New fluorogenic probes for neutral and alkaline ceramidases

New fluorogenic ceramidase substrates derived from the N-acyl modification of our previously reported probes (RBM14) are reported. While none of the new probes were superior to the known RBM14C12 as acid ceramidase substrates, the corresponding nervonic acid amide (RBM14C24:1) is an efficient and selective substrate for the recombinant human neutral ceramidase, both in cell lysates and in intact cells. A second generation of substrates, incorporating the natural 2-(N-acylamino)-1,3-diol-4-ene framework (compounds RBM15) is also reported. Among them, the corresponding fatty acyl amides with an unsaturated N-acyl chain can be used as substrates to determine alkaline ceramidase (ACER)1 and ACER2 activities. In particular, compound RBM15C18:1 has emerged as the best fluorogenic probe reported so far to measure ACER1 and ACER2 activities in a 96-well plate format. Keywords: ceramides; sphingolipids; substrate; umbelliferone

Pharmacological Elevation of Cellular Dihydrosphingomyelin Provides a Novel Antiviral Strategy against West Nile Virus Infection

The flavivirus life cycle is strictly dependent on cellular lipid metabolism. Polyphenols like gallic acid and its derivatives are promising lead compounds for new therapeutic agents as they can exert multiple pharmacological activities, including the alteration of lipid metabolism. The evaluation of our collection of polyphenols against West Nile virus (WNV), a representative medically relevant flavivirus, led to the identification of N,N'-(dodecane-1,12-diyl)bis(3,4,5-trihydroxybenzamide) and its 2,3,4-trihydroxybenzamide regioisomer as selective antivirals with low cytotoxicity and high antiviral activity (half-maximal effective concentrations [EC50s] of 2.2 and 0.24 μM, respectively, in Vero cells; EC50s of 2.2 and 1.9 μM, respectively, in SH-SY5Y cells). These polyphenols also inhibited the multiplication of other flaviviruses, namely, Usutu, dengue, and Zika viruses, exhibiting lower antiviral or negligible antiviral activity against other RNA viruses. The mechanism underlying their antiviral activity against WNV involved the alteration of sphingolipid metabolism. These compounds inhibited ceramide desaturase (Des1), promoting the accumulation of dihydrosphingomyelin (dhSM), a minor component of cellular sphingolipids with important roles in membrane properties. The addition of exogenous dhSM or Des1 blockage by using the reference inhibitor GT-11 {N-[(1R,2S)-2-hydroxy-1-hydroxymethyl-2-(2-tridecyl-1-cyclopropenyl)ethyl]octanamide} confirmed the involvement of this pathway in WNV infection. These results unveil the potential of novel antiviral strategies based on the modulation of the cellular levels of dhSM and Des1 activity for the control of flavivirus infection.We thank Theodore C. Pierson (National Institutes of Health, USA) for the subgenomic replicon of WNV. This work was supported by the Spanish Ministry of Science and Innovation AEI/10.13039/501100011033 under grants PID2019-105117RR-C21 (to M.A.M.-A.), PID2019-105117RR-C22 (to M.-J.P.-P.), and PID2020-119195RJ-I00 (to N.J.d.O.) and by the AECSIC under grant PIE-201980E100 (to M.-J.P.-P. and A.S.-F.). This research work was also funded by the European Commission-NextGenerationEU (regulation EU 2020/2094) through CSIC’s Global Health Platform (PTI Salud Global). P.M.-C. was supported by an FPI fellowship (PRE2020-093374) from AEI/10.13039/501100011033. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.Peer reviewe

