Ceramide Derivatives Research Articles

Targeting dihydroceramide desaturase 1 (Des1): Syntheses of ceramide analogues with a rigid scaffold, inhibitory assays, and AlphaFold2-assisted structural insights reveal cyclopropenone PR280 as a potent inhibitor

Dihydroceramide desaturase 1 (Des1) catalyzes the formation of a CC double bond in dihydroceramide to furnish ceramide. Inhibition of Des1 is related to cell cycle arrest and programmed cell death. The lack of the Des1 crystalline structure, as well as that of a close homologue, hampers the detailed understanding of its inhibition mechanism and difficults the design of new inhibitors, thus making Des1 a strategic target. Based on previous structure-activity studies, different ceramides containing rigid scaffolds were designed. The synthesis and evaluation of these compounds as Des1 inhibitors allowed the identification of PR280 as a better Des 1 inhibitor in vitro (IC50 = 700 nM) than GT11 and XM462, the current reference inhibitors. This cyclopropenone ceramide was obtained in a 6-step synthesis with a 24 % overall yield. The highly confident 3D structure of Des1, recently predicted by AlphaFold2, served as the basis for conducting docking studies of known Des1 inhibitors and the ceramide derivatives synthesized by us in this study. For this purpose, a complete holoprotein structure was previously constructed. This study has allowed a better knowledge of key ligand-enzyme interactions for Des1 inhibitory activity. Furthermore, it sheds some light on the inhibition mechanism of GT11.

Lethal Phenotype-Based Database Screening Identifies Ceramide as a Negative Regulator of Primitive Streak Formation.

In early embryogenesis, the primitive streak (PrS) generates the mesendoderm and is essential for organogenesis. However, because the PrS is a minute and transient tissue, elucidating the mechanism of its formation has been challenging. We performed comprehensive screening of 2 knockout mouse databases based on the fact that failure of PrS formation is lethal. We identified 812 genes involved in various cellular functions and responses that might be linked to PrS formation, with the category of greatest abundance being "Metabolism." In this study, we focused on genes of sphingolipid metabolism and investigated their roles in PrS formation using an in vitro mouse ES cell differentiation system. We show here that elevated intracellular ceramide negatively regulates gene expression essential for PrS formation and instead induces neurogenesis. In addition, sphingosine-1-phosphate (a ceramide derivative) positively regulates neural maturation. Our results indicate that ceramide regulates both PrS formation and the induction of neural differentiation.

Azido-Ceramides, a Tool to Analyse SARS-CoV-2 Replication and Inhibition-SARS-CoV-2 Is Inhibited by Ceramides.

Recently, we have shown that C6-ceramides efficiently suppress viral replication by trapping the virus in lysosomes. Here, we use antiviral assays to evaluate a synthetic ceramide derivative α-NH2-ω-N3-C6-ceramide (AKS461) and to confirm the biological activity of C6-ceramides inhibiting SARS-CoV-2. Click-labeling with a fluorophore demonstrated that AKS461 accumulates in lysosomes. Previously, it has been shown that suppression of SARS-CoV-2 replication can be cell-type specific. Thus, AKS461 inhibited SARS-CoV-2 replication in Huh-7, Vero, and Calu-3 cells up to 2.5 orders of magnitude. The results were confirmed by CoronaFISH, indicating that AKS461 acts comparable to the unmodified C6-ceramide. Thus, AKS461 serves as a tool to study ceramide-associated cellular and viral pathways, such as SARS-CoV-2 infections, and it helped to identify lysosomes as the central organelle of C6-ceramides to inhibit viral replication.

