Neutral Sphingomyelinase Inhibitor Research Articles

Enantiocontrolled synthesis of the epoxycyclohexenone moieties of scyphostatin, a potent and specific inhibitor of neutral sphingomyelinase

The epoxycyclohexenone moieties 2 and 3b of scyphostatin ( 1 ), a potent and specific inhibitor of neutral sphingomyelinase, were synthesized in enantiomerically pure forms starting from (−)-quinic acid ( 11 ). The synthetic method features (i) the preparation of the olefin masked enones 25 and 29 , the precursors for the key aldol-type coupling reaction, (ii) the efficient and stereocontrolled aldol-type coupling reactions between 25 (or 29 ) and benzaldehyde ( 8 ) and Garner's aldehyde analogue 9 to deliver alcohols 23 and 24 , respectively, both of which possess the requisite asymmetric quaternary carbon center at the C6 position, and (iii) the stereospecific S N2-type epoxide ring formation of the mesylates 35 and 47 under mild basic conditions to produce the targeted compounds 2 and 3b , respectively.

Endothelial apoptosis induced by inhibition of integrins αvβ3 and αvβ5 involves ceramide metabolic pathways

AbstractMatrix ligation of integrins αvβ3/αvβ5 is critical for endothelial survival and angiogenesis. We have previously shown that ceramide, a proapoptotic lipid second messenger, increases during endothelial anoikis (detachment-induced apoptosis). We now show that RGDfV, an integrin αvβ3/αvβ5 cyclic function-blocking peptide, increased ceramide and decreased sphingomyelin in human brain microvascular endothelial cells (HBMECs) plated on vitronectin, suggesting that sphingomyelin hydrolysis contributes to RGDfV-induced ceramide increase. Desipramine and imipramine, inhibitors of acid sphingomyelinase (ASMase), suppressed RGDfV-induced ceramide increase. Importantly, desipramine, imipramine, and a third ASMase inhibitor, SR33557, but not inhibitors of neutral sphingomyelinase, suppressed RGDfV-induced apoptosis, suggesting that ASMase was required for integrin-mediated apoptosis. Myriocin, an inhibitor of de novo ceramide synthesis, had no effect on RGDfV-induced HBMEC apoptosis. Interestingly, ASMase inhibitors also suppressed the RGDfV-induced loss of spreading on vitronectin. RGDfV induced a similar increase in ceramide and apoptosis in HBMECs on poly-l-lysine or vitronectin, although cells detached only from vitronectin, indicating that cell detachment was not required for RGDfV-induced apoptosis. Our results suggest involvement of ASMase and ceramide in endothelial apoptosis induced by inhibition of integrins αvβ3/αvβ5, and propose a novel molecular mechanism for the antiangiogenic effect of RGDfV.

Synthesis and Antiapoptotic Activity of a Novel Analogue of the Neutral Sphingomyelinase Inhibitor Scyphostatin

The enantioselective synthesis of an analogue of scyphostatin, a potent inhibitor of the neutral sphingomyelinase, is described. The synthesis starts with cyclohexanone and a protected D-serine derivative. The key step is an asymmetric hydroxylation to access a hydroxycyclohexanone, which is transformed into a substituted hydroxycyclohexenone. This is converted into the scyphostatin analogue 14, a chemically and metabolically stabilised compound lacking the epoxy function of the natural congener and carrying a palmitic acid group instead of the native trienoyl residue. An evaluation of the biological activity of 14 revealed neutral sphingomyelinase inhibition in several in vivo test systems (monocytes, macrophages, hepatocytes) monitoring antiapoptotic effects and the inversion of phorbolester-induced translocation of green fluorescent protein labelled kinase (protein kinase C-alpha).

