts further revealed that AD2765 was taken up by cells and inhibited the hydrolysis of BODIPY-conjugated sphingomyelin in situ. In situ and in vitro studies also showed that this compound inhibited the synthesis of sphingomyelin from BODIPY-conjugated ceramide. The specificity of AD2765 for enzymes involved in sphingomyelin metabolism was demonstrated by the fact that it had no effect on the hydrolysis of BODIPY-conjugated ceramide by acid ceramidase or on the synthesis of BODIPY-conjugated glucosylceramide from BODIPY-conjugated ceramide. The overall effect of AD2765 on sphingomyelin metabolism was concentration-dependent, and treatment of normal human skin fibroblasts or cancer cells with this compound at concentrations > 10 μM led to an increase in cellular ceramide and cell death. Thus, AD2765 might be used to manipulate sphingomyelin metabolism in various ways, potentially to reduce substrate accumulation in cells from types A and B Niemann-Pick disease patients, and/or to affect the growth of human cancer cells. Sphingomyelin is an important component of cell membranes and a major source of ceramide involved in signal transduction (1Levade T. Jaffrezou J.P. Signalling sphingomyelinases: which, where, how and why?.Biochim. Biophys. Acta. 1999; 1438: 1-17Crossref PubMed Scopus (283) Google Scholar). Human acid sphingomyelinase (ASM; sphingomyelin phosphodiesterase, EC 3.1.4.12) is one enzyme that catalyzes the hydrolysis of sphingomyelin, resulting in the formation of ceramide and phosphocholine (2Barenholz Y. Roitman R. Gatt S. Enzymatic hydrolysis of sphingolipids. II. Hydrolysis of sphingomyelin by an enzyme from rat brain.J. Biol. Chem. 1966; 241: 3731-3737PubMed Google Scholar). ASM is found predominantly within lysosomes, but other enzyme forms, including a secreted, Zn2+-dependant form (3Tabas I. Secretory sphingomyelinase.Chem. Phys. Lipids. 1999; 102: 123-130Crossref PubMed Scopus (96) Google Scholar) and a plasma membrane form localized to raft structures (4Liu P. Anderson R.G. Compartmentalized production of ceramide at the cell surface.J. Biol. Chem. 1995; 270: 27179-27185Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 5Gulbins E. Regulation of death receptor signaling and apoptosis by ceramide.Pharmacol. Res. 2003; 47: 393-399Crossref PubMed Scopus (134) Google Scholar), have been identified. Each of these enzyme forms is derived from the same gene, located on the short arm of chromosome 11 and designated sphingomyelin phosphodiesterase-1 (SMPD-1) (6Da Veiga Pereira L. Desnick R.J. Adler D.A. Disteche C.M. Schuchman E.H. Regional assignment of the human acid sphingomyelinase gene (SMPD1) by PCR analysis of somatic cell hybrids and in situ hybridization to 11p15.1-p15.4.Genomics. 1991; 9: 229-234Crossref PubMed Scopus (65) Google Scholar, 7Callahan J.W. Khalil M. Philippart M. Sphingomyelinases in human tissues. II. Absence of a specific enzyme from liver and brain of Niemann-Pick disease, type C.Pediatr. Res. 1975; 9: 908-913Crossref PubMed Scopus (49) Google Scholar, 8Yamanaka T. Suzuki K. Acid sphingomyelinase of human brain: purification to homogeneity.J. Neurochem. 1982; 38: 1753-1764Crossref PubMed Scopus (60) Google Scholar, 9Quintern L.E. Weitz G. Nehrkorn H. Tager J.M. Schram A.W. Sandhoff K. Acid sphingomyelinase from human urine: purification and characterization.Biochim. Biophys. Acta. 1987; 922: 323-336Crossref PubMed Scopus (113) Google Scholar). The full-length cDNA encoding ASM has been isolated (10Quintern L.E. Schuchman E.H. Levran O. Suchi M. Ferlinz K. Reinke H. Sandhoff K. Desnick R.J. Isolation of cDNA clones encoding human acid sphingomyelinase: occurrence of alternatively processed transcripts.EMBO J. 1989; 8: 2469-2473Crossref PubMed Scopus (136) Google Scholar, 11Schuchman E.H. Suchi M. Takahashi T. Sandhoff K. Desnick R.J. Human acid sphingomyelinase. Isolation, nucleotide sequence and expression of the full-length and alternatively spliced cDNAs.J. Biol. Chem. 1991; 266: 8531-8539Abstract Full Text PDF PubMed Google Scholar), leading to the production of recombinant enzyme in overexpressing CHO cells (12He X. Miranda S.R. Xiong X. Dagan A. Gatt S. Schuchman E.H. Characterization of human acid sphingomyelinase purified from the media of overexpressing Chinese hamster ovary cells.Biochim. Biophys. Acta. 1999; 1432: 251-264Crossref PubMed Scopus (84) Google Scholar) and insect cells (13Bartelsen O. Lansmann S. Nettersheim M. Lemm T. Ferlinz K. Sandhoff K. Expression of recombinant human acid sphingomyelinase in insect Sf21 cells: purification, processing and enzymatic characterization.J. Biotechnol. 