Neutral ceramidase-active site inhibitor chemotypes and binding modes.

Ceramides impact a diverse array of biological functions and have been implicated in disease pathogenesis. The enzyme neutral ceramidase (nCDase) is a zinc-containing hydrolase and mediates the metabolism of ceramide to sphingosine (Sph), both in cells and in the intestinal lumen. nCDase inhibitors based on substrate mimetics, for example C6-urea ceramide, have limited potency, aqueous solubility, and micelle-free fraction. To identify non-ceramide mimetic nCDase inhibitors, hit compounds from an HTS campaign were evaluated in biochemical, cell based and in silico modeling approaches. A majority of small molecule nCDase inhibitors contained pharmacophores capable of zinc interaction but retained specificity for nCDase over zinc-containing acid and alkaline ceramidases, as well as matrix metalloprotease-3 and histone deacetylase-1. nCDase inhibitors were refined by SAR, were shown to be substrate competitive and were active in cellular assays. nCDase inhibitor compounds were modeled by in silico DOCK screening and by molecular simulation. Modeling data supports zinc interaction and a similar compound binding pose with ceramide. nCDase inhibitors were identified with notably improved activity and solubility in comparison with the reference lipid-mimetic C6-urea ceramide.

Straightforward access to novel cytotoxic phytosphingosine-like aminotriols from l-erythrose chiron.

A divergent approach to a small library of long-chain 6-amino-1,4,5-triols as novel phytosphingosine-type entities, together with their preliminary cytotoxic evaluation, was achieved. Construction of the target compounds addressed two key aspects. First, the installation of a carbon-nitrogen bond via two prototypes of [3,3]-sigmatropic rearrangements and second the introduction of an alkyl side chain unit by using a late stage olefin cross-metathesis process. As shown in cell viability experiments, the corresponding HCl salts proved to be the most cytotoxic derivatives among all the tested substances, with IC50 values in the lower micromolar range on the Jurkat, HeLa and HCT-116 cell lines.

Nonalcoholic Fatty Liver Disease Patients Exhibit Reduced CD47 and Increased Sphingosine, Cholesterol, and Monocyte Chemoattractant Protein-1 Levels in the Erythrocyte Membranes.

Background: Nonalcoholic fatty liver disease (NAFLD) constitutes a significant cause of deaths, liver transplantations, and economic costs worldwide. Despite extended research, investigations on the role of erythrocytes are scarce. Red blood cells from experimental animals and human patients with NAFLD present phosphatidylserine exposure, which is then recognized by Kupffer cells. This event leads to erythrophagocytosis and amplification of inflammation through iron disposition. In addition, it has been shown that erythrocytes from NAFLD patients release the chemokine monocyte chemoattractant protein-1 (MCP1), leading to increased tumor necrosis factor alpha release from macrophages RAW 264.7. However, erythrophagocytosis can also be caused by reduced CD47 levels. Moreover, increased MCP1 release could be either signal-induced or caused by higher MCP1 levels on the erythrocyte membrane. Finally, erythrocyte efferocytosis could provide additional inflammatory metabolites. Methods: In this study, we measured the erythrocyte membrane levels of CD47 and MCP1 by enzyme-linked immunosorbent assay, and cholesterol and sphingosine with thin-layer chromatography. Eighteen patients (8 men and 10 women, aged 56.7 ± 11.5 years) and 14 healthy controls (7 men and 7 women, aged 39.3 ± 15.6 years) participated in our study. Results: The erythrocyte CD47 levels were decreased in the erythrocyte membranes of NAFLD patients (844 ± 409 pg/mL) compared with healthy controls (2969 ± 1936 pg/mL) with P = 0.012. Levels of MCP1 increased in NAFLD patients (389 ± 255 pg/mL) compared with healthy controls (230 ± 117 pg/mL) with P = 0.0274, but low statistical power. Moreover, in erythrocyte membranes, there was a statistically significant accumulation of sphingosine and cholesterol in NAFLD patients compared with healthy controls. Conclusions: Our results imply that erythrocytes release chemotactic "find me" signals (MCP1) while containing reduced "do not eat me" signals (CD47). These molecules can lead to erythrophagocytosis. Next, increased "goodbye" signals (sphingosine and cholesterol) could augment inflammation by metabolic reprogramming.