Stimulation of CD95-induced apoptosis in T-cells by a subtype specific neutral sphingomyelinase inhibitor

Neutral sphingomyelinase (nSMase) has been supposed to be involved in the activation of anti-apoptotic genes and, thus, could well sustain autoimmune reactions by preventing activation induced death of autoreactive T-cells. When screening cellular extracts for SMase activity in the range between pH 6.5 and 8.5 various murine tissue samples as well as cell lines of murine and human origin displayed peaks of activity, both, at pH 7.0 and 8.0. In contrast, T-cells (human T-cell lymphoma and PHA stimulated murine lymph node cells) and monocytic leukemia cells were lacking SMase activity at pH 8.0. Only one peak of activity was found at pH 7.0. Recently we described an inhibitory compound, C11AG which selectively suppresses nSMase activity. In dose–response assays using cellular extracts the pH 7.0 nSMase turned out to be almost 100-fold more sensitive to the inhibitor than the pH 8.0 nSMase. In Jurkat T-cell lymphoma cells lacking the pH 8.0 nSMase, treatment with C11AG enhanced sensitivity to apoptosis: the concentration of CD95-specific antibody anti-APO1 could be lowered by six-fold in order to induce cell death. Concomitantly the expression of the anti-apoptotic protein A1 was found to be down-regulated. In the joints of arthritic mice, apoptosis of T-cells was stimulated after application of C11AG. Accordingly, C11AG displayed curative effects on experimental arthritis: swelling and inflammation were found to be significantly alleviated.

Total Synthesis of (+)‐Scyphostatin (I), a Potent and Specific Inhibitor of Neutral Sphingomyelinase.

Total Synthesis of (+)‐Scyphostatin (I), a Potent and Specific Inhibitor of Neutral Sphingomyelinase.

Total synthesis of (+)-scyphostatin, a potent and specific inhibitor of neutral sphingomyelinase.

Total synthesis of (+)-scyphostatin, a potent and specific inhibitor of neutral sphingomyelinase.

Total Synthesis of (+)‐Scyphostatin, a Potent and Specific Inhibitor of Neutral Sphingomyelinase

Total Synthesis of (+)‐Scyphostatin, a Potent and Specific Inhibitor of Neutral Sphingomyelinase

Inflammatory mediator and β-amyloid (25–35)-induced ceramide generation and iNOS expression are inhibited by vitamin E

To investigate the putative role of β-amyloid peptide (Aβ) in inducing oxidative stress damage in Alzheimer disease (AD), we studied the effects of proinflammatory cytokines and Aβ peptide on the induction of inducible nitric oxide synthase (iNOS). Aβ(25–35) upregulated the cytokine (TNF-α/IL-1 β)-induced expression of iNOS and the production of nitric oxide (NO) in astrocytes, which were inhibited by vitamin E. Aβ treatment of C6 glial cells (together with LPS and IFN-γ), in addition to inducing iNOS, enhanced the oxidative stress as measured by increased expression of manganese superoxide dismutase and an increase in 2,7′-dichlorofluorescein diacetate fluorescence. We also observed that LPS, IFN-γ, and Aβ(25–35) treatment led to the activation of the sphingomyelin–ceramide (SM-Cer) cascade with an increase in cellular ceramide. Inhibition of the SM-Cer cascade either by vitamin E treatment or by the neutral sphingomyelinase inhibitor 3-O-methyl sphingomyelin also resulted in alteration of the transcriptional binding activities of C/EBP, NFκB, AP-1, and CREB in C6 glial cells. Hence, these findings suggest a role for ceramide in iNOS induction and NO production in Aβ-induced AD pathobiology and provide a possible explanation for the beneficial effects of vitamin E therapy.

Neutral sphingomyelinase inhibitor scyphostatin prevents and ceramide mimics mechanotransduction in vascular endothelium.

