James A. Shayman

Improved Inhibitors of Glucosylceramide Synthase

Previous work has led to the identification of inhibitors of glucosylceramide synthase, the enzyme catalyzing the first glycosylation step in the synthesis of glucosylceramide-based glycosphingolipids. These inhibitors have two identified sites of action: the inhibition of glucosylceramide synthase, resulting in the depletion of cellular glycosphingolipids, and the inhibition of 1-O-acylceramide synthase, resulting in the elevation of cell ceramide levels. A new series of glucosylceramide synthase inhibitors based on substitutions in the phenyl ring of a parent compound, 1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4), was made. For substitutions of single functional groups, the potency of these inhibitors in blocking glucosylceramide synthase was primarily dependent upon the hydrophobic and electronic properties of the substituents. An exponential relationship was found between the IC50 of each inhibitor and the sum of derived hydrophobic (π) and electronic (ς) parameters. This relationship demonstrated that substitutions that increased the electron-donating characteristics and decreased the lipophilic characteristics of the homologues enhanced the potency of these compounds in blocking glucosylceramide formation. A novel compound was subsequently designed and observed to be even more active in blocking glucosylceramide formation. This compound,d-threo-4′-hydroxy-P4, inhibited glucosylceramide synthase at an IC50 of 90 nm. In addition, a series of dioxane substitutions was designed and tested. These included 3′,4′-methylenedioxyphenyl-, 3′,4′-ethylenedioxyphenyl-, and 3′4′-trimethylenedioxyphenyl-substituted homologues.d-threo-3′,4′-Ethylenedioxy-P4-inhibited glucosylceramide synthase was comparably active to thep-hydroxy homologue. 4′-Hydroxy-P4 and ethylenedioxy-P4 blocked glucosylceramide synthase activity at concentrations that had little effect on 1-O-acylceramide synthase activity. These novel inhibitors resulted in the inhibition of glycosphingolipid synthesis in cultured cells at concentrations that did not significantly raise intracellular ceramide levels or inhibit cell growth.

(1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol as an inhibitor of ceramidase

In this study, we have examined the cellular and biochemical activities of the ceramide analog (1S,2R)-Derythro-2-(N-myristoylamino)-1-phenyl-1-propanol (Derythro-MAPP). Addition of 5 μMD-e-MAPP to HL-60 human promyelocytic leukemia cells resulted in a concentration- and time-dependent growth suppression accompanied by an arrest in the G/G phase of the cell cycle; thus mimicking the action of exogenous ceramides. Its enantiomer L-e-MAPP was without effect. Two lines of evidence suggested that D-e-MAPP may not function as a direct analog of ceramide. First, D-e-MAPP possesses a stereochemical configuration opposite to that of D-erythro-ceramide. Second, D-e-MAPP failed to activate ceramide-activated protein phosphatase in vitro. Therefore, we examined if D-e-MAPP functioned indirectly by modulating endogenous ceramide levels. The addition of D-e-MAPP to cells, but not L-e-MAPP, caused a time- and concentration-dependent elevation in endogenous ceramide levels reaching greater than 3-fold over baseline following 24 h of treatment. Both D-e-MAPP and L-e-MAPP underwent similar uptake by HL-60 cells. D-e-MAPP was poorly metabolized, and remained intact in cells, whereas L-e-MAPP underwent a time- and concentration-dependent metabolism; primarily through N-deacylation. In vitro, L-e-MAPP was metabolized by alkaline ceramidase to an extent similar to that seen with C-ceramide. D-e-MAPP was not metabolized. Instead, D-e-MAPP inhibited alkaline ceramidase activity in vitro with an IC of 1-5 μM. D-e-MAPP did not modulate the activity of other ceramide metabolizing enzymes in vitro or in cells, and it was a poor inhibitor of acid ceramidase (IC > 500 μM). Finally, D-e-MAPP inhibited the metabolism of L-e-MAPP in cells. These studies demonstrate that D-e-MAPP functions as an inhibitor of alkaline ceramidase in vitro and in cells resulting in elevation in endogenous levels of ceramide with the consequent biologic effects of growth suppression and cell cycle arrest. These studies point to an important role for ceramidases in the regulation of endogenous levels of ceramide.