Structural insights into sphingosine-1-phosphate receptor activation.

As a critical sphingolipid metabolite, sphingosine-1-phosphate (S1P) plays an essential role in immune and vascular systems. There are five S1P receptors, designated as S1PR1 to S1PR5, encoded in the human genome, and their activities are governed by endogenous S1P, lipid-like S1P mimics, or nonlipid-like therapeutic molecules. Among S1PRs, S1PR1 stands out due to its nonredundant functions, such as the egress of T and B cells from the thymus and secondary lymphoid tissues, making it a potential therapeutic target. However, the structural basis of S1PR1 activation and regulation by various agonists remains unclear. Here, we report four atomic resolution cryo-electron microscopy (cryo-EM) structures of Gi-coupled human S1PR1 complexes: bound to endogenous agonist d18:1 S1P, benchmark lipid-like S1P mimic phosphorylated Fingolimod [(S)-FTY720-P], or nonlipid-like therapeutic molecule CBP-307 in two binding modes. Our results revealed the similarities and differences of activation of S1PR1 through distinct ligands binding to the amphiphilic orthosteric pocket. We also proposed a two-step "shallow to deep" transition process of CBP-307 for S1PR1 activation. Both binding modes of CBP-307 could activate S1PR1, but from shallow to deep transition may trigger the rotation of the N-terminal helix of Gαi and further stabilize the complex by increasing the Gαi interaction with the cell membrane. We combine with extensive biochemical analysis and molecular dynamic simulations to suggest key steps of S1P binding and receptor activation. The above results decipher the common feature of the S1PR1 agonist recognition and activation mechanism and will firmly promote the development of therapeutics targeting S1PRs.

Characterization of a Bacterial Kinase That Phosphorylates Dihydrosphingosine to Form dhS1P.

Like other members of the phylum Bacteroidetes, the oral anaerobe Porphyromonas gingivalis synthesizes a variety of sphingolipids, similar to its human host. Studies have shown that synthesis of these lipids (dihydroceramides [DHCs]) is involved in oxidative stress resistance, the survival of P. gingivalis during stationary phase, and immune modulation. Here, we constructed a deletion mutant of P. gingivalis strain W83 with a deletion of the gene encoding DhSphK1, a protein that shows high similarity to a eukaryotic sphingosine kinase, an enzyme that phosphorylates sphingosine to form sphingosine-1-phosphate. Our data show that deletion of the dhSphK1 gene results in a shift in the sphingolipid composition of P. gingivalis cells; specifically, the mutant synthesizes higher levels of phosphoglycerol DHCs (PG-DHCs) than the parent strain W83. Although PG1348 shows high similarity to the eukaryotic sphingosine kinase, we discovered that the PG1348 enzyme is unique, since it preferentially phosphorylates dihydrosphingosine, not sphingosine. Besides changes in lipid composition, the W83 ΔPG1348 mutant displayed a defect in cell division, the biogenesis of outer membrane vesicles (OMVs), and the amount of K antigen capsule. Taken together, we have identified the first bacterial dihydrosphingosine kinase whose activity regulates the lipid profile of P. gingivalis and underlies a regulatory mechanism of immune modulation. IMPORTANCE Sphingoid base phosphates, such as sphingosine-1-phosphate (S1P) and dihydrosphingosine-1-phosphate (dhS1P), act as ligands for S1P receptors, and this interaction is known to play a central role in mediating angiogenesis, vascular stability and permeability, and immune cell migration to sites of inflammation. Studies suggest that a shift in ratio to higher levels of dhS1P in relation to S1P alters downstream signaling cascades due to differential binding and activation of the various S1P receptor isoforms. Specifically, higher levels of dhS1P are thought to be anti-inflammatory. Here, we report on the characterization of a novel kinase in Porphyromonas gingivalis that phosphorylates dihydrosphingosine to form dhS1P.