Inactivation of potassium channels by ceramide in rat pancreatic β-cells

Lipid regulation of ion channels is a fundamental mechanism in physiological processes as of neurotransmitter release and hormone secretion. Ceramide is a bioactive lipid proposed as a regulator of several voltage-gated ion channels including potassium channels (Kv). It is generated either de novo or by sphingomyelin (SM) hydrolysis in membranes of mammalian cells. In pancreatic β-cells, ceramide is the main sphingolipid associated with lipotoxicity and likely involved in cell dysfunction. Despite of the wealth of information regarding regulation of potassium channels by ceramides, the regulation of Kv channels by accumulated ceramide in native pancreatic β-cells has not been investigated. To do so, we used either the C2-ceramide, a cell-permeable short-chain analogue, or a sphingomyelinase (SMase C), a hydrolase causing ceramide to elevate from an endogenous production, in pancreatic β-cells of rat. C2-ceramide markedly accelerates steady-state current inactivation according to kinetic changes in the channel machinery. Interestingly, only C2-ceramide accelerates current inactivation while SMase C decreases both, peak-current and step-current amplitude supporting differential effects of ceramide derivatives. A specific inhibitor of the Kv2.1 channel (GxTX-1E), readily inhibits a fraction of the Kv channel current while no further inhibition by C2-ceramide superfusion can be observed supporting Kv2.1 channel involvement in the ceramide inhibition. Thus, intramembrane ceramide accumulation, as a lipidic metabolite released under cell-stress conditions, may alter pancreatic β-cell repolarization and secretion. These results may provide a new insight regarding lipid-protein regulation and advance our understanding in ceramide actions on Kv channels in pancreatic β-cells.

Chemical Synthesis and Molecular Interaction Analysis of α-Galactosyl Ceramide Derivatives as CD1d Ligands.

CD1d is a non-classical major histocompatibility complex (MHC) protein, responsible for lipid antigen presentation, which presents lipids to natural killer T (NKT) cells. Various CD1d lipid ligands have been reported, including microbial and endogenous glycolipids/phospholipids. Among them, an α-galactosylceramide (α-GalCer), a representative CD1d ligand, is one of the most potent ligands and its derivatives have been developed. In this chapter, the chemistry of α-GalCer and its derivatives are described with an emphasis on their chemical syntheses and molecular interaction analysis with CD1d are described.

Secondary metabolites from Detarium microcarpum Guill. and Perr. (Fabaceae).

The chemical investigation of the ethanol/water (7:3) extract of the roots of Detarium microcarpum (Fabaceae) led to the isolation of one new labdane diterpenoid, microcarpin (1) and one new ceramide derivative, microcarpamide (2), along with eight known secondary metabolites (3-10)including, 5-(carboxymethyl)-5,6,8a-trimethyl-3,4,4a,5,6,7,8,8a-octahydronaphthalene-1-carboxylic acid (3), microcarposide (4), rhinocerotinoic acid (5), 1,7-dihydroxy-6-methylxanthone (6), ursolic acid (7), 3β,23-dihydroxylup-20(29)-en-28-oic acid (8), alphitolic acid (9), and stigmasterol glucoside (10). The structures of these compounds were elucidated based on their spectroscopic data. Although compounds 3 and 4 are known, their crystalline structures are reported here for the first time. These compounds were evaluated in vitro for their antisalmonella activity. The results obtained showed that, microcarpamide (2), microcarposide (4), and rhinocerotinoic acid (5) were moderately active against three salmonella strains: Salmonella typhi, Salmonella enteritidis and Salmonella typhimirium, with minimum inhibition concentration values of 76.7 and 153.5μM.

Antibacterial and cytotoxic activities of undescribed cassiaric acid and other constituents from Cassia arereh stem barks

A new lupane-type triterpene, 2α,3β-dihydroxylupan-29-oic acid (1), and one new ceramide derivative: (2S*,2'R*,3S*,4R*,5R*,7'E,11E,12'E,20E)-N-[2'-hydroxyoctadeca-6,11-dienoyl]-2-aminohexacosa-11,20-diene-1,3,4,5-tetrol (2) were isolated from the ethyl acetate fraction obtained from the methanol extract of the stem barks of Cassia arereh together with seven known compounds. Their structures were characterized using two-dimensional NMR, mass spectrometry, and compared with reported data. To date, this is the first report of the isolation of a multiple double bonds sphingolipid type (2) from this genus. The ethyl acetate extract and selected isolates were examined for antimicrobial and cytotoxic activities in vitro. Betulinaldehyde (5) has shown to be active against all bacterial strains whereas, cassiaric acid (1) and betulinic acid (6) have demonstrated to be moderately active. In addition, cassiaric acid (1) showed the best cytotoxic result against HeLa and MCF-7 cell lines tested with IC50 75.00 µM, while lupeol (3) and betulinic acid (6) displayed weak cytotoxicity at 100.00 µM.