Reduction of globotriaosylceramide in Fabry disease mice by substrate deprivation

We used a potent inhibitor of glucosylceramide synthase to test whether substrate deprivation could lower globotriaosylceramide levels in alpha-galactosidase A (alpha-gal A) knockout mice, a model of Fabry disease. C57BL/6 mice treated twice daily for 3 days with D-threo-1-ethylendioxyphenyl-2-palmitoylamino-3-pyrrolidi no-propanol (D-t-EtDO-P4) showed a concentration-dependent decrement in glucosylceramide levels in kidney, liver, and spleen. A single intraperitoneal injection of D-t-EtDO-P4 resulted in a 55% reduction in renal glucosylceramide, consistent with rapid renal glucosylceramide metabolism. A concentration-dependent decrement in renal and hepatic globotriaosylceramide levels was observed in alpha-Gal A(-) males treated for 4 weeks with D-t-EtDO-P4. When 8-week-old alpha-Gal A(-) males were treated for 8 weeks with 10 mg/kg twice daily, renal globotriaosylceramide fell to below starting levels, consistent with an alpha-galactosidase A-independent salvage pathway for globotriaosylceramide degradation. Complications observed with another glucosylceramide synthase inhibitor, N-butyldeoxynojirimycin, including weight loss and acellularity of lymphatic organs, were not observed with D-t-EtDO-P4. These data suggest that Fabry disease may be amenable to substrate deprivation therapy.

Globotriaosylceramide induces oxidative stress and up-regulates cell adhesion molecule expression in Fabry disease endothelial cells

Fabry disease, an X-linked systemic vasculopathy, is caused by a deficiency of alpha-galactosidase A resulting in globotriaosylceramide (Gb(3)) storage in cells. The pathogenic role of Gb(3) in the disease is not known. Based on previous work, we tested the hypothesis that accumulation of Gb(3) in the vascular endothelium of Fabry disease is associated with increased production of reactive oxygen species (ROS) and increased expression of cell adhesion molecules. Gb(3)-loading resulted in increased intracellular ROS production in cultured vascular endothelial cells in a dose-dependent manner. Increased Gb(3) also induced expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin. Reduction of endogenous Gb(3) by treatment of the cells with an inhibitor of glycosphingolipid synthase or alpha-galactosidase A led to decreased expression of adhesion molecules. Plasma from Fabry patients significantly increased ROS generation in endothelial cells when compared with plasma from non-Fabry controls. This effect was not influenced by reduction of intracellular Gb(3). This study provided direct evidence that excess intracellular Gb(3) induces oxidative stress and up-regulates the expression of cellular adhesion molecules in vascular endothelial cells. In addition, other factors in patients plasma may also contribute to oxidative stress in Fabry vascular endothelial cells.

Cloning and characterization of a lysosomal phospholipase A2, 1-O-acylceramide synthase

Recently, a novel enzyme, 1-O-acylceramide synthase (ACS), was purified and characterized from bovine brain. This enzyme has both calcium-independent phospholipase A2 and transacylase activities. The discovery of this enzyme led us to propose a new pathway for ceramide metabolism in which the sn-2-acyl group of either phosphatidylethanolamine or phosphatidylcholine is transferred to the 1-hydroxyl group of ceramide. In this study, the partial amino acid sequences from the purified enzyme revealed that the enzyme contains amino acid sequences identical to those of human lecithin:cholesterol acyltransferase-like lysophospholipase (LLPL). The coding sequences of the mouse, bovine, and human genes were obtained from the respective kidney cDNAs by PCR. The open reading frames of LLPL were cloned into pcDNA3 to generate carboxyl-terminally tagged proteins. The expression of mouse LLPL in COS-7 cells demonstrated that transfected cells had higher transacylase and phospholipase A2 activities than did non-transfected cells. Immunoprecipitation confirmed that LLPL had ACS activity. There were no significant lecithin:cholesterol acyltransferase and lysophospholipase activities in the mouse LLPL-transfected cells under either acidic or neutral conditions. Amino acid sequences from cDNAs of mouse, human, and bovine LLPLs demonstrated a signal peptide cleavage site, one lipase motif (AXSXG), and several N-linked glycosylation sites in each LLPL molecule. The replacement of serine with alanine in the lipase motif of mouse LLPL resulted in elimination of enzyme activity, indicating that the serine residue is part of the catalytic site. Deglycosylation of mouse, human, and bovine LLPLs yielded core proteins with a molecular mass of 42 kDa without change in enzyme activities. LLPL was post-translationally modified by signal peptide cleavage and N-linked glycosylation, and each mature LLPL had the same size core protein. Subcellular fractionation demonstrated that ACS activity co-localized withN-acetylglucosaminidase. Therefore, LLPL encodes a novel lysosomal enzyme, ACS.