Bioactive Lipid O-cyclic phytosphingosine-1-phosphate Promotes Differentiation of Human Embryonic Stem Cells into Cardiomyocytes via ALK3/BMPR Signaling.

Adult human cardiomyocytes have an extremely limited proliferative capacity, which poses a great barrier to regenerative medicine and research. Human embryonic stem cells (hESCs) have been proposed as an alternative source to generate large numbers of clinical grade cardiomyocytes (CMs) that can have potential therapeutic applications to treat cardiac diseases. Previous studies have shown that bioactive lipids are involved in diverse cellular responses including cardiogenesis. In this study, we explored the novel function of the chemically synthesized bioactive lipid O-cyclic phytosphingosine-1-phosphate (cP1P) as an inducer of cardiac differentiation. Here, we identified cP1P as a novel factor that significantly enhances the differentiation potential of hESCs into cardiomyocytes. Treatment with cP1P augments the beating colony number and contracting area of CMs. Furthermore, we elucidated the molecular mechanism of cP1P regulating SMAD1/5/8 signaling via the ALK3/BMP receptor cascade during cardiac differentiation. Our result provides a new insight for cP1P usage to improve the quality of CM differentiation for regenerative therapies.

Effects of (R)- and (S)-α-Hydroxylation of Acyl Chains in Sphingosine, Dihydrosphingosine, and Phytosphingosine Ceramides on Phase Behavior and Permeability of Skin Lipid Models.

Ceramides (Cers) with α-hydroxylated acyl chains comprise about a third of all extractable skin Cers and are required for permeability barrier homeostasis. We have probed here the effects of Cer hydroxylation on their behavior in lipid models comprising the major SC lipids, Cer/free fatty acids (C 16-C 24)/cholesterol, and a minor component, cholesteryl sulfate. Namely, Cers with (R)-α-hydroxy lignoceroyl chains attached to sphingosine (Cer AS), dihydrosphingosine (Cer AdS), and phytosphingosine (Cer AP) were compared to their unnatural (S)-diastereomers and to Cers with non-hydroxylated lignoceroyl chains attached to sphingosine (Cer NS), dihydrosphingosine (Cer NdS), and phytosphingosine (Cer NP). By comparing several biophysical parameters (lamellar organization by X-ray diffraction, chain order, lateral packing, phase transitions, and lipid mixing by infrared spectroscopy using deuterated lipids) and the permeabilities of these models (water loss and two permeability markers), we conclude that there is no general or common consequence of Cer α-hydroxylation. Instead, we found a rich mix of effects, highly dependent on the sphingoid base chain, configuration at the α-carbon, and permeability marker used. We found that the model membranes with unnatural Cer (S)-AS have fewer orthorhombically packed lipid chains than those based on the (R)-diastereomer. In addition, physiological (R)-configuration decreases the permeability of membranes, with Cer (R)-AdS to theophylline, and increases the lipid chain order in model systems with natural Cer (R)-AP. Thus, each Cer subclass makes a distinct contribution to the structural organization and function of the skin lipid barrier.

Sphingosine 1-Phosphate Liposomes for Targeted Nitric Oxide Delivery to Mediate Anticancer Effects against Brain Glioma Tumors.

Specifically targeting glioblastoma multiforme (GBM) blood vessels and actively enhancing the permeability of the brain-blood-tumor barrier (BBTB) are two extremely difficult challenges currently hindering the development of effective therapies against GBM. Herein, a liposome drug delivery system (S1P/JS-K/Lipo) is described, which delivers the nitric oxide (NO) prodrug JS-K, O2 -(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl) piperazin-1-yl] diazen-1-ium-1,2-diolate, to GBM tumors using sphingosine-1-phosphate (S1P)-signaling molecules as active targeting lipid ligands. It is revealed that S1P/JS-K/Lipo actively penetrates the BBTB, aided by caveolin-1-mediated transcytosis, and it is demonstrated that the system specifically interacts with S1P receptors (S1PRs), which are highly expressed on GBM cells. Nondestructive ultrasound imaging in GBM mouse models is also utilized to observe microsized NO bubble production from JS-K, as catalyzed by the glutathione S-transferases (GSTs) resident in GBM cells. Given that these NO bubbles strongly promote GBM cell death in vivo, the S1PR-targeted liposome delivery system-which successfully achieves BBTB penetration and tumor targeted delivery of a complex multicomponent drug regimen-represents a promising approach for targeted therapies against GBM and other carcinomas characterized by elevated S1PR expression.