Recently, we showed that neutral sphingomyelinase (N-SMase) is concentrated at the endothelial cell surface in caveolae and is activated to produce ceramide in an acute and transient manner by increase in flow rate and pressure in rat lung vasculature (Czarny M, Liu J, Oh P, and Schnitzer JE, J Biol Chem 278: 4424-4430, 2003). Here, we report further on our investigations of this new acute mechanotransduction pathway. We employed three experimental models to explore the role of N-SMase and ceramides in mechanosignaling: 1) a cell-free, in vitro model using isolated luminal plasma membranes of rat lung endothelium; 2) a fluid shear stress model using monolayers of intact bovine aorta endothelial cell in culture; and 3) an in situ model using controlled perfusion of the rat lung vasculature. Scyphostatin, which specifically inhibited N-SMase but not acid SMase activity, prevented mechanoactivation of N-SMase as well as downstream tyrosine and mitogen-activated protein kinases. Cell-permeable ceramide analogs (N-acetylsphingosine, C2-ceramide, and N-hexanoylsphingosine, C6-ceramide) but not the inactive dihydroderivatives D2-ceramide and D6-ceramide (N-acetylsphinganine and N-hexanoylsphinganine, respectively) mimic rapid mechano-induced tyrosine phosphorylation of cell surface proteins as well as mechanoactivation of Src-like kinases and the extracellular regulated kinase pathway. The responses common to ceramide and mechanical stress were inhibited by genistein, herbamycin A, and PP2, but not PP3, which suggests an obligate role of Src-like kinases in ceramide-mediated mechanotransduction. Ceramides also induced serine/threonine phosphorylation to activate the Akt/endothelial nitric oxide synthase pathway. Thus N-SMase at the plasma membrane in caveolae may be an upstream initiating mechanosensor, which acutely triggers mechanotransduction by generation of the lipid second messenger ceramide.

Inflammatory mediator and $beta;-amyloid (25?35)-induced ceramide generation and iNOS expression are inhibited by vitamin E

To investigate the putative role of β-amyloid peptide (Aβ) in inducing oxidative stress damage in Alzheimer disease (AD), we studied the effects of proinflammatory cytokines and Aβ peptide on the induction of inducible nitric oxide synthase (iNOS). Aβ(25–35) upregulated the cytokine (TNF-α/IL-1 β)-induced expression of iNOS and the production of nitric oxide (NO) in astrocytes, which were inhibited by vitamin E. Aβ treatment of C6 glial cells (together with LPS and IFN-γ), in addition to inducing iNOS, enhanced the oxidative stress as measured by increased expression of manganese superoxide dismutase and an increase in 2,7′-dichlorofluorescein diacetate fluorescence. We also observed that LPS, IFN-γ, and Aβ(25–35) treatment led to the activation of the sphingomyelin–ceramide (SM-Cer) cascade with an increase in cellular ceramide. Inhibition of the SM-Cer cascade either by vitamin E treatment or by the neutral sphingomyelinase inhibitor 3-O-methyl sphingomyelin also resulted in alteration of the transcriptional binding activities of C/EBP, NFκB, AP-1, and CREB in C6 glial cells. Hence, these findings suggest a role for ceramide in iNOS induction and NO production in Aβ-induced AD pathobiology and provide a possible explanation for the beneficial effects of vitamin E therapy.

Total Synthesis of (+)‐Macquarimicin A.

AbstractFor Abstract see ChemInform Abstract in Full Text.

The role of neutral sphingomyelinase produced ceramide in lipopolysaccharide-mediated expression of inducible nitric oxide synthase.

Lipopolysaccharide (LPS) and interferon-gamma (IFN) treatment of C6 rat glioma cells increased the intracellular ceramide level and the expression of the inducible nitric oxide synthase (iNOS) gene. To delineate the possible role of ceramide in the induction of iNOS, we examined the source of intracellular ceramide and associated signal transduction pathway(s) with the use of inhibitors of intracellular ceramide generation. The inhibitor of neutral sphingomyelinase (3-O-methylsphingomyelin, MSM) inhibited the induction of iNOS, whereas inhibitor of acidic sphingomyelinase (SR33557) or that of ceramide de novo synthesis (fumonisin B1) had no effect on the induction of iNOS. MSM-mediated inhibition of iNOS induction was reversed by the supplementation of exogenous C8-ceramide, suggesting that ceramide production by neutral sphingomyelinase (nSMase) is a key mediator in the induction of iNOS. The MSM-mediated inhibition of iNOS gene expression correlated with the decrease in the activity of ras. Inhibition of co-transfected iNOS promoter activity by dominant negative ras supported the role of ras in the nSMase-dependent regulation of iNOS gene. NF-kappaB DNA binding activity and its transactivity were also reduced by MSM pretreatment, and were completely reversed by the supplementation of C8-ceramide. As the dominant negative ras also reduced NF-kappaB transactivity, NF-kappaB activation may be downstream of ras. Our results suggest that ceramide generated by nSMase may be a critical mediator in the regulation of iNOS gene expression via ras-mediated NF-kappaB activation under inflammatory conditions.

