Design, synthesis of ceramide 1-phosphate analogs and their affinity for cytosolic phospholipase A2 as evidenced by surface plasmon resonance.

Ceramide 1-phosphate (C1P) is a lipid mediator that specifically binds and activates cytosolic phospholipase A2α (cPLA2α). To elucidate the structure-activity relationship of the affinity of C1P for cPLA2α in lipid environments, we prepared a series of C1P analogs containing structural modifications in the hydrophilic parts and subjected them to surface plasmon resonance (SPR). The results suggested the presence of a specific binding site for cPLA2α on the amide, 3-OH and phosphate groups in C1P structure. Especially, dihydro-C1P exhibited enhanced affinity for cPLA2α, suggesting the hydrogen bonding ability of 3-hydroxy group is important for interactions with cPLA2α. This study helps to understand the influence of specific structural moieties of C1P on the interaction with cPLA2α at the atomistic level and may lead to the design of drugs that regulate cPLA2α activation.

Discovery of novel ceramide analogs with favorable pharmacokinetic properties and combination with AKT inhibitor against colon cancer.

Ceramides have emerged as potential therapeutic option with novel mechanism to affect the proliferation, differentiation, senescence, and apoptosis of cancer cells through regulation of multiple signal transduction. Aiming at the improvement of the apoptosis activity and pharmacokinetic profiles of ceramides, a novel series of ceramide analogs were developed through structure simplification and conformation restriction. Among them, compound 12 bearing an alkoxyl naphthyl motif, with favorable rat pharmacokinetic properties, showed better anti-proliferative activity against various colon cancer cells (IC50 ∼20 µM) than other ceramide analogues, as well as the synergistic effect combined with AKT inhibitor MK2206. Additionally, we demonstrated that this combination therapy promoted caspase 3-dependent apoptotic pathway and intensified cell cycle arrest in the G0/G1 phase. Furthermore, the combination of compound 12 and MK2206 displayed synergistic anti-tumor effect in vivo.

Development of an in vitro submerged culture system to synthesize epidermal ceramides in canine keratinocytes.

Ceramides (CERs) in the stratum corneum (SC) are known to play a crucial role in determining skin barrier function in dogs. We aimed to develop an in vitro culture system that synthesized epidermal CER classes to better understand the synthesis of CER classes in canine SC-. Canine keratinocyte cells (MSCEK) at appropriate confluency were incubated with high Ca2+ (1.8 mM CaCl2) supplemented serum-free medium. Eight days post Ca2+ application, the surface of cultured MSCEK was broadly stained with anti-loricrin antibody implying that the keratinocytes had stratified into stratum granulosum. MSCEK cells synthesized heterogenous epidermal CERs, similar to those seen during the stratification of canine keratinocytes. CER fractions obtained from MSCEK cells were comparable to those from canine SC, including CER[EOS] (combination of ω-hydroxy fatty acids and sphingosines), CER[NP] (combination of non-hydroxy fatty acids and phytosphingosines), and CER[EOP] (combination of ω-hydroxy fatty acids and phytosphingosines), all of which are lowered in the SC during canine atopic dermatitis. Thus, the present study provides a simple culture system as a tool for in-depth analysis of CER production in canine keratinocytes.

Chiral combinatorial preparation and biological evaluation of unique ceramides for inhibition of sphingomyelin synthase.

Enantiomers or diastereomers of chiral bioactive compounds often exhibit different biological and toxicological properties. Here, we report the efficient synthesis of four stereoisomers of sphingosine and derivatization of unique chiral ceramides through a combinatorial chemistry by solid-phase activated resin ester. In addition, to test the effectivity of stereochemistry of ceramide, we demonstrated a cell-based assay of sphingomyelin synthase inhibition in the presence ofchiral unique ceramides, which suggested that libraries of this sort will be a rich source of biologically active synthetic molecules.

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.

Mega-stokes pyrene ceramide conjugates for STED imaging of lipid droplets in live cells.

