Antonio Delgado

Synthesis and biological properties of Pachastrissamine (jaspine B) and diastereoisomeric jaspines

The synthesis of isomeric jaspines (anhydro phytosphingosines), arising from intramolecular cyclization of the corresponding phytosphingosines with different configurations at C3 and C4 positions of the sphingoid backbone, is reported. Natural jaspine B is the most cytotoxic isomer on A549 cells and it induces cell death in a dose-dependent manner. The cytotoxicity of jaspine B has been correlated with a significant increase of intracellular dihydroceramides, which seem to play an active role in autophagy.

Synthesis and Biological Activity of a Novel Inhibitor of Dihydroceramide Desaturase

A novel mechanism‐based dihydroceramide desaturase inhibitor (XM462) in which the substrate C5 methylene group is replaced by a sulfur atom is reported. Dihydroceramide desaturase inhibition occurred both in vitro and in cultured cells with IC50 values of 8.2 and 0.78 μM, respectively, at a substrate concentration of 10 μM. In vitro experiments showed that XM462 produced a mixed‐type inhibition (Ki=2 μM, α=0.83). LC‐MS analyses showed that accumulation of endogenous dihydroceramides occurred in cells upon treatment with XM462 in serum‐free medium, whereas ceramides built up in controls. In addition, XM462 was found to be metabolised to its 1‐glucosyl and 1‐phosphocholine derivatives, and to the products of N‐deacylation and reacylation with palmitoyl and stearoyl groups. In Jurkat A3 cells cultured in serum‐free medium, viability, as the percentage of trypan blue unstained cells in total cells, was reduced upon XM462 treatment (5 μM, 24 h), but not in controls. The interest of this compound is discussed.

Chemical tools to investigate sphingolipid metabolism and functions

Sphingolipids comprise an important group of biomolecules, some of which have been shown to play important roles in the regulation of many cell functions. From a structural standpoint, they all share a long 2‐amino‐1,3‐diol chain, which can be either saturated (sphinganine), hydroxylated at C4 (phytosphingosine), or unsaturated at C4 (sphingosine) as in most mammalian cells. N‐acylation of sphingosine leads to ceramide, a key intermediate in sphingolipd metabolism that can be enzymatically modified at the C1‐OH position to other biologically important sphingolipids, such as sphingomyelin or glycosphingolipids. In addition, both ceramide and sphingosine can be phosphorylated at C1‐OH to give ceramide‐1‐phosphate and sphingosine‐1‐phosphate, respectively. To better understand the biological and biophysical roles of sphingolipids, many efforts have been made to design synthetic analogues as chemical tools able to unravel their structure–activity relationships, and to alter their cellular levels. This last approach has been thoroughly studied by the development of specific inhibitors of some key enzymes that play an important role in biosynthesis or metabolism of these intriguing lipids. With the above premises in mind, the aim of this review is to collect, in a systematic way, the recent efforts described in the literature leading to the development of new chemical entities specifically designed to achieve the above goals.

Cytotoxicity and acid ceramidase inhibitory activity of 2-substituted aminoethanol amides.

The acid ceramidase (AC) inhibitory activity of octanoylamides, p-tert-butylbenzamides and pivaloylamides of several 2-substituted aminoethanols is reported. All the aminoethanol amides bearing a hexadecyl substituent (C16), as well as (S)-N-(1-(hexadecylthio)-3-hydroxypropan-2-yl)pivaloylamide (SC16-tb) were inhibitory in cell lysates overexpressing AC, while all other compounds were not inhibitors. Kinetic experiments with (R,E)-N-(1-hydroxyoctadec-3-en-2-yl)pivaloylamide (E-tb) and SC16-tb showed that inhibition was competitive, with K(i) values of 34 and 94.0 microM, respectively. None of the compounds inhibited neutral ceramidase. Compounds E-tb and E-c7 (the octanoylamide of the unsaturated base E), which elicited a dose-response inhibition with IC(50) values around 15 microM, were the only AC inhibitors in intact cells. Both compounds were toxic to A549 cells with LD(50) values nearly 40 microM. Flow cytometry studies with E-tb evidenced that this compound induced a concentration-dependent cell cycle arrest at G(1) and a 20-25% apoptosis/late apoptosis/necrosis after a 24-h incubation at 50 microM. In agreement with its activity as acidic ceramidase inhibitor, this effect was accompanied with an increase in the amounts of C14, C16 and C18 ceramides (LC-MS analyses), which suggested that these lipids may be responsible for the cytotoxic activity of E-tb.