1998; 63: 29-40Crossref PubMed Scopus (30) Google Scholar). Other neutral and alkaline sphingomyelinase activities also exist in mammalian cells, but these are distinct enzymes derived from different genes (1Levade T. Jaffrezou J.P. Signalling sphingomyelinases: which, where, how and why?.Biochim. Biophys. Acta. 1999; 1438: 1-17Crossref PubMed Scopus (283) Google Scholar). In humans, inherited mutations in the SMPD-1 gene result in types A and B Niemann-Pick disease (NPD) (14Schuchman E.H. Desnick R.J. Niemann-Pick disease type A and B: acid-sphingomyelinase deficiencies.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Disease. 8th edition. McGraw-Hill, New York2001: 2601-2624Google Scholar). In addition, a recent report showed that deletion of the gene (Smpd3) encoding one of the neutral sphingomyelinases in mice results in osteogenesis and dentiogenesis imperfecta (15Aubin I. Adams C.P. Opsahl S. Septier D. Bishop C.E. Auge N. Salvayre R. Begre-Salvayre A. Goldberg M. Guenet J.L. et al.A deletion in the gene encoding sphingomyelin phosphodiesterase 3 (Smpd3) results in osteogenesis and dentinogenesis imperfecta in the mouse.Nat. Genet. 2005; 37: 803-805Crossref PubMed Scopus (138) Google Scholar). As noted above, the reaction product of ASM activity, ceramide, is a potent lipid second messenger involved in diverse cellular functions, including cell growth, differentiation, and death (16Ruvolo P.P. Intracellular signal transduction pathways activated by ceramide and its metabolites.Pharmacol. Res. 2003; 47: 383-392Crossref PubMed Scopus (295) Google Scholar, 17Gulbins E. Kolesnick R. Raft ceramide in molecular medicine.Oncogene. 2003; 22: 7070-7077Crossref PubMed Scopus (351) Google Scholar). Ceramide production has been implicated in the pathogenesis of diseases such as atherosclerosis and acquired immunodeficiency syndrome, as well as in liver damage and acute lung injury (18Barnes P.J. Ceramide lances the lungs.Nat. Med. 2004; 10: 130-131Crossref PubMed Scopus (18) Google Scholar, 19Mari M. Colell A. Morales A. Paneda C. Varela-Nieto I. Garcia-Ruiz C. Fernandez-Checa J.C. Acidic sphingomyelinase downregulates the liver-specific methionine adenosyltransferase 1A, contributing to tumor necrosis factor-induced lethal hepatitis.J. Clin. Invest. 2004; 113: 895-904Crossref PubMed Scopus (71) Google Scholar). The need for greater understanding of sphingomyelin and ceramide metabolism, as well as the potential clinical significance of modulating this pathway, has prompted the development of several classes of sphingomyelinase inhibitors. For example, various synthetic sphingomyelin analogues (20Yokomatsu T. Murano T. Akiyama T. Koizumi J. Shibuya S. Tsuji Y. Soeda S. Shimeno H. Synthesis of non-competitive inhibitors of sphingomyelinases with significant activity.Bioorg. Med. Chem. Lett. 2003; 13: 229-236Crossref PubMed Scopus (66) Google Scholar, 21Hakogi T. Monden Y. Taichi M. Iwama S. Fujii S. Ikeda K. Katsumura S. Synthesis of sphingomyelin carbon analogues as sphingomyelinase inhibitors.J. Org. Chem. 2002; 67: 4839-4846Crossref PubMed Scopus (44) Google Scholar, 22Taguchi M. Goda K. Sugimoto K. Akama T. Yamamoto K. Suzuki T. Tomishima Y. Nishiguchi M. Arai K. Takahashi K. et al.Biological evaluation of sphingomyelin analogues as inhibitors of sphingomyelinase.Bioorg. Med. Chem. Lett. 2003; 13: 3681-3684Crossref PubMed Scopus (25) Google Scholar), as well as analogues of naturally occurring inhibitors, such as α-mangostin, a natural product derived from Garcinia mangostana, have been developed (23Hamada M. Iikubo K. Ishikawa Y. Ikeda A. Umezawa K. Nishiyama S. Biological activities of alpha-mangostin derivatives against acidic sphingomyelinase.Bioorg. Med. Chem. Lett. 2003; 13: 3151-3153Crossref PubMed Scopus (33) Google Scholar). In addition, the lipid phosphatidylinositol-3,5-bisphosphate has been shown to inhibit ASM activity (24Kolzer M. Arenz C. Ferlinz K. Werth N. Schulze H. Klingenstein R. Sandhoff K. Phosphatidylinositol-3,5-bisphosphate is a potent and selective inhibitor of acid sphingomyelinase.Biol. Chem. 2003; 384: 1293-1298Crossref PubMed Scopus (59) Google Scholar). Such sphingomyelinase inhibitors may be useful for a wide range of applications. For example, sphingomyelinase inhibitors may potentially be used to alter the growth of cancer cells, to modulate the effects of inflammation, or, in the case of ASM inhibitors, as molecular chaperones for the treatment of types A and B NPD (25Desnick R.J. Schuchman E.H. Enzyme replacement and enhancement therapies: lessons from lysosomal disorders.Nat. Rev. Genet. 2002; 3: 954-966Crossref PubMed Scopus (236) Google Scholar, 26Cohen F.E. Kelly J.W. Therapeutic approaches to protein-misfolding diseases.Nature. 2003; 426: 905-909Crossref PubMed Scopus (724) Google Scholar). This latter approach was pioneered by the work of Fan and colleagues for the treatment of another lysosomal storage disorder, Fabry disease (27Fan J.Q. Ishii S. Asano N. Suzuki Y. Accelerated transport and maturation of lysosomal alpha-galactosidase A in Fabry lymphoblasts by an enzyme inhibitor.Nat. Med. 1999; 5: 112-115Crossref PubMed Scopus (571) Google Scholar, 28Asano N. Ishii S. Kizu H. Ikeda K. Yasuda K. Kato A. Martin O.R. Fan J.Q. In vitro inhibition and intracellular enhancement of lysosomal alpha-galactosidase A activity in Fabry lymphoblasts by 1-deoxygalactonojirimycin and its derivatives.Eur. J. Biochem. 2000; 267: 4179-4186Crossref PubMed Scopus (215) Google Scholar). Although many sphingomyelinase inhibitors are known, the broader effects these compounds have on sphingomyelin metabolism in living cells remain mostly unknown. Here, we have developed a new sphingomyelin analogue (designated AD2765) and extensively characterized it in vitro and in living cells. We reveal that this compound has broad effects on sphingomyelin metabolism, including the inhibition of ASM and Mg2+-dependent neutral sphingomyelinase activities, as well as on the inhibition of sphingomyelin synthesis. There were no effects on ceramide hydrolysis or on the synthesis of glucosylceramide. In addition, treatment of cells with AD2765 at concentrations > 10 μM led to an increase in cellular ceramide and cell death. The potential therapeutic applications of this compound are discussed. Human skin fibroblasts were maintained in DMEM supplemented with 10% (v/v) FBS, 1% (v/v) penicillin-streptomycin, and l-glutamine (Invitrogen, Carlsbad, CA). Human lymphoblasts and Jurkat lymphoma cells were maintained in RPMI 1640 (Cellgro, Herndon, VA) supplemented with 10% heat-inactivated FBS and 1% (v/v) penicillin-streptomycin (Invitrogen). M059J Glioma cells (American Tissue Culture Collection) were maintained in DMEM/Ham's F12 (1:1, v/v) supplemented with 10% FBS, 15 mM HEPES, and 1% penicillin-streptomycin (Invitrogen). HL-60 cells were grown in DMEM supplemented with 10% FBS, 15 mM HEPES, and 1% penicillin-streptomycin (Invitrogen). Twenty milligrams of sphingosylphosphorylcholine was dissolved in 5.0 ml of 0.5 N sodium bicarbonate in a 10 ml Erlenmeyer flask. To the stirred solution was added 20 mg of tert-butyl-isothiocyanate. The solution was left to stir for 48 h and transferred to a small separatory funnel, and 8.0 ml of dichloromethane, 4.0 ml of methanol, and 3.0 ml of water were added. The funnel was vigorously shaken, and the phases were then left to separate. The organic phase was washed with 4.0 ml of water, then dried over MgSO4, filtered, and evaporated to dryness. The product was purified by preparative TLC using dichloromethane-methanol-water (65:35:5) for development, identified by an ultraviolet