Ceramides and Sphingosino-1-Phosphate in Obesity.

Obesity is a growing worldwide problem, especially in developed countries. This disease adversely affects the quality of life and notably contributes to the development of type 2 diabetes, metabolic syndrome, and cardiovascular disorders. It is characterised by excessive lipids accumulation in the subcutaneous and visceral adipose tissue. Considering the secretory function of adipose tissue, this leads to impaired adipokines and cytokines release. Changes in adipose tissue metabolism result in chronic inflammation, pancreatic islets dysfunction and peripheral insulin resistance. In addition to saturating various adipocytes, excess lipids are deposited into non-adipose peripheral tissues, which disturbs cell metabolism and causes a harmful effect known as lipotoxicity. Fatty acids are metabolised into bioactive lipids such as ceramides, from which sphingolipids are formed. Ceramides and sphingosine-1-phosphate (S1P) are involved in intracellular signalling, cell proliferation, migration, and apoptosis. Studies demonstrate that bioactive lipids have a crucial role in regulating insulin signalling pathways, glucose homeostasis and ß cell death. Data suggests that ceramides may have an opposite cellular effect than S1P; however, the role of S1P remains controversial. This review summarises the available data on ceramide and sphingolipid metabolism and their role in obesity.

Detection of 2-hydroxy-fatty acids and 2-hydroxy-fatty acid-containing ceramides in a gliding marine bacterium Aureispira marina.

The cellular fatty acid composition of Aureispira marina IAM 15389T (JCM 23197T), a gliding bacterium isolated from the coastline of Thailand, was re-examined by using a standard MIDI method based on alkaline hydrolysis, and two other methods. The direct transesterification using 5% HCl/methanol or 4 M HCl hydrolysis followed by methyl esterification revealed that 2-hydroxy-15-methyl-hexadecanoic acid (2-OH-iso-C17:0) and 2-hydroxy-15-methyl-hexadecenoic acid (2-OH-iso-C17:1), which were not reported in a previous paper, were found to be major cellular fatty acids of this bacterium, and the amount of 2-OH-iso-C17:1 was even higher than that of arachidonic acid (C20:4), a characteristic polyunsaturated fatty acid present in this bacterium. These 2-hydroxy-fatty acids were contained in two cellular lipids that were relatively stable against alkaline hydrolysis. One of them was analyzed by mass spectrometry, 1H-nuclear magnetic resonance, and other chemical methods, and identified as a ceramide composed of 2-hydroxy-fatty acid and sphingosine of 19 carbons with three double bonds. A minor ceramide containing 18 carbon sphingosine with three double bonds was also detected.

Unravelling the structural complexity of glycolipids with cryogenic infrared spectroscopy.

Glycolipids are complex glycoconjugates composed of a glycan headgroup and a lipid moiety. Their modular biosynthesis creates a vast amount of diverse and often isomeric structures, which fulfill highly specific biological functions. To date, no gold-standard analytical technique can provide a comprehensive structural elucidation of complex glycolipids, and insufficient tools for isomer distinction can lead to wrong assignments. Herein we use cryogenic gas-phase infrared spectroscopy to systematically investigate different kinds of isomerism in immunologically relevant glycolipids. We show that all structural features, including isomeric glycan headgroups, anomeric configurations and different lipid moieties, can be unambiguously resolved by diagnostic spectroscopic fingerprints in a narrow spectral range. The results allow for the characterization of isomeric glycolipid mixtures and biological applications.