Caveolin-1-mediated sphingolipid oncometabolism underlies a metabolic vulnerability of prostate cancer

Plasma and tumor caveolin-1 (Cav-1) are linked with disease progression in prostate cancer. Here we report that metabolomic profiling of longitudinal plasmas from a prospective cohort of 491 active surveillance (AS) participants indicates prominent elevations in plasma sphingolipids in AS progressors that, together with plasma Cav-1, yield a prognostic signature for disease progression. Mechanistic studies of the underlying tumor supportive onco-metabolism reveal coordinated activities through which Cav-1 enables rewiring of cancer cell lipid metabolism towards a program of 1) exogenous sphingolipid scavenging independent of cholesterol, 2) increased cancer cell catabolism of sphingomyelins to ceramide derivatives and 3) altered ceramide metabolism that results in increased glycosphingolipid synthesis and efflux of Cav-1-sphingolipid particles containing mitochondrial proteins and lipids. We also demonstrate, using a prostate cancer syngeneic RM-9 mouse model and established cell lines, that this Cav-1-sphingolipid program evidences a metabolic vulnerability that is targetable to induce lethal mitophagy as an anti-tumor therapy.

Advances in determining signaling mechanisms of ceramide and role in disease

Ceramide is a critical bioactive lipid involved in diverse cellular processes. It has been proposed to regulate cellular processes by influencing membrane properties and by directly interacting with effector proteins. Advances over the past decade have improved our understanding of ceramide as a bioactive lipid. Generation and characterization of ceramide-metabolizing enzyme KO mice, development of specific inhibitors and ceramide-specific antibodies, use of advanced microscopy and mass spectrometry, and design of synthetic ceramide derivatives have all provided insight into the signaling mechanisms of ceramide and its implications in disease. As a result, the role of ceramide in biological functions and disease are now better understood, with promise for development of therapeutic strategies to treat ceramide-regulated diseases.

Preparation and Membrane Distribution of Fluorescent Derivatives of Ceramide.

Ceramide is a bioactive lipid with significant roles in several biological processes including cell proliferation, apoptosis, and raft formation. Although fluorescent derivatives of ceramide are required to probe the behavior of ceramide in cells and cell membranes, commercial fluorescent ceramide derivatives do not reproduce the membrane behavior of native ceramide because of the introduction of bulky fluorophores in the acyl chain. Recently, we developed novel fluorescent analogs of sphingomyelin in which the hydrophilic fluorophores, ATTO488 and ATTO594, are attached to the polar head of sphingomyelin via a nonaethylene glycol linker and demonstrated that their partition and dynamic behaviors in bilayer membranes are similar to native sphingomyelin. In this report, by extending the concept used for the development of fluorescent analogs of sphingomyelin, we prepared novel fluorescent ceramides that exhibit membrane behaviors similar to native ceramide and succeeded in visualizing ceramide-rich membrane domains segregated from ceramide-poor domains.

Untargeted Metabolomics Reveal Lipid Alterations upon 2-Deoxyglucose Treatment in Human HaCaT Keratinocytes.