Lipid droplets are dynamic subcellular organelles that participate in a range of physiological processes including metabolism, regulation and lipid storage. Their role in disease, such as cancer, where they are involved in metabolism and in chemoresistance, has emerged over recent years. Thus, the value of lipid droplets as diagnostic markers is increasingly apparent where number and size of droplets can be a useful prognostic. Although diverse in size, LDs are typically too small to be easily enumerated by conventional microscopy. The advent of super-resolution microscopy methods offers the prospect of detailed insights but there are currently no commercial STED probes suited to this task and STED, where this method has been used to study LDs it has relied on fixed samples. Here, we report a pyrene-based ceramide conjugate PyLa-C17Cer, that stains lipid droplets with exceptionally high precision in living cells and shows excellent performance in stimulated emission depletion microscopy. The parent compound PyLa comprises a pyrene carboxyl core appended with 3,4-dimethylaminophenyl. The resulting luminophore exhibits high fluorescent quantum yield, mega-Stokes shift and low cytotoxicity. From DFT calculations the Stokes shifted fluorescent state arises from a dimethylaminophenyl to pyrene charge-transfer transition. While the parent compound is cell permeable, it is relatively promiscuous, emitting from both protein and membranous structures within the living mammalian cell. However, on conjugation of C17 ceramide to the free carboxylic acid, the resulting PyLa-C17Cer, remains passively permeable to the cell membrane but targets lipid droplets within the cell through a temperature dependent mechanism, with high selectivity. Targeting was confirmed through colocalisation with the commercial lipid probe Nile Red. PyLa-C17Cer offers outstanding contrast of LDs both in fluorescence intensity and lifetime imaging due to its large Stokes shift and very weak emission from aqueous media. Moreover, because the compound is exceptionally photochemically stable with no detectable triplet emission under low temperature conditions, it can be used as an effective probe for fluorescence correlation spectroscopy (FCS). These versatile fluorophores are powerful multimodal probes for combined STED/FCS/lifetime studies of lipid droplets and domains in live cells.

Click reactions with functional sphingolipids.

Sphingolipids and glycosphingolipids can regulate cell recognition and signalling. Ceramide and sphingosine-1-phosphate are major players in the sphingolipid pathways and are involved in the initiation and regulation of signalling, apoptosis, stress responses and infection. Specific chemically synthesised sphingolipid derivatives containing small functionalities like azide or alkyne can mimic the biological properties of natural lipid species, which turns them into useful tools for the investigation of the highly complex sphingolipid metabolism by rapid and selective 'click chemistry' using sensitive tags like fluorophores. Subsequent analysis by various fluorescence microscopy techniques or mass spectrometry allows the identification and quantification of the corresponding sphingolipid metabolites as well as the research of associated enzymes. Here we present an overview of recent advances in the synthesis of ceramide and sphingosine analogues for bioorthogonal click reactions to study biosynthetic pathways and localization of sphingolipids for the development of novel therapeutics against lipid-dependent diseases.

STED Imaging of Golgi Dynamics with Cer-SiR: A Two-Component, Photostable, High-Density Lipid Probe for Live Cells.

Long time-lapse super-resolution imaging in live cells requires a labeling strategy that combines a bright, photostable fluorophore with a high-density localization probe. Lipids are ideal high-density localization probes, as they are >100 times more abundant than most membrane-bound proteins and simultaneously demark the boundaries of cellular organelles. Here, we describe Cer-SiR, a two-component, high-density lipid probe that is exceptionally photostable. Cer-SiR is generated in cells via a bioorthogonal reaction of two components: a ceramide lipid tagged with trans-cyclooctene (Cer-TCO) and a reactive, photostable Si-rhodamine dye (SiR-Tz). These components assemble within the Golgi apparatus of live cells to form Cer-SiR. Cer-SiR is benign to cellular function, localizes within the Golgi at a high density, and is sufficiently photostable to enable visualization of Golgi structure and dynamics by 3D confocal or long time-lapse STED microscopy.

Stereoselective preparation of quaternary 2-vinyl sphingosines and ceramides and their effect on basal sphingolipid metabolism.

The dicyclohexylborane-mediated addition of allene 1 to (E)-2-tridecenal affords a quaternary protected 2-amino-2-vinyl-1,3-diol in good yield as a single diastereomer. This compound is readily transformed into the four stereoisomers of the quaternary (E)-2-vinyl analogs of sphingosine. The metabolic fate and the effect of these compounds on the basal sphingolipid metabolism in human A549 lung adenocarcinoma cells has been studied, together with the ceramide analog of the most relevant vinylsphingosine derivative.

Synthesis of specific deuterated derivatives of the long chained stratum corneum lipids [EOS] and [EOP] and characterization using neutron scattering.

The synthesis of specific deuterated derivatives of the long chained ceramides [EOS] and [EOP] is described. The structural differences with respect to the natural compounds are founded in the substitution of the 2 double bonds containing linoleic acid by a palmitic acid branched with a methyl group in 10-position. The specific deuteration is introduced both in the branched and in the terminal methyl group, which was realized by common methods of successive deuteration of carboxylic groups in 3 steps. These modified fatty acids resp. the corresponding ceramides [EOS] and [EOP] were prepared for neutron scattering investigations. First results of these investigations were presented in this manuscript showing that the deuterated compounds could be detected in the stratum corneum lipid model membranes. The deuterated ceramides [EOS] and [EOP] are valuable tools to investigate the influence of these long chained ceramide species on the nanostructure of stratum corneum lipid model membranes.