Ceramide and glutathione define two independently regulated pathways of cell death initiated by p53 in Molt-4 leukaemia cells.

The tumour suppressor p53 induces cell death by launching several pathways that are either dependent on or independent of gene transcription. Accumulation of the sphingolipid ceramide and reactive oxygen species are among these pathways. Crossregulation of these two pathways is possible owing to the demonstrated inhibition of neutral sphingomyelinase by glutathione, the predominant cellular antioxidant, and has been observed in some cytokine-dependent cell-death models. In a model of irradiation-induced cell death of Molt-4 leukaemia cells, it was found that ceramide accumulation and glutathione depletion were dependent on p53 up-regulation. The loss of p53 owing to expression of the papilloma virus E6 protein inhibited both pathways after irradiation. However, in this model, these two pathways appeared to be independently regulated on the basis of the following observations: (1) glutathione supplementation or depletion did not alter irradiation-induced ceramide accumulation, (2) exogenous ceramide treatment did not induce glutathione depletion, (3) glutathione depletion was dependent on new protein synthesis, whereas ceramide accumulation was independent of it and (4) caspase activation was required for ceramide accumulation but not for glutathione depletion. Furthermore, caspase 9 activation, which is dependent on the release of mitochondrial cytochrome c, was not required for ceramide accumulation. This suggested that a caspase, other than caspase 9, was necessary for ceramide accumulation. Interestingly, Bcl-2 expression inhibited these pathways, indicating a possible role for mitochondria in regulating both pathways. These findings indicate that these two pathways exhibit cross-regulation in cytokine-dependent, but not in p53-dependent, cell-death models.

Total synthesis of (+)-macquarimicin A.

The first total synthesis of (+)-macquarimicin A (1), a novel inhibitor of neutral sphingomyelinase (N-SMase) with antiinflammatory activity, has been accomplished. The present work determined the absolute configuration of (+)-1 and revised the C(2)-C(3) geometry to be Z. The synthesis features a transannular Diels-Alder reaction, which constructed the tetracyclic framework stereoselectively, and a convergent and efficient synthetic pathway, which afforded (+)-macquarimicin A (1) in 27 steps (longest linear sequence) with 9.9% overall yield.

Synthesis and evaluation of three novel scyphostatin analogues as neutral sphingomyelinase inhibitors.

The development of new therapies for various inflammatory and autoimmune diseases could be aided by the efficient synthesis and study of three novel analogues of scyphostatin (1), an inhibitor of sphingomyelinases. Unlike previous analogues, compounds such as 2 lack the epoxy moiety of scyphostatin. This could help resolve the contribution of the various reactive functionalities of the inhibitors to the observed biological activity.

Ceramide is involved in r(+)-methanandamide-induced cyclooxygenase-2 expression in human neuroglioma cells.

Cannabinoids have recently been shown to induce the expression of the cyclooxygenase-2 (COX-2) isoenzyme in H4 human neuroglioma cells. Using this cell line, the present study investigates the contribution of the second messenger ceramide to this signaling pathway. Incubation of cells with the endocannabinoid analog R(+)-methanandamide (R(+)-MA) was associated with an increase of intracellular ceramide levels. Enhancement of ceramide formation by R(+)-MA was abolished by fumonisin B1, a ceramide synthase inhibitor, whereas inhibitors of neutral sphingomyelinase (spiroepoxide, glutathione) and serine palmitoyltransferase (l-cycloserine, ISP-1) were inactive in this respect. R(+)-MA caused a biphasic activation of the p38 and p42/44 mitogen-activated protein kinases (MAPKs), with phosphorylation peaks occurring after 15-min and 4- to 8-h treatments, respectively. Inhibition of ceramide synthesis with fumonisin B1 was associated with a suppression of R(+)-MA-induced delayed phosphorylations of p38 and p42/44 MAPKs and subsequent COX-2 expression. The involvement of ceramide in COX-2 expression was corroborated by findings showing that C2-ceramide and neutral sphingomyelinase from Bacillus cereus caused concentration-dependent increases of COX-2 expression that were suppressed in the presence of 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)imidazol (SB203580, a p38 MAPK inhibitor) or 2'-amino-3'-methoxyflavone (PD98059, a p42/44 MAPK activation inhibitor). In contrast, dihydro-C2-ceramide being used as a negative control did not induce MAPK phosphorylation and COX-2 expression. Collectively, our results demonstrate that R(+)-MA induces COX-2 expression in human neuroglioma cells via synthesis of ceramide and subsequent activation of p38 and p42/44 MAPK pathways. Induction of COX-2 expression via ceramide represents a hitherto unknown mechanism by which cannabinoids mediate biological effects within the central nervous system.