The glucose analogue 2-deoxyglucose (2-DG) impedes cancer progression in animal models and is currently being assessed as an anticancer therapy, yet the mode of action of this drug of high clinical significance has not been fully delineated. In an attempt to better characterize its pharmacodynamics, an integrative UPLC-Q-Exactive-based joint metabolomic and lipidomic approach was undertaken to evaluate the metabolic perturbations induced by this drug in human HaCaT keratinocyte cells. R-XCMS data processing and subsequent multivariate pattern recognition, metabolites identification, and pathway analyses identified eight metabolites that were most significantly changed upon a 3 h 2-DG exposure. Most of these dysregulated features were emphasized in the course of lipidomic profiling and could be identified as ceramide and glucosylceramide derivatives, consistently with their involvement in cell death programming. Even though metabolomic analyses did not generally afford such clear-cut dysregulations, some alterations in phosphatidylcholine and phosphatidylethanolamine derivatives could be highlighted as well. Overall, these results support the adequacy of the proposed analytical workflow and might contribute to a better understanding of the mechanisms underlying the promising effects of 2-DG.

Isolation and Structure Determination of Antiproliferative Secondary Metabolites from the Potato Earthball Mushroom, Scleroderma bovista (Agaricomycetes)

Mycochemical examination of a methanol extract of Scleroderma bovista Fr. (Agaricomycetes) led to the isolation of 7 compounds, which were, to our knowledge, identified for the first time in this species. The chemical structures of these compounds were determined through extensive spectroscopic methods (nuclear magnetic resonance and mass spectrometry). The fungal metabolites were identified as steroids based on ergostane (compounds 1-4) and lanostane (compounds 6 and 7) skeletons, whereas compound 5 was a ceramide derivative. We evaluated the antiproliferative activity of compounds 4-7 against human cancer cell lines (HeLa, A2780, MDA-MB-231, and MCF-7) using the MTT assay. The lanostane-type derivatives (compounds 6 and 7) and ergosterol peroxide 3-glucoside (compound 4) exerted significant antiproliferative property on 1 or more human cancer cell lines.

Preparation and Membrane Properties of Oxidized Ceramide Derivatives.

Ceramide is a bioactive lipid with important roles in several biological processes including cell proliferation and apoptosis. Although 3-ketoceramides that contain a keto group in place of the 3-OH group of ceramide occur naturally, ceramide derivatives oxidized at the primary 1-OH group have not been identified to date. To evaluate how the oxidative state of the 1-OH group affects the physical properties of membranes, we prepared novel ceramide derivatives in which the 1-OH group was oxidized to a carboxylic acid (PCerCOOH) or methylester (PCerCOOMe) and examined the rigidity of their monolayers and the formation of gel domains in palmitoyloleoylphosphatidylcholine (POPC) or sphingomyelin (SM) bilayers. As a result, PCerCOOH and PCerCOOMe exhibited membrane properties similar to those of native ceramide, although the deprotonated form of PCerCOOH, PCerCOO-, exhibited markedly lower rigidity and higher miscibility with POPC and SM. This was attributed to the electrostatic repulsion of the negative charge, which hampered the formation of the ceramide-enriched gel domain. The similarities in the properties of PCerCOOMe and ceramide revealed the potential to introduce various functional groups onto PCerCOOH via ester or amide linkages; therefore, these derivatives will also provide a new strategy for developing molecular probes, such as fluorescent ceramides, and inhibitors of ceramide-related enzymes.

Acid Ceramidase Deficiency is characterized by a unique plasma cytokine and ceramide profile that is altered by therapy

Acid Ceramidase Deficiency (Farber disease, FD) is an ultra-rare Lysosomal Storage Disorder that is poorly understood and often misdiagnosed as Juvenile Idiopathic Arthritis (JIA). Hallmarks of FD are accumulation of ceramides, widespread macrophage infiltration, splenomegaly, and lymphocytosis. The cytokines involved in this abnormal hematopoietic state are unknown. There are dozens of ceramide species and derivatives, but the specific ones that accumulate in FD have not been investigated. We used a multiplex assay to analyze cytokines and mass spectrometry to analyze ceramides in plasma from patients and mice with FD, controls, Farber patients treated by hematopoietic stem cell transplantation (HSCT), JIA patients, and patients with Gaucher disease. KC, MIP-1α, and MCP-1 were sequentially upregulated in plasma from FD mice. MCP-1, IL-10, IL-6, IL-12, and VEGF levels were elevated in plasma from Farber patients but not in control or JIA patients. C16-Ceramide (C16-Cer) and dhC16-Cer were upregulated in plasma from FD mice. a-OH-C18-Cer, dhC12-Cer, dhC24:1-Cer, and C22:1-Cer-1P accumulated in plasma from patients with FD. Most cytokines and only a-OH-C18-Cer returned to baseline levels in HSCT-treated Farber patients. Sphingosines were not altered. Chitotriosidase activity was also relatively low. A unique cytokine and ceramide profile was seen in the plasma of Farber patients that was not observed in plasma from HSCT-treated Farber patients, JIA patients, or Gaucher patients. The cytokine profile can potentially be used to prevent misdiagnosis of Farber as JIA and to monitor the response to treatment. Further understanding of why these signaling molecules and lipids are elevated can lead to better understanding of the etiology and pathophysiology of FD and inform development of future treatments.