Metabolisme dels Esfingolípids. Noves metodologies i efecte sobre l´autofàgia

[cat] Els esfingolípids són lípids complexes derivats de l’esfingosina que a més de servir de base estructural de les membranes cel·lulars, regulen múltiples vies de senyalització, com per exemple l’autofàgia. Entre tots els esfingolípids, són la ceramida (Cer), la dihidroceramida (dhCer) i l’esfingosina-1-fosfat (S1P) les molècules amb un paper més determinant en l’homeòstasi cel·lular. És per això que els enzims involucrats en la formació d’aquests esfingolípids són clau per la determinació del destí cel·lular. Entre aquests enzims destaca la dihidroceramida dessaturasa 1 (Des1), les ceramidases (CDases) i finalment l’esfingosina-1-fosfat liasa (S1PL). Tot i que s’han descrits mètodes per determinar l’activitat CDasa i S1PL, la recerca de nous moduladors enzimàtics requereix tècniques de cribratge d’alt rendiment (HTS). Tenint en compte que actualment aquestes metodologies són escasses, el primer objectiu de la tesi va consistir en determinar la capacitat de les CDases alcalines per hidrolitzar els substrats fluorogènics RBM14. Es va determinar l’activitat enzimàtica en diferents models cel·lulars, on les diferents CDases alcalines estaven sobreexpressades o silenciades, mesurant la fluorescència del substrat RBM14 quan és hidrolitzat. En cèl·lules que sobreexpressaven l’ACER3 es va alliberar més fluorescència dels substrats en comparació amb les cèl·lules control. En canvi en cèl·lules on l’ACER2 o l’ACER1 estaven sobreexpressades no es va detectar activitat enzimàtica. Per tant, de totes les CDases alcalines, s’ha demostrat que només l’ACER3, i no l’ACER1 ni l’ACER2, pot hidrolitzar els substrats RBM14. El substrat fluorogènic RBM13 permet analitzar l’activitat S1PL aplicant procediments HTS, però no es pot emprar en cèl·lules ja que no pot travessar la membrana plasmàtica. Amb l’objectiu de millorar la incorporació dins les cèl·lules dels substrats RBM13, i els seus anàlegs millorats RBM77 i RBM148, es va decidir encapsular els compostos en liposomes catiònics. Amb els substrats lliures, sense ser encapsulats, no s’observà activitat enzimàtica. No obstant, una vegada encapsulats es va detectar fluorescència i aquesta va resultar ser dependent de la concentració del substrat i, tal com s’esperava, era més elevada en les cèl·lules que sobreexpressaven la S1PL. L’autofàgia és un procés en el que la cèl·lula degrada proteïnes i orgànuls en els autofagolisosomes. El propòsit és reciclar material cel·lular per mantenir el metabolisme actiu durant l’escassetat de nutrients o evitar l’acumulació de material danyat. L’últim objectiu de la tesi va consistir en estudiar la relació entre les dhCer i l’autofàgia, i la seva repercussió en la mort o en la supervivència cel·lular. Es va examinar la capacitat de diferents inhibidors de Des1 d’estimular l’autofàgia i el seu efecte en el destí cel·lular en dues línies de glioma humà, les T98G i les U87MG. En primer lloc es va comprovar que el celecoxib (CCX), fenoxodiol (PXD), resveratrol (RV), γ-tocotrienol (γ-TE) i XM462 provocaven una acumulació de dhCer. Això era degut a la inhibició de l’enzim Des1 juntament amb l’estimulació de la via de biosíntesi de novo dels esfingolípids. En les mateixes condicions en les que es va analitzar l’esfingolipidoma, els compostos van induir autofàgia. No obstant, l’autofàgia va ser també activada en absència de dhCer, indicant l’existència d’un mecanisme d’inducció d’autofàgia per part dels compostos independent de dhCer. Quan es va inhibir l’autofàgia en aquestes cèl·lules, els compostos eren menys tòxics, suggerint que la via d’inducció d’autofàgia independent de dhCer és citotòxica. D’altra banda quan es va inhibir l’autofàgia en les cèl·lules amb nivells normals de dhCer, els compostos eren més tòxics demostrant que la via d’inducció d’autofàgia dependent de dhCer és un mecanisme de supervivència cel·lular. Els resultats suggereixen que els compostos indueixen l’autofàgia per ambdues vies dependent i independent de dhCer, i és l’equilibri entre elles el que influeix en el destí cel·lular.[eng] Sphingolipids are molecules that, in addition to their structural function in cell membranes, regulate multiple signalling pathways and contribute to various cellular functions. Among the enzymes of sphingolipid metabolism, dihydroceramide desaturase 1 (Des1), ceramidases (CDases) and sphingosine-1-phosphate lyase (S1PL) play a decisive role in the cell fate. Although several methods for CDase and S1PL activity determination have been described, most of them do not allow the application of HTS assays. The first objective of the thesis was to determine the ability of the alkaline CDases to hydrolyze the RBM14 fluorogenic substrates. Different cell models with the alkaline CDases overexpressed or silenced were used. It was shown that among all alkaline CDases, only ACER3, and not ACER1 or ACER2, could hydrolyse RBM14 substrates. In order to improve the incorporation into the cells of the S1PL substrates RBM13 and their improved analogues RBM77 and RBM148, we decided to encapsulate the compounds into cationic liposomes. With the free substrates, no enzymatic activity was observed. However, once encapsulated, the probes were hydrolysed by S1PL and the resulting fluorescence was dependent on the substrate concentration and, as expected, it was higher in cells overexpressing S1PL. The last objective of the thesis was to study the relationship between dihydroceramides (dhCer) and autophagy, and its repercussion on the cell fate of two glioblastoma cell lines, T98G and U87MG. First of all it was verified that celecoxib (CCX), phenoxodiol (PXD), resveratrol (RV), γ-tocotrienol (γ-TE) and XM462 induced autophagy concomitantly with dihydroceramide (dhCer) buildup due to stimulation of ceramide synthesis de novo and decreased Des1 activity. However, autophagy activation was also induced in the absence of dhCer accumulation, indicating the existence of a dhCer-independent autophagy induction mechanism. In this context, the compounds were less toxic, suggesting that this pathway is cytotoxic. On the other hand, when autophagy was inhibited in cells with normal dhCer levels, compounds were more toxic showing that this pathway is a mechanism of cell survival. We propose that Des1 inhibitors activate autophagy via both dhCer-dependent and independent pathways and the balance between the two pathways influences the final cell fate