Evaluation of pyrrolidine-based analog of jaspine B as potential SphK1 inhibitors against rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovitise, and its pathogenesis is complicated. Sphingosine-1-phosphate (S1P) is a lipid produced by sphingosine kinase 1 and 2 (SphK1/2), which participate in some of most-spread skeletal diseases such as rheumatoid arthritis or osteoarthritis. To explore the anti-inflammatory activity of 2-epi-jaspine B analogs as SphKs inhibitors, we used LPS-induced rheumatoid arthritis fibroblast-like synovial cells (HFLS-RA) as the research object to evaluate the anti-inflammatory activity of 16 2-epi-jaspine B analogs and the newly synthesized salt CHJ01. We found that 2-epi-jaspine B analog CHJ01 in hydrochloride salt form has excellent SphK1 inhibitory effect and better anti-RA effect. CHJ01 showed an anti-inflammatory effect similar to that of MTX in vitro, its IC50 value is 8.64 µM. Moreover, the anti-RA effect of CHJ01 was also studied by using a Complete Freund's Adjuvant (CFA)-induced arthritis (AIA) in a rat mode. Pharmacological experiments show that CHJ01 can help to significantly improve the symptoms of rheumatoid arthritis by reducing the swelling volume, arthritis score, spleen index and the level of IL-1ß, TNF-α, IL-6 of AIA rats. Therefore, CHJ01 holds high potential for the treatment of RA.

The Sphingosine and Acyl Chains of Ceramide [NS] Show Very Different Structure and Dynamics That Challenge Our Understanding of the Skin Barrier.

The lipid phase of the uppermost human skin layer is thought to comprise highly rigid lipids in an orthorhombic phase state to protect the body against the environment. By synthesizing sphingosine-d28 deuterated N-lignoceroyl-d-erythro-sphingosine (ceramide [NS]), we compare the structure and dynamics of both chains of that lipid in biologically relevant mixtures using X-ray diffraction, 2 H NMR analysis, and infrared spectroscopy. Our results reveal a substantial fraction of sphingosine chains in a fluid and dynamic phase state at physiological temperature. These findings prompt revision of our current understanding of the skin lipid barrier, where an extended ceramide [NS] conformation is preferred and a possible domain structure is proposed. Mobile lipid chains may be crucial for skin elasticity and the translocation of physiologically important molecules.

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.

Enantiomers of phospholipids and cholesterol: A key to decipher lipid-lipid interplay in membrane.

Most phospholipids constituting biological membranes are chiral molecules with a hydrophilic head group and hydrophobic alkyl chains, rendering biphasic property characteristic of membrane lipids. Some lipids assemble into small domains via chirality-dependent homophilic and heterophilic interactions, the latter of which sometimes include cholesterol to form lipid rafts and other microdomains. On the other hand, lipid mediators and hormones derived from chiral lipids are recognized by specific membrane or nuclear receptors to induce downstream signaling. It is crucial to clarify the physicochemical properties of the lipid self-assembly for the study of the functions and behavior of biological membranes, which often become elusive due to effects of membrane proteins and other biological events. Three major lipids with different skeletal structures were discussed: sphingolipids including ceramides, phosphoglycerolipids, and cholesterol. The physicochemical properties of membranes and physiological functions of lipid enantiomers and diastereomers were described in comparison to natural lipids. When each enantiomer formed a self-assembly or interacted with achiral lipids, both lipid enantiomers exhibited identical membrane physicochemical properties, while when the enantiomer interacted with chiral lipids or with the opposite enantiomer, mixed membranes exhibited different properties. For example, racemic membranes comprising native sphingomyelin and its antipode exhibited phase segregation due to their strong homophilic interactions. Therefore, lipid enantiomers and diastereomers can be good probes to investigate stereospecific lipid-lipid and lipid-protein interactions occurring in biological membranes.

Synthesis and anticancer profile of novel sphingoid base-like compounds with a quaternary stereocentre.

The synthesis of novel sphingoid base-like compounds with a quaternary stereocentre was achieved in a sequence featuring [3,3]-sigmatropic rearrangements and olefin cross-metathesis transformation as the key reaction steps, which were accompanied by the rational selection of suitable functional group transformations. The stereochemistry of the desired tetra-substituted carbon bearing nitrogen functionality was determined via NOESY experiments of the advanced oxazolidine-2-thiones. Cell viability experiments revealed significant antiproliferative/cytotoxic activity of the target compounds 7, ent-7 and 29 against the Jurkat cell line, with the IC50 values of 6.6 µM, 5.6 µM and 6.1 µM, respectively.