Isolated Polar Amino Acid Residues Modulate Lipid Binding in the Large Hydrophobic Cavity of CD1d.

The CD1d protein is a nonpolymorphic MHC class I-like protein that controls the activation of natural killer T (NKT) cells through the presentation of self- and foreign-lipid ligands, glycolipids, or phospholipids, leading to the secretion of various cytokines. The CD1d contains a large hydrophobic lipid binding pocket: the A' pocket of CD1d, which recognizes hydrophobic moieties of the ligands, such as long fatty acyl chains. Although lipid-protein interactions typically rely on hydrophobic interactions between lipid chains and the hydrophobic sites of proteins, we showed that the small polar regions located deep inside the hydrophobic A' pocket could be used for the modulation of the lipid binding. A series of the ligands, α-galactosyl ceramide (α-GalCer) derivatives containing polar groups in the acyl chain, was synthesized, and the structure-activity relationship studies demonstrated that simple modification from a methylene to an amide group in the long fatty acyl chain, when introduced at optimal positions, enhanced the CD1d recognition of the glycolipid ligands. Formation of hydrogen bonds between the amide group and the polar residues was supported by molecular dynamics (MD) simulations and WaterMap calculations. The computational studies suggest that localized hydrating water molecules may play an important role in the ligand recognition. Here, the results showed that confined polar residues in the large hydrophobic lipid binding pockets of the proteins could be potential targets to modulate the affinity for its ligands.

Anti-inflammatory Cerebrosides from Cultivated Cordyceps militaris.

Cordyceps militaris (bei-chong-chaw, northern worm grass) is a precious and edible entomopathogenic fungus, which is widely used in traditional Chinese medicine (TCM) as a general booster for the nervous system, metabolism, and immunity. Saccharides, nucleosides, mannitol, and sterols were isolated from this fungus. The biological activity of C. militaris was attributed to the saccharide and nucleoside contents. In this study, the aqueous methanolic fraction of C. militaris fruiting bodies exhibited a significant anti-inflammatory activity. Bioactivity-guided fractionation of the active fraction led to the isolation of eight compounds, including one new and two known cerebrosides (ceramide derivatives), two nucleosides, and three sterols. Cordycerebroside A (1), the new cerebroside, along with soyacerebroside I (2) and glucocerebroside (3) inhibited the accumulation of pro-inflammatory iNOS protein and reduced the expression of COX-2 protein in LPS-stimulated RAW264.7 macrophages. This is the first study on the isolation of cerebrosides with anti-inflammatory activity from this TCM.

A Series of Ceramide Analogs Modified at the 1-Position with Potent Activity Against the Intracellular Growth of Chlamydia Trachomatis

Chlamydia trachomatis is an intracellular pathogen that requires different nutrients, including sphingolipids, for survival. Components for the transport and biosynthesis of sphingolipids thus may have a potential as antichlamydial targets. In this study, we synthesized a collection of 24 ceramide derivatives. Three derivatives show pronounced activity with submicromolar IC50. The potency of these compounds was one order of magnitude higher than that of the antibiotic chloramphenicol. We show a detailed structure-activity relationship study for this novel compound class exhibiting a significant effect on the growth of C. trachomatis L2 without penetrating the bacteria itself. The structure-activity relationship presented here defines an important step toward the molecular target of this compound class, which is still elusive.