Synthesis and Characterization of Fluorescent Probes for the Development of a Ceramide Synthase FRET-Based Assay

Ceramides are recognized to occupy a pivotal position in sphingolipid metabolism, playing a fundamental role as membrane structural components, as well as second messengers. Ceramide synthases (CerS) are a family of enzymes that catalyze the N-acylation of sphingosine and dihydrosphingosine, thus controlling the levels of intracellular ceramides. In this work, we present the synthesis and the fluorescent properties of new probes for the development of a FRET-based assay for CerS activity. Based on our previous discovery of spisulosine as a suitable probe for CerS activity in cells, the use of a modified NBD-spisulosine with a clickable fatty acid as CerS substrates may allow the ultimate formation of a bichromophoric reporter for a FRET-based analysis of CerS activity in cells

Synthesis and characterization of bichromophoric 1-deoxyceramides as FRET probes

The suitability as FRET probes of two bichromophoric 1-deoxydihydroceramides containing a labelled spisulosine derivative as a sphingoid base and two differently ω-labelled fluorescent palmitic acids has been evaluated. The ceramide synthase (CerS) catalyzed metabolic incorporation of ω-azido palmitic acid into the above labeled spisulosine to render the corresponding ω-azido 1-deoxyceramide has been studied in several cell lines. In addition, the strain-promoted click reaction between this ω-azido 1-deoxyceramide and suitable fluorophores has been optimized to render the target bichromophoric 1-deoxydihydroceramides. These results pave the way for the development of FRET-based assays as a new tool to study sphingolipid metabolism.Partial financial support from the Project CTQ2017-85378-R (AEI/FEDER, UE) of the Spanish Ministry of Science, Innovation and Universities is acknowledged. We are grateful to Prof. Anthony H. Futerman (Weizmann Institute of Science, Rehovot, Israel) for providing the CerS5 plasmid and to Prof. Vicente Marchán (UB) for technical support. Experimental contributions from Mr Alexandre García and Mr Pedro Rayo are also acknowledged. About the source of the cells, we purchased from ATCC (https://www.lgcstandards-atcc.org/en.aspx). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Peer reviewe