lamp, scraped, and eluted with dichloromethane-methanol-water (1:2:1) in a small column. The yield of product from this reaction was ∼80%, and the purity as judged by TLC was >95%. A fluorescent analogue of AD2765 was synthesized as described above except that FITC was used instead of the tert-butyl-isothiocyanate. The product was similarly purified. ASM activity was assessed in vitro using fluorescent and radioactive sphingomyelin derivatives. Fluorescence-based assays were performed as described previously (29He X. Chen F. Dagan A. Gatt S. Schuchman E.H. A fluorescence-based, high-performance liquid chromatographic assay to determine acid sphingomyelinase activity and diagnose types A and B Niemann-Pick disease.Anal. Biochem. 2003; 314: 116-120Crossref PubMed Scopus (45) Google Scholar). Briefly, equal volumes of cell extracts (prepared by three cycles of freeze-thaw in 10 mM Tris-HCl, pH 7.0, containing 0.2% Triton X-100) or purified enzyme solution (in 20 mM Tris-HCl, pH 7.0, containing 0.2% BSA) were mixed with 200 μM BODIPY®-labeled C12-sphingomyelin (B12SM; Molecular Probes, Eugene, OR) diluted in assay buffer (0.1 M sodium acetate, pH 5.0, containing 0.1 mM ZnCl2 and 0.2% Igepal CA-630). The reaction mixtures were then incubated at 37°C for 60 min and terminated by the addition of ethanol, and the hydrolytic product [BODIPY®-labeled C12-ceramide (B12CER)] was detected and quantified by HPLC analysis using a reversed-phase column (Aquasil C-18; Keystone Scientific, Inc., St. Marys, PA). The radioactive assay system was as described above with the following modifications. B12SM was replaced with 150,000 dpm of [14C]sphingomyelin (Amersham Biosciences, Piscataway, NJ), and 0.6% Triton X-100 was used in the assay buffer in place of Igepal CA-630. The reaction was subsequently terminated after a 2 h incubation at 37°C by the addition of 50 μl of chloroform-methanol (1:1, v/v) and 22.5 μl of water. Samples were then thoroughly mixed and centrifuged for 2 min at 2,000 g to achieve a phase split. The upper aqueous phase containing the [14C]phosphocholine reaction product was then removed for scintillation counting with an ISODATA γ counter (Polymedco, Inc.). The activity of Mg2+-dependent neutral sphingomyelinase was assessed in vitro using the fluorescence-based HPLC method described above for the ASM assay except that the assay buffer used contained 200 mM Tris-HCl (pH 7.4), 5 mM magnesium chloride, and 0.6% Triton X-100. Acid ceramidase activity was assessed in vitro essentially as described previously (30He X. Li C.M. Park J.H. Dagan A. Gatt S. Schuchman E.H. A fluorescence-based high-performance liquid chromatographic assay to determine acid ceramidase activity.Anal. Biochem. 1999; 274: 264-269Crossref PubMed Scopus (29) Google Scholar). Briefly, the standard 10 μl reaction mixture consisted of 5 μl of enzyme source (prepared by three cycles of freeze-thaw in 0.25 M sucrose) plus 5 μl of 0.2 M citrate/phosphate buffer (pH 4.5) containing 200 μM B12CER, 300 mM NaCl, 0.1% BSA, and 0.2% Igepal CA-630. Assays were carried out at 37°C for 60 min. The reaction was terminated by the addition of ethanol, and the fluorescent fatty acid product was detected and quantified by chromatographic analysis using a reversed-phase column (BetaBasic-18, 4.6 × 30 mm; Keystone Scientific, Inc., Bellefonte, PA). Jurkat lymphoma or HL-60 cells (2 × 106) were dispersed in 1 ml of the following homogenization buffer: 50 mM Tris-HCl, pH 7.4, 50 mM NaCl, and 5 mM EDTA. The tubes were kept in liquid nitrogen for 4 min and then in a 37°C water bath for 3 min. This freezing and thawing was repeated two more times. For experiments testing the effects of AD2765, varying concentrations of the compound (dissolved in 0.1% DMSO) were added, and the tubes were preincubated for 5 min at 37°C. BODIPY-C3 or -C12 ceramide (as a solution in 0.1% DMSO) was then added, and incubation at 37°C was continued for 30 min. Two milliliters of dichloromethane-methanol (1:1) was added to stop the reactions, the tubes were vortexed and centrifuged, and the upper phase was