Inhibition of glucosylceramide accumulation results in effective blockade of polycystic kidney disease in mouse models

Polycystic kidney disease (PKD) represents a family of genetic disorders characterized by renal cystic growth and progression to kidney failure. No treatment is currently available for people with PKD, although possible therapeutic interventions are emerging. Despite genetic and clinical heterogeneity, PKDs have in common defects of cystic epithelia, including increased proliferation, apoptosis and activation of growth regulatory pathways. Sphingolipids and glycosphingolipids are emerging as major regulators of these cellular processes. We sought to evaluate the therapeutic potential for glycosphingolipid modulation as a new approach to treat PKD. Here we demonstrate that kidney glucosylceramide (GlcCer) and ganglioside GM3 levels are higher in human and mouse PKD tissue as compared to normal tissue, regardless of the causative mutation. Blockade of GlcCer accumulation with the GlcCer synthase inhibitor Genz-123346 effectively inhibits cystogenesis in mouse models orthologous to human autosomal dominant PKD (Pkd1 conditional knockout mice) and nephronophthisis (jck and pcy mice). Molecular analysis in vitro and in vivo indicates that Genz-123346 acts through inhibition of the two key pathways dysregulated in PKD: Akt protein kinase–mammalian target of rapamycin signaling and cell cycle machinery. Taken together, our data suggest that inhibition of GlcCer synthesis represents a new and effective treatment option for PKD.

Lysosomal Phospholipase A2 and Phospholipidosis

ABSTRACT A lysosomal phospholipase A2, LPLA2, was recently characterized and shown to have substrate specificity for phosphatidylcholine and phosphatidylethanolamine. LPLA2 is ubiquitously expressed but is most highly expressed in alveolar macrophages. Double conditional gene targeting was employed to elucidate the function of LPLA2. LPLA2-deficient mice (Lpla2−/−) were generated by the systemic deletion of exon 5 of the Lpla2 gene, which encodes the lipase motif essential for the phospholipase A2 activity. The survival of the Lpla2−/− mice was normal. Lpla2−/− mouse mating pairs yielded normal litter sizes, indicating that the gene deficiency did not impair fertility or fecundity. Alveolar macrophages from wild-type but not Lpla2−/− mice readily degraded radiolabeled phosphatidylcholine. A marked accumulation of phospholipids, in particular phosphatidylethanolamine and phosphatidylcholine, was found in the alveolar macrophages, the peritoneal macrophages, and the spleens of Lpla2−/− mice. By 1 year of age, Lpla2−/− mice demonstrated marked splenomegaly and increased lung surfactant phospholipid levels. Ultrastructural examination of Lpla2−/− mouse alveolar and peritoneal macrophages revealed the appearance of foam cells with lamellar inclusion bodies, a hallmark of cellular phospholipidosis. Thus, a deficiency of lysosomal phospholipase A2 results in foam cell formation, surfactant lipid accumulation, splenomegaly, and phospholipidosis in mice.