Discovery of deoxyceramide analogs as highly selective ACER3 inhibitors in live cells

Acid (AC), neutral (NC) and alkaline ceramidase 3 (ACER3) are the most ubiquitous ceramidases and their therapeutic interest as targets in cancer diseases has been well sustained. This supports the importance of discovering potent and specific inhibitors for further use in combination therapies. Although several ceramidase inhibitors have been reported, most of them target AC and a few focus on NC. In contrast, well characterized ACER3 inhibitors are lacking. Here we report on the synthesis and screening of two series of 1-deoxy(dihydro)ceramide analogs on the three enzymes. Activity was determined using fluorogenic substrates in recombinant human NC (rhNC) and both lysates and intact cells enriched in each enzyme. None of the molecules elicited a remarkable AC inhibitory activity in either experimental setup, while using rhNC, several compounds of both series were active as non-competitive inhibitors with Ki values between 1 and 5 μM. However, a dramatic loss of potency occurred in NC-enriched cell lysates and no activity was elicited in intact cells. Interestingly, several compounds of Series 2 inhibited ACER3 dose-dependently in both cell lysates and intact cells with IC50‘s around 20 μM. In agreement with their activity in live cells, they provoked a significant increase in the amounts of ceramides. Overall, this study identifies highly selective ACER3 activity blockers in intact cells, opening the door to further medicinal chemistry efforts aimed at developing more potent and specific compounds.This work has been partly funded by the Spanish Ministry of Economy, Industry and Competitiveness (grant CTQ2017-85378-R) and Fundación BBVA (grant 35_2018). We thank Dr. Gemma Triola for critically reading the manuscript, and Alexandre Garcia, Pedro Rayo, Neus Roca and Eva Dalmau for their excellent technical assistance.Peer reviewe

Jaspine B induces nonapoptotic cell death in gastric cancer cells independently of its inhibition of ceramide synthase

Sphingolipids (SLs) have been extensively investigated in biomedical research due to their role as bioactive molecules in cells. Here, we describe the effect of a SL analog, jaspine B (JB), a cyclic anhydrophytosphingosine found in marine sponges, on the gastric cancer cell line, HGC-27. JB induced alterations in the sphingolipidome, mainly the accumulation of dihydrosphingosine, sphingosine, and their phosphorylated forms due to inhibition of ceramide synthases. Moreover, JB provoked atypical cell death in HGC-27 cells, characterized by the formation of cytoplasmic vacuoles in a time and dose-dependent manner. Vacuoles appeared to originate from macropinocytosis and triggered cytoplasmic disruption. The pan-caspase inhibitor, z-VAD, did not alter either cytotoxicity or vacuole formation, suggesting that JB activates a caspase-independent cell death mechanism. The autophagy inhibitor, wortmannin, did not decrease JB-stimulated LC3-II accumulation. In addition, cell vacuolation induced by JB was characterized by single-membrane vacuoles, which are different from double-membrane autophagosomes. These findings suggest that JB-induced cell vacuolation is not related to autophagy and it is also independent of its action on SL metabolism. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.This work was supported by the Ministerio de Ciencia e Innovaci?n (Project CTQ2014-54743-R) and a predoctoral contract from Generalitat de Catalunya to F.C. A.H.F. is the Joseph Meyerhoff Professor of Biochemistry at the Weizmann Institute of Science. The authors thank Prof. R. Ghidoni and Dr. V. Gagliostro (University of Milan) for preliminary LC3-II data and Prof. A. Delgado for helpful discussions. The authors also thank E. Dalmau for excellent technical assistance.Peer reviewe

Activity of neutral and alkaline ceramidases on fluorogenic N -acylated coumarin-containing aminodiols