Inhibition of acid ceramidase by a 2-substituted aminoethanol amide synergistically sensitizes prostate cancer cells to N-(4-hydroxyphenyl) retinamide.

The purpose of this study was to determine whether the therapeutic efficacy of fenretinide (4‐HPR), a ceramide‐generating anticancer agent, could be enhanced in prostate cancer cells by inclusion of a novel synthetic acid ceramidase (AC) inhibitor, DM102, a pivaloylamide of a 2‐substituted aminoethanol. In prostate cancer, AC plays a role in progression and resistance to chemotherapy.

Aminocyclitols as pharmacological chaperones for glucocerebrosidase, a defective enzyme in Gaucher disease.

3 pages, 4 figures.-- PMID: 17479993 [PubMed].-- Supporting information (11 pages) available at: http://www.wiley-vch.de/contents/jc_2452/2007/z700061_s.pdf

Short and enantioselective syntheses of (R)- and (S)-2-hydroxymethyl-1,4-benzodioxan

Abstract Two straightforward and highly enantioselective syntheses of ( R )- and ( S )-2-hydroxymethyl-1,4-benzodioxan from readily available chiral glycidol derivatives are described.

Acid ceramidase as a therapeutic target in metastatic prostate cancer

Acid ceramidase (AC) catalyzes the hydrolysis of ceramide into sphingosine, in turn a substrate of sphingosine kinases that catalyze its conversion into the mitogenic sphingosine-1-phosphate. AC is expressed at high levels in several tumor types and has been proposed as a cancer therapeutic target. Using a model derived from PC-3 prostate cancer cells, the highly tumorigenic, metastatic, and chemoresistant clone PC-3/Mc expressed higher levels of the AC ASAH1 than the nonmetastatic clone PC-3/S. Stable knockdown of ASAH1 in PC-3/Mc cells caused an accumulation of ceramides, inhibition of clonogenic potential, increased requirement for growth factors, and inhibition of tumorigenesis and lung metastases. We developed de novo ASAH1 inhibitors, which also caused a dose-dependent accumulation of ceramides in PC-3/Mc cells and inhibited their growth and clonogenicity. Finally, immunohistochemical analysis of primary prostate cancer samples showed that higher levels of ASAH1 were associated with more advanced stages of this neoplasia. These observations confirm ASAH1 as a therapeutic target in advanced and chemoresistant forms of prostate cancer and suggest that our new potent and specific AC inhibitors could act by counteracting critical growth properties of these highly aggressive tumor cells.

Sphingosine 1-Phosphate Regulation of Extracellular Signal-Regulated Kinase-1/2 in Embryonic Stem Cells

Recent evidence suggests that sphingosine 1-phosphate (S1P) regulates self-renewal of human embryonic stem (ES) cells and differentiation of mouse embryoid bodies (derived from mouse ES cells) to cardiomyocytes. We have investigated the role of S1P in regulating ERK-1/2 signaling in mouse ES cells. In this regard, we found that both mouse ES-D3 and CGR8 cells express S1P(1), S1P(2), S1P(3), and S1P(5) but lack S1P(4). The treatment of ES cells with S1P induced the activation of ERK-1/2 via a mechanism that was not mediated by S1P(1), S1P(2), or S1P(3). This was based on: (i) the failure of S1P(1), S1P(2), or S1P(3) antagonists to inhibit S1P-stimulated ERK-1/2 activation and (ii) the failure of SEW 2871 (S1P(1) receptor agonist) to stimulate ERK-1/2 activation. The treatment of ES cells with phytosphingosine 1-phosphate (phyto-S1P), which we show here is an agonist of the S1P(5) receptor, stimulated ERK-1/2 activation. These findings therefore suggest that S1P(5) may mediate the effects of S1P in terms of regulating ERK-1/2 signaling in ES cells. The S1P-dependent activation of ERK-1/2 was sensitive to inhibition by pertussis toxin (uncouples the G-protein, G(i) from GPCR), bisindolylmaleimide I (PKC inhibitor), and PP2 (c-Src inhibitor), but was not reduced by LY29004 (PI3K inhibitor) suggesting that S1P uses G(i)-, PKC-, and c-Src-dependent mechanisms to activate the ERK-1/2 pathway in ES cells.

Synthesis of a fluorogenic analogue of sphingosine-1-phosphate and its use to determine sphingosine-1-phosphate lyase activity

Illuminating an ER enzyme: We report on the design and synthesis of a fluorogenic chemical sensor (1) to measure sphingosine‐1‐phosphate lyase activity in high‐throughput screening formats, as well as its validation using lyase knockout (Sgpl1−/−) cells.