Disruption of the glucosylceramide biosynthetic pathway in Aspergillus nidulans and Aspergillus fumigatus by inhibitors of UDP-Glc:ceramide glucosyltransferase strongly affects spore germination, cell cycle, and hyphal growth

The opportunistic mycopathogen Aspergillus fumigatus expresses both glucosylceramide and galactosylceramide (GlcCer and GalCer), but their functional significance in Aspergillus species is unknown. We here identified and characterized a GlcCer from Aspergillus nidulans, a non‐pathogenic model fungus. Involvement of GlcCer in fungal development was tested on both species using a family of compounds known to inhibit GlcCer synthase in mammals. Two analogs, D‐threo‐1‐phenyl‐2‐palmitoyl‐3‐pyrrolidinopropanol (P4) and D‐threo‐3′,4′‐ethylenedioxy‐P4, strongly inhibited germination and hyphal growth. Neutral lipids from A. fumigatus cultured in the presence of these inhibitors displayed a significantly reduced GlcCer/GalCer ratio. These results suggest that synthesis of GlcCer is essential for normal development of A. fumigatus and A. nidulans.

A role for glycosphingolipid accumulation in the renal hypertrophy of streptozotocin-induced diabetes mellitus.

Glucosylceramide (GlcCer) and related glycosphingolipids have been implicated as causal elements in both the growth of cells and in the regulation of hormonal signaling. We therefore studied whether the renal hypertrophy induced by diabetes was associated with enhanced synthesis of glycosphingolipids. 16 d after the induction of diabetes, increases in renal size and concentration of glucocerebroside and ganglioside GM3 were observed paralleling an increase in UDP-Glc concentration. GlcCer synthase and beta-glucosidase-specific activities were no different between control and diabetic kidneys. The apparent Km of the GlcCer synthase with respect to UDP-Glc was 250 microM and was unchanged in the diabetic kidneys. The observed concentrations of UDP-Glc were 149 and 237 microM in control and diabetic kidneys, respectively. The UDP-Glc level is thus rate limiting with regard to GlcCer synthesis. To determine whether the changes in glycolipid content were functionally significant, diabetic and control groups were treated with the GlcCer synthase inhibitor, D-threo-1-phenyl-2-decanoyl-amino-3-morpholino-1- propanol, 2 wk after the induction of diabetes. Kidney weights in the diabetic rats treated with D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol were no different than the control groups. Morphometric analysis of glomerular volumes paralleled changes in renal growth. Glycosphingolipid formation may therefore represent a significant pathway for glucose utilization in early diabetic nephropathy.

Dexamethasone modulates rat renal brush border membrane phosphate transporter mRNA and protein abundance and glycosphingolipid composition.

Glucocorticoids are important regulators of renal phosphate transport. This study investigates the role of alterations in renal brush border membrane (BBM) sodium gradient-dependent phosphate transport (Na-Pi cotransporter) mRNA and protein abundance in the dexamethasone induced inhibition of Na-Pi cotransport in the rat. Dexamethasone administration for 4 d caused a 1.5-fold increase in the Vmax of Na-Pi cotransport (1785 +/- 119 vs. 2759 +/- 375 pmol/5 s per mg BBM protein in control, P < 0.01), which was paralleled by a 2.5-fold decrease in the abundance of Na-Pi mRNA and Na-Pi protein. There was also a 1.7-fold increase in BBM glucosylceramide content (528 +/- 63 vs. 312 +/- 41 ng/mg BBM protein in control, P < 0.02). To determine whether the alteration in glucosylceramide content per se played a functional role in the decrease in Na-Pi cotransport, control rats were treated with the glucosylceramide synthase inhibitor, D-threo-1-phenyl-2-decanoyl-amino-3-morpholino-1-propanol (PDMP). The resultant 1.5-fold decrease in BBM glucosylceramide content (199 +/- 19 vs. 312 +/- 41 ng/mg BBM protein in control, P < 0.02) was associated with a 1.4-fold increase in Na-Pi cotransport activity (1422 +/- 73 vs. 1048 +/- 85 pmol/5 s per mg BBM protein in control, P < 0.01), and a 1.5-fold increase in BBM Na-Pi protein abundance. Thus, dexamethasone-induced inhibition of Na-Pi cotransport is associated with a decrease in BBM Na-Pi cotransporter abundance, and an increase in glucosylceramide. Since primary alteration in BBM glucosylceramide content per se directly and selectively modulates BBM Na-Pi cotransport activity and Na-Pi protein abundance, we propose that the increase in BBM glucosylceramide content plays an important role in mediating the inhibitory effect of dexamethasone on Na-Pi cotransport activity.