From the configurational preference of dihydroceramide desaturase-1 towards Δ6-unsaturated substrates to the discovery of a new inhibitor.

Dihydroceramide desaturase 1 (Des1) catalyzes the last step of ceramide synthesis de novo, thus regulating the physiologically relevant balance between dihydrosphingolipids and sphingolipids. Here we report on the configurational preference of Des1 towards isomeric Δ6-unsaturated dihydroceramide analogs and the discovery of a potent Des1 inhibitor.

Synthesis and evaluation of a tag-free photoactive phospho-ceramide analogue-1 (PCERA-1) probe to study immunomodulation in macrophages.

Phospho-ceramide analogue-1 (PCERA-1), a synthetic analogue of ceramide-1-phosphate (C1P), has been previously shown to act as a potent modulator of macrophage activity and inflammation. We have developed an efficient synthesis of PCERA-1 from readily available starting materials, and designed and prepared derivatives of this analogue, including a photoaffinity probe to tag and identify putative proteins that bind PCERA-1.

Synthesis of specifically deuterated ceramide [AP]-C18 and its biophysical characterization using neutron diffraction.

The very heterogeneous group of ceramides is known to be mandatory for proper barrier functions of the outermost layer of mammalian skin, referred to as stratum corneum (SC). The synthesis of a specifically deuterated ceramide [AP]-C18 variant is described. The synthesized ceramide contains the racemic forms of the α hydroxy fatty acid. For the biophysical implementation, the received diastereomeric ceramide was applied in a neutron diffraction experiment. Therefore, a SC lipid model membrane was prepared containing the described ceramide (CER), cholesterol (CHOL), stearic acid (SA), and cholesterol sulfate (ChS) in a ratio of 55/25/15/5wt%. Thus, we were able to localize the deuterated molecule part within the bilayers. In the process, a short-periodicity phase (SPP) was observed with a unit cell scale of about 44Å. For the first time, we were able to confirm former ideas concerning the arrangement of the CER within this quaternary lipid model membrane.

Synthesis of ceramides NS and NP with perdeuterated and specifically ω deuterated N-acyl residues.

The synthesis of 12 deuterated ceramides with either a deuteration at the last carbon atom of the amide bound fatty acid or a perdeuterated fatty acid chain is described. The ceramides were prepared starting from sphingosine or phytosphingosine and ω deuterated or perdeuterated fatty acids with PyBOP® as activating agent in high yields. For the synthesis of the specifically deuterated fatty acids, dicarboxylic acids were transformed into ω deuterated alkyl bromide, which was chain elongated with blocked ω bromo alcohols by copper catalyzed Grignard coupling. Oxidation of regenerated alcohol function yields the ω deuterated fatty acids.

Ceramide mediates FasL-induced caspase 8 activation in colon carcinoma cells to enhance FasL-induced cytotoxicity by tumor-specific cytotoxic T lymphocytes.

FasL-mediated cytotoxicity is one of the mechanisms that CTLs use to kill tumor cells. However, human colon carcinoma often deregulates the Fas signaling pathway to evade host cancer immune surveillance. We aimed at testing the hypothesis that novel ceramide analogs effectively modulate Fas function to sensitize colon carcinoma cells to FasL-induced apoptosis. We used rational design and synthesized twenty ceramide analogs as Fas function modulators. Five ceramide analogs, IG4, IG7, IG14, IG17, and IG19, exhibit low toxicity and potent activity in sensitization of human colon carcinoma cells to FasL-induced apoptosis. Functional deficiency of Fas limits both FasL and ceramide analogs in the induction of apoptosis. Ceramide enhances FasL-induced activation of the MAPK, NF-κB, and caspase 8 despite induction of potent tumor cell death. Finally, a sublethal dose of several ceramide analogs significantly increased CTL-mediated and FasL-induced apoptosis of colon carcinoma cells. We have therefore developed five novel ceramide analogs that act at a sublethal dose to enhance the efficacy of tumor-specific CTLs, and these ceramide analogs hold great promise for further development as adjunct agents in CTL-based colon cancer immunotherapy.

Total synthesis and in vitro bioevaluation of clavaminols A, C, H & deacetyl clavaminol H as potential chemotherapeutic and antibiofilm agents.