Activation of sphingosine 1-phosphate receptor 2 attenuates chemotherapy-induced neuropathy.

Platinum-based therapeutics are used to manage many forms of cancer, but frequently result in peripheral neuropathy. Currently, the only option available to attenuate chemotherapy-induced neuropathy is to limit or discontinue this treatment. Sphingosine 1-phosphate (S1P) is a lipid-based signaling molecule involved in neuroinflammatory processes by interacting with its five cognate receptors: S1P1-5 In this study, using a combination of drug pharmacodynamic analysis in human study participants, disease modeling in rodents, and cell-based assays, we examined whether S1P signaling may represent a potential target in the treatment of chemotherapy-induced neuropathy. To this end, we first investigated the effects of platinum-based drugs on plasma S1P levels in human cancer patients. Our analysis revealed that oxaliplatin treatment specifically increases one S1P species, d16:1 S1P, in these patients. Although d16:1 S1P is an S1P2 agonist, it has lower potency than the most abundant S1P species (d18:1 S1P). Therefore, as d16:1 S1P concentration increases, it is likely to disproportionately activate proinflammatory S1P1 signaling, shifting the balance away from S1P2 We further show that a selective S1P2 agonist, CYM-5478, reduces allodynia in a rat model of cisplatin-induced neuropathy and attenuates the associated inflammatory processes in the dorsal root ganglia, likely by activating stress-response proteins, including ATF3 and HO-1. Cumulatively, the findings of our study suggest that the development of a specific S1P2 agonist may represent a promising therapeutic approach for the management of chemotherapy-induced neuropathy.

Synthesis and biological evaluation of 2-epi-jaspine B analogs as selective sphingosine kinase 1 inhibitors.

2-Epi-jaspine B is an isomer of the natural product jaspine B and shows certain selectivity for SphK1 and potent antitumor activity. Based on the crystal structure of SphK1, we transformed the structure of 2-epi-jaspine B and modified the hydrophobic side chain to obtain a series of 2-epi-jaspine B analogs. The MTT assay was used to examine the antitumor activities of these analogs. We identified a novel 2-epi-jaspine B analog YHR17, which has potent antiproliferative activities for tested cell lines with IC50 values that ranged from 0.68 to 5.68 µM and inhibited the proliferation of the A375 cell line by affecting the cell cycle and apoptosis. Furthermore, YHR17 inhibited SphK1 with more than 125-fold selectivity over SphK2.

Molecular Mechanism of S1P Binding and Activation of the S1P1 Receptor.

Sphingosine-1-phosphate (S1P) is a lipidic mediator in mammals that functions either as a second messenger or as a ligand. In the latter case, it is transported by its HDL-associated apoM carrier and circulated in blood where it binds to specific S1P receptors on cell membranes and induces downstream reactions. Although S1P signaling pathways are essential for many biological processes, they are poorly understood at the molecular level. Here, the solved crystal structures of the S1P1 receptor were used to evaluate molecular dynamics (MD) simulations to generate greater detailed molecular insights into the mechanism of S1P signaling. The MD simulations provided observations at the coarse-grained and atomic levels indicating that S1P may access the receptor binding pocket directly from solvents. Lifting of the bulky N-terminal cap region of the receptor precedes initial S1P binding. Glu1213.29 guides S1P penetration, and together with Arg2927.34 is responsible for the stabilization of S1P in the binding pocket, which is consistent with experimental predictions. The complete binding of S1P is followed by receptor activation, wherein Trp2696.48 moves toward the transmembrane helix (TM) 7, resulting in the formation of an enhanced hydrogen bond network in the lower region of TM7. The distance between TM3 and TM6 is subsequently increased, resulting in the opening of the intracellular binding pocket that enables G protein binding. Further analysis of the force distribution network in the receptor yielded a detailed molecular understanding of the signal transmission network that is activated upon agonist binding.