Biological evaluation of sphingomyelin analogues as inhibitors of sphingomyelinase.

Seeking neutral sphingomyelinase inhibitors, we designed and synthesized hydrolytically stable analogues of sphingomyelin. These novel analogues replace the phosphodiester moiety of sphingomyelin with carbamate and urea moiety, resulting in inhibition of neutral sphingomyelinase. Compound 1 prevented ceramide generation and apoptotic neuronal cell death in a model of ischemia based on organotypic hippocampal slice cultures.

Sialic acids linked to glycoconjugates of Fas regulate the caspase-9-dependent and mitochondria-mediated pathway of Fas-induced apoptosis in Jurkat T cell lymphoma

To clarify the functions of sialic acids linked to glycoconjugates of Fas in Fas-induced apoptosis, Jurkat T cells, untreated and treated with neuraminidase, were incubated with anti-Fas monoclonal antibody, CH11. Apoptosis of Jurkat T cells induced by incubation with CH11 was enhanced by the pre-treatment with neuraminidase. By flow cytometry sialylated glycoconjugates were detected on the cell surface of Jurkat T cells using LFA lectin, which specifically reacts with sialic acid, and pre-treatment with Vibrio Cholerae neuraminidase resulted in desialylation of Jurkat cell surface glycoconjugates. The enhancement of Fas-induced apoptosis by pre-treatment with neuraminidase was inhibited by z-VAD-fmk, a broad caspase inhibitor, and Ac-LEHD-CHO, an inhibitor of caspase-9, but not by Ac-IETD-CHO an inhibitor of caspase-8 or 6, imipramine, an inhibitor of acidic sphingomyelinase, glutathione, an inhibitor of neutral sphingomyelinase and Fumonisin B1, an inhibitor of ceramide synthase. Mitochondrial membrane potentials (Deltapsim) measured with a Mitocapture assay kit demonstrated that the loss of Deltapsim involved in Fas-induced apoptosis was enhanced by pre-treatment with neuraminidase. Furthermore, Western blot analysis using polyclonal antibody (C-20) against Fas detected Fas at about 45 kDa, and pre-treatment with neuraminidase resulted in a reduction of the molecular weight of Fas of about 8 kDa. These data suggest that the enhancement of Fas-induced apoptosis by pre-treatment with neuraminidase was mediated by a caspase-9 dependent pathway closely associated with the loss of Deltapsim, not by activation of caspase-8, -6 or acidic and neutral sphingomyelinases, and that sialic acid linked to glycoconjugates of Fas may regulate Fas-induced apoptosis in human T cell lymphoma.

Sphingomyelin analogues as inhibitors of sphingomyelinase.

To search for neutral sphingomyelinase inhibitors we designed and synthesized hydrolytically stable analogues of sphingomyelin. The novel compounds 8 and 9 which were replaced the phosphodiester moiety of sphingomyelin with the carbamate moiety showed inhibitory activity with an IC(50) value of micro M on neutral sphingomyelinase in rat brain microsomes. Compound 8i showed a selective neutral sphingomyelinase inhibitory activity.

Stereoselective reactions of a (-)-quinic acid-derived enone: application to the synthesis of the core of scyphostatin.

[reaction: see text] A (-)-quinic acid-derived enone, with the trans-1,2-diol protected as a 2,3-dimethoxybutanediyldioxy ketal, provides an excellent template for further highly stereoselective elaboration as exemplified by its conversion into the core of scyphostatin, a potent inhibitor of neutral sphingomyelinase.