A highly concise and expedient total synthesis of bioactive clavaminols (1-4) has been executed using commercially available achiral compound decanol. The synthetic strategy relied on trans-Wittig olefination, Sharpless asymmetric epoxidation, regioselective azidolysis and in situ detosylation followed by reduction as key reactions with good overall yield. Based on biological evaluation studies of all the synthesized compounds, it was observed that the clavaminol A (1) exhibited good cytotoxicity against DU145 and SKOV3 cell lines with IC50 value of 10.8 and 12.5 µM, respectively. Clavaminol A (1) and deacetyl clavaminol H (3) displayed selective promising inhibition towards Gram-positive pathogenic bacterial strains and showed good antifungal activity against the tested Candida strains. In addition, compounds 1 and 3 have demonstrated significant bactericidal activity. Compound 3 was found to be equipotent to the standard drug Miconazole displaying MFC value of 15.6 µg/mL against Candida albicans MTCC 854, C. albicans MTCC 1637, C. albicans MTCC 3958 and Candida glabrata MTCC 3019. Compounds 1 and 3 were also able to inhibit the biofilm formation of Micrococcus luteus MTCC 2470 and Staphylococcus aureus MLS16 MTCC 2940. Clavaminol A (1) increased the levels of reactive oxygen species (ROS) accumulation in M. luteus MTCC 2470.

Non-glycosidic compounds can stimulate both human and mouse iNKT cells.

Invariant natural killer T (iNKT) cells recognize CD1d/glycolipid complexes and upon activation with synthetic agonists display immunostimulatory properties. We have previously described that the non-glycosidic CD1d-binding lipid, threitolceramide (ThrCer) activates murine and human iNKT cells. Here, we show that incorporating the headgroup of ThrCer into a conformationally more restricted 6- or 7-membered ring results in significantly more potent non-glycosidic analogs. In particular, ThrCer 6 was found to promote strong anti-tumor responses and to induce a more prolonged stimulation of iNKT cells than does the canonical α-galactosylceramide (α-GalCer), achieving an enhanced T-cell response at lower concentrations compared with α-GalCer both in vitro, using human iNKT-cell lines and in vivo, using C57BL/6 mice. Collectively, these studies describe novel non-glycosidic ThrCer-based analogs that have improved potency in iNKT-cell activation compared with that of α-GalCer, and are clinically relevant iNKT-cell agonists.

Synthesis and photochemical properties of PEGylated coumarin-caged ceramides for cell studies.

Caged ceramide analogues (C6-, C16-, C18-, C22- and C24-Cer) have been prepared by introducing a hydrophilic coumarin-based cage bearing a short polyethylene glycol (PEG) chain. (6-Bromo-7-mTEGylated-coumarin-4-yl)methyl (Btc) caged ceramide showed efficient photo-uncaging to release the parent ceramide upon direct exposure to 350 nm UV light; in contrast (7-mTEGylated-coumarin-4-yl)methyl (Tc) caged ceramide was photolysed more slowly. In preliminary experiments, Btc-caged ceramides were taken up by cells and their photolysis led to decreases in cell viability, but not to activation of caspase enzymes, suggesting that either reactive oxygen species or an alternate caspase-independent pathway may be responsible for the decreases in cell viability caused by photolysis of caged ceramides.

A series of ceramide analogs modified at the 1-position with potent activity against the intracellular growth of Chlamydia trachomatis.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: The structure-activity relationship presented here defines an important step toward the molecular target of this compound class, which is still elusive.

Synthesis and Evaluation of Hybrid Structures Composed of Two Glucosylceramide Synthase Inhibitors.

Glucosylceramide metabolism and the enzymes involved have attracted significant interest in medicinal chemistry, because aberrations in the levels of glycolipids that are derived from glucosylceramide are causative in a range of human diseases including lysosomal storage disorders, type 2 diabetes, and neurodegenerative diseases. Selective modulation of one of the glycoprocessing enzymes involved in glucosylceramide metabolism-glucosylceramide synthase (GCS), acid glucosylceramidase (GBA1), or neutral glucosylceramidase (GBA2)-is therefore an attractive research objective. In this study we took two established GCS inhibitors, one based on deoxynojirimycin and the other a ceramide analogue, and merged characteristic features to obtain hybrid compounds. The resulting 39-compound library does not contain new GCS inhibitors; however, a potent (200 nm) GBA1 inhibitor was identified that has little activity toward GBA2 and might therefore serve as a lead for further biomedical development as a selective GBA1 modulator.