removed. The dichloromethane was evaporated under a stream of air, and the residue was dissolved in 50 μl of dichloromethane-methanol (1:1) and applied to a thin-layer chromatography plate. Standards of BODIPY-C3 or -C12 sphingomyelin and BODIPY-C3 or -C12 glucosylceramide were also applied. The plate was developed in dichloromethane-methanol-water (90:10:1), and the fluorescence of the respective sphingomyelin and glucosylceramide products was quantified using a Fuji FLA-2000 fluorometer. Protein levels were quantified with the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA) using BSA as a standard. Pulse/chase labeling of human skin fibroblasts was accomplished as described previously (31Erlich S. Miranda S.R. Visser J.W. Dagan A. Gatt S. Schuchman E.H. Fluorescence-based selection of gene-corrected hematopoietic stem and progenitor cells from acid sphingomyelinase-deficient mice: implications for Niemann-Pick disease gene therapy and the development of improved stem cell gene transfer procedures.Blood. 1999; 93: 80-86Crossref PubMed Google Scholar). Briefly, B12SM was incorporated into liposomes by mixing with dioleoyl phosphatidylcholine (DOPC; Sigma, St. Louis, MO) at a molar ratio of 1:4. The solvent was evaporated and the lipid mixture was resuspended in PBS, followed by sonication. Human skin fibroblasts were incubated with B12SM/DOPC liposomes (final concentration of 2–3 nmol/ml) at 37°C for 5 h. Labeling was terminated by removal and replacement of the medium with fresh growth medium lacking B12SM/DOPC liposomes. The cells were then incubated (i.e., “chased”) for a further 24–48 h before processing. After pulse/chase labeling, the fibroblasts were harvested by trypsinization and centrifuged at 2,000 g, and the pellets were resuspended in phosphate-buffered saline. Aliquots of cells were taken for protein determination, and the remaining sample was processed as follows. Cells were centrifuged at 2,000 g, and the cell pellet was resuspended in 150 μl of chloroform-methanol (1:2, v/v). The resulting cell suspension was then sonicated for 5 min, followed by incubation at 55°C for 10 min and a further 2 min sonication. The solvent was evaporated, and the dried lipid extract was resuspended in 50 μl of ethanol. The hydrolytic product, B12CER, was then detected and quantified by HPLC analysis using a reversed-phase column (Aquasil C-18; Keystone Scientific, Inc.) as described above for the ASM activity assays. To assess sphingomyelin and glucosylceramide synthesis in situ, the methods described by Dagan et al. (32Dagan A. Wang C. Fibach E. Gatt S. Synthetic, non-natural sphingolipid analogs inhibit the biosynthesis of sphingolipids, elevate ceramide and induce apoptotic cell death.Biochim. Biophys. Acta. 2003; 1633: 161-169Crossref PubMed Scopus (53) Google Scholar) were used. A 2 ml suspension of Jurkat lymphoma cells (0.75 × 106/ml) was incubated with or without AD2765 for 1 h in medium lacking fetal calf serum. B12CER (2.5 μM) was then added, and the incubation continued for an additional 3 h. Cells were sedimented by centrifugation and washed twice with PBS. To the cell pellet was added 1 ml of dichloromethane-ethanol (1:2), and after vortexing and centrifugation, the liquid supernatant was collected. One milliliter of dichloromethane-ethanol (1:1) was added to the residual pellet, which was then vortexed and recentrifuged, and the solvent was evaporated under a stream of air. The residue was dissolved in dichloromethane-methanol (1:1) and applied to a thin-layer chromatography silica gel plate. The latter was developed in dichloromethane-methanol-water (85:15:1.5), and the fluorescence of the B12SM and B12 glucosylceramide products was identified by comigration with standards and quantified using a Fuji FLA-2000 scanner. The total ceramide content in cells was determined by a previously published method (33He X. Dagan A. Gatt S. Schuchman E.H. Simultaneous, quantitative analysis of ceramide and sphingosine in mouse blood by naphthalene 2,3-dicarboxyaldehyde derivatization after hydrolysis with ceramidase.Anal. Biochem. 