Ceramidases catalyze the cleavage of ceramides into sphingosine and fatty acids. Previously, we reported on the use of the RBM14 fluorogenic ceramide analogs to determine acidic ceramidase activity. In this work, we investigated the activity of other amidohydrolases on RBM14 compounds. Both bacterial and human purified neutral ceramidases (NCs), as well as ectopically expressed mouse neutral ceramidase hydrolyzed RBM14 with different selectivity, depending on the N -acyl chain length. On the other hand, microsomes from alkaline ceramidase (ACER)3 knockdown cells were less competent at hydrolyzing RBM14C12, RBM12C14, and RBM14C16 than controls, while microsomes from ACER2 and ACER3 overexpressing cells showed no activity toward the RBM14 substrates. Conversely, N -acylethanolamine-hydrolyzing acid amidase (NAAA) overexpressing cells hydrolyzed RBM14C14 and RBM14C16 at acidic pH. Overall, NC, ACER3, and, to a lesser extent, NAAA hydrolyze fluorogenic RBM14 compounds. Although the selectivity of the substrates toward ceramidases can be modulated by the length of the N -acyl chain, none of them was specific for a particular enzyme. Despite the lack of specificity, these substrates should prove useful in library screening programs aimed at identifying potent and selective inhibitors for NC and ACER3. Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.This work was supported, in whole or in part, by the Spanish Ministry of Economy and Competitiveness (Grants SAF2011-22444 and CTQ2014-54743-R to G.F.), the Fundació la Marató de TV3 (Grants 112130 to J.C. and 112132), Agència de Gestió d’Ajuts Universitaris i de Recerca of Generalitat de Catalunya (Grant 2009SGR1072 to G.F.), National Cancer Institute (Grant NCI PO1 97132 to Y.A.H.), and National Institutes of Health (Grant R01CA163825 to C.M.). L.C. and F.C. were supported by predoctoral fellowships and C.B. was supported by postdoctoral contracts awarded by the Generalitat de Catalunya.Peer reviewe

Methuosis Contributes to Jaspine-B-Induced Cell Death

Methuosis is a type of programmed cell death in which the cytoplasm is occupied by fluid-filled vacuoles that originate from macropinosomes (cytoplasmic vacuolation). A few molecules have been reported to behave as methuosis inducers in cancer cell lines. Jaspine B (JB) is a natural anhydrous sphingolipid (SL) derivative reported to induce cytoplasmic vacuolation and cytotoxicity in several cancer cell lines. Here, we have investigated the mechanism and signalling pathways involved in the cytotoxicity induced by the natural sphingolipid Jaspine B (JB) in lung adenocarcinoma A549 cells, which harbor the G12S K-Ras mutant. The effect of JB on inducing cytoplasmic vacuolation and modifying cell viability was determined in A549 cells, as well as in mouse embryonic fibroblasts (MEF) lacking either the autophagy-related gene ATG5 or BAX/BAK genes. Apoptosis was analyzed by flow cytometry after annexin V/propidium iodide staining, in the presence and absence of z-VAD. Autophagy was monitored by LC3-II/GFP-LC3-II analysis, and autophagic flux experiments using protease inhibitors. Phase contrast, confocal, and transmission electron microscopy were used to monitor cytoplasmic vacuolation and the uptake of Lucifer yellow to assess macropinocyosis. We present evidence that cytoplasmic vacuolation and methuosis are involved in Jaspine B cytotoxicity over A549 cells and that activation of 5' AMP-activated protein kinase (AMPK) could be involved in Jaspine-B-induced vacuolation, independently of the phosphatidylinositol 3-kinase/protein kinase B/mechanistic target of rapamycin complex 1 (PI3K/Akt/mTORC1) axis.This research was funded by MCIN/AEI/10.13039/501100011033 and by the European Union (“ERDF A way of making Europe”), grants number CTQ2017-85378-R and PID2020-113813RB-I00 (to G.F.) and PID2019-107561RB-I00 (to J.M.L.).Peer reviewe