GluN2B-containing NMDA receptors are upregulated in plasma membranes by the sphingosine-1-phosphate analog FTY720P

Sphingosine-1-phosphate (S1P) is a ceramide derivative serving not only as a regulator of immune properties but also as a modulator of brain functions. To better understand the mechanism underlying the effects of S1P on brain functions, we investigated the potential impact of S1P receptor (S1PR) activation on NMDA receptor subunits. We used acute rat hippocampal slices as a model system, and determined the effects of the active phosphorylated S1P analog, fingolimod (FTY720P) on various NMDA receptors. Treatment with FTY720P significantly increased phosphorylation of GluN2B-containing NMDA receptors at Tyr1472. This effect appears rather specific, as treatment with FTY720P did not modify GluN2B-Tyr1336, GluN2B-Ser1480, GluN2A-Tyr1325 or GluN1-Ser897 phosphorylation. Pre-treatment of hippocampal slices with the compounds W146 and PP1 indicated that FTY720P-induced GluN2B phosphorylation at Tyr1472 epitopes was dependent on activation of S1PR subunit 1 (S1PR1) and Src/Fyn kinase, respectively. Cell surface biotinylation experiments indicated that FTY720P-induced GluN2B phosphorylation at Tyr1472 was also associated with increased levels of GluN1 and GluN2B subunits on membrane surface, whereas no change was observed for GluN2A subunits. We finally demonstrate that FTY720P is inclined to favor Tau and Fyn accumulation on plasma membranes. These results suggest that activation of S1PR1 by FTY720P enhances GluN2B receptor phosphorylation in rat hippocampal slices, resulting in increased levels of GluN1 and GluN2B receptor subunits in neuronal membranes through a mechanism probably involving Fyn and Tau.

Sphingolipids in cardiovascular diseases and metabolic disorders.

Many investigations suggest the pivotal role of sphingolipids in the pathogenesis of lifestyle diseases such as myocardial infarction, hypertension, stroke, diabetes mellitus type 2 and obesity. Some studies suggest that sphingolipids are important factors in cellular signal transduction. They serve as biologically active components of cell membrane and are involved in many processes such as proliferation, maturation and apoptosis. Recently, ceramide and sphingosine-1-phosphate have become the target of many investigations. Ceramide is generated in three metabolic pathways and many factors induce its production as a cellular stress response. Ceramide has proapoptotic properties and acts as a precursor for many other sphingolipids. Sphingosine-1-phosphate is a ceramide derivative, acting antiapoptotically and mitogenically and it is importantly involved in cardioprotection. Further research on the involvement of sphingolipids in cellular pathophysiology may improve the prevention and therapy of lifestyle diseases.

Improved plaque assay identifies a novel anti-Chlamydia ceramide derivative with altered intracellular localization.

Chlamydia trachomatis is a medically important human pathogen causing different diseases, including trachoma, the leading cause of preventable blindness in developing countries, and sexually transmitted infections that can lead to infertility and ectopic pregnancies. There is no vaccine against C. trachomatis at present. Broad-spectrum antibiotics are used as standard therapy to treat the infection but have unwanted side effects, such as inducing persistent or recurring infections and affecting the host microbiome, necessitating the development of novel anti-Chlamydia therapies. Here, we describe the establishment of a robust, fast, and simple plaque assay using liquid overlay medium (LOM) for the identification of anti-Chlamydia compounds. Using the LOM plaque assay, we identified nitrobenzoxadiazole (NBD)-labeled 1-O-methyl-ceramide-C16 as a compound that efficiently inhibits C. trachomatis replication without affecting the viability of the host cell. Further detailed analyses indicate that 1-O-methyl-NBD-ceramide-C16 acts outside the inclusion. Thereby, 1-O-methyl-NBD-ceramide-C16 represents a lead compound for the development of novel anti-Chlamydia drugs and furthermore constitutes an agent to illuminate sphingolipid trafficking pathways in Chlamydia infections.