2005; 340: 113-122Crossref PubMed Scopus (55) Google Scholar). Briefly, a total lipid extract was prepared using organic solvents, air-dried, and resuspended in the nonionic detergent 0.2% Igepal CA-630. The ceramide present in these lipid extracts was then fully hydrolyzed into sphingosine using recombinant acid ceramidase, and the sphingosine was derivatized with naphthalene-2,3-dialdehyde and quantified by reverse-phase high-performance liquid chromatography. A fluorescent analogue of AD2765, FITC-AD2765, was added to the medium of normal skin fibroblasts grown on glass chamber slides at a concentration of 0.25 μM for 2 h. Cells were then fixed in 4% paraformaldehyde for 25 min at 4°C before three 5 min washes with PBS. Cells were then either immediately mounted in Vectashield® mounting medium with 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA) and analyzed or processed further for lysosomal-associated membrane protein-2 (LAMP-2) colocalization. To minimize the nonspecific reactivity of the anti-LAMP-2 antibodies, the fixed cells were blocked for 2 h at room temperature in PBS containing 10% normal goat serum and 0.1% Tween 20. After a brief rinse with PBS, cells were incubated with LAMP-2-specific primary antibody (purified mouse anti-human CD107B monoclonal antibody; BD Biosciences Pharmingen, San Diego, CA) for 2 h at room temperature at a concentration of 1.25 μg/ml in PBS containing 5% normal goat serum and 0.1% Tween 20. The slides were then washed twice for 5 min each in PBS containing 0.1% Tween 20, followed by two 5 min washes in PBS, before incubation for 1 h at room temperature with secondary antibody conjugated with Alexa Fluor 594 (Molecular Probes) at a concentration of 10 μg/ml. Slides were then washed three times for 5 min each in PBS containing 0.1% Tween 20, followed by two 5 min washes in PBS, before mounting as described above and analyzed. Cell proliferation and viability were assessed through cell counting and trypan blue staining. Briefly, a small sample of a cell suspension was diluted 1:5 in 0.4% (w/v) trypan blue. The resulting suspension was applied to a hemocytometer, and cells were counted according to the manufacturer's instructions. Cell viability was assessed as the percentage of cells incorporating the trypan blue dye. A novel thiourea derivative of sphingomyelin (Fig. 1B)was synthesized, and its ability to inhibit the hydrolysis of B12SM by recombinant human ASM was tested in vitro. As illustrated by the Dixon plots in Fig. 2A, this compound, designated AD2765, inhibited this reaction with an apparent inhibition constant (Ki) of ∼70 μM. These plots also suggested that under these nonphysiological conditions, the inhibition of ASM was noncompetitive. Studies performed using 14C-labeled sphingomyelin as the substrate confirmed the inhibitory effect of AD2765 on the activity of pure ASM in vitro (data not shown).Fig. 2A: Dixon plots showing the inhibition of human recombinant acid sphingomyelinase (ASM) activity by AD2765. All reactions contained 0.256 ng of purified ASM and varying concentrations of AD2765. Curves were generated at three different substrate [BODIPY®-labeled C12-sphingomyelin (B12SM)] concentrations (ranging from 0.01 to 0.1 mM). In vitro assays were performed as described in Materials and Methods. B: Inhibition of ASM activity in cell extracts. Normal human skin fibroblasts were incubated in the presence of AD2765 at the indicated concentrations for 48 h before harvesting and measurement of ASM activity in vitro. All reactions contained 1.5–3 μg of human fibroblast cell extract protein and 100 μM substrate. Data are presented as means ± SD (n = 3). * P < 0.05 by Student's t-test. Ki, inhibition constant.View Large Image Figure ViewerDownload (PPT) We next incubated normal human skin fibroblasts with varying amounts of AD2765 for 48 h, washed the cells extensively, and then used cell extracts to determine ASM activity. As shown in Fig. 2B, significant inhibition of ASM activity was observed in the cell extracts, revealing that AD2765 had entered the cells and was present in the extracts. Because cells incubated with only 2 μM AD2765 for 48 h had a 50% inhibition of ASM activity, and the apparent Ki of this compound using the pure enzyme was ∼70 μM (Fig. 2A), this suggested that AD2765 was concentrating in the cells over the 48 h incubation period. Similar experiments also were performed to determine the effects of AD2765 on the activities of Mg2+-dependent neutral sphingomyelinase and acid ceramidase. As shown in Fig. 3, the activity of Mg2+-dependent neutral sphingomyelinase (Fig. 3A), but not acid ceramidase (Fig. 3B), was significantly inhibited by this compound. We next carried out experiments to evaluate the ability of AD2765 to inhibit the hydrolysis of B12SM in situ. A pulse/chase experiment was conducted whereby the cells were pulsed with B12SM to allow the accumulation of the fluorescent substrate within the cells, followed by a 48 h chase period in which the medium was changed to medium lacking B12SM. Quantification of the product (B12CER) formed in situ was determined by extraction of total lipids and analysis using the HPLC-based detection method described (33He X. Dagan A. Gatt S. Schuchman E.H. Simultaneous, quantitative analysis of ceramide and sphingosine in mouse blood by naphthalene 2,3-dicarboxyaldehyde derivatization after hydrolysis with ceramidase.Anal. Biochem. 2005; 340: 113-122Crossref PubMed Scopus (55) Google Scholar). As shown in Fig. 4, when 10 μM AD2765 was included in the chase medium, the amount of B12CER formed was reduced substantially, confirming the ability of this compound to enter cells and inhibit sphingomyelinase activity in situ. Because the experiment described above monitored the hydrolysis of B12SM only, and previous studies have indicated that this fluorescent lipid is trafficked principally to lysosomes (34Pagano R.E. Watanabe R. Wheatley C. Chen C.S. Use of N-[5-(5,7-dimethyl boron dipyrromethene difluoride]-sphingomyelin to study membrane traffic along the endocytic pathway.Chem. Phys. Lipids. 1999; 102: 55-63Crossref PubMed Scopus (17) Google Scholar), the fact that AD2765 could inhibit this reaction suggested that this compound was delivered, at least in part, to this compartment. To evaluate this further, a FITC derivative of sphingomyelin was prepared, and its uptake and localization in cells were examined by fluorescence microscopy (Fig. 5). Although the FITC derivative was not chemically identical to AD2765 (Fig. 1), the results revealed the rapid uptake of this compound and delivery to LAMP-2-positive compartments (i.e., lysosomes and/or late endosomes), consistent with the cell experiments described above. It is important to note that because of the high fluorescence of the FITC compound, low concentrations (0.25 μM) were used for the microscopy experiments. It is possible that at higher concentrations the compound might be delivered to additional compartments, but this could not be confirmed. To further investigate the effects of AD2765, we next performed an experiment in which normal skin fibroblasts (data not shown) and Jurkat lymphoma cells were incubated with BODIPY-conjugated C3 ceramide, and the ability to form BODIPY-C3 sphingomyelin was evaluated after 4 h. Surprisingly, as shown in Fig. 6A, this synthetic reaction also was inhibited when 10 μM AD2765 was included in the chase medium. In fact, even low concentrations (2 μM) of AD2765 inhibited the formation of sphingomyelin by ∼70% (data not sho" @default.
• W2107082487 created "2016-06-24" @default.
• W2107082487 creator A5011401228 @default.
• W2107082487 creator A5014981769 @default.
• W2107082487 creator A5023868452 @default.
• W2107082487 creator A5034341624 @default.
• W2107082487 creator A5060988714 @default.
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• W2107082487 date "2005-11-01" @default.
• W2107082487 modified "2023-10-10" @default.
• W2107082487 title "A lipid analogue that inhibits sphingomyelin hydrolysis and synthesis, increases ceramide, and leads to cell death" @default.
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