Meritxell Egido-Gabás

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.

Dihydroxyacetone Phosphate Aldolase Catalyzed Synthesis of Structurally Diverse Polyhydroxylated Pyrrolidine Derivatives and Evaluation of their Glycosidase Inhibitory Properties

The chemoenzymatic synthesis of a collection of pyrrolidine-type iminosugars generated by the aldol addition of dihydroxyacetone phosphate (DHAP) to C-alpha-substituted N-Cbz-2-aminoaldehydes derivatives, catalyzed by DHAP aldolases is reported. L-fuculose-1-phosphate aldolase (FucA) and L-rhamnulose-1-phosphate aldolase (RhuA) from E. coli were used as biocatalysts to generate configurational diversity on the iminosugars. Alkyl linear substitutions at C-alpha were well tolerated by FucA catalyst (i.e., 40-70 % conversions to aldol adduct), whereas no product was observed with C-alpha-alkyl branched substitutions, except for dimethyl and benzyl substitutions (20 %). RhuA was the most versatile biocatalyst: C-alpha-alkyl linear groups gave the highest conversions to aldol adducts (60-99 %), while the C-alpha-alkyl branched ones gave moderate to good conversions (50-80 %), with the exception of dimethyl and benzyl substituents (20 %). FucA was the most stereoselective biocatalyst (90-100 % anti (3R,4R) adduct). RhuA was highly stereoselective with (S)-N-Cbz-2-aminoaldehydes (90-100 % syn (i.e., 3R,4S) adduct), whereas those with R configuration gave mixtures of anti/syn adducts. For iPr and iBu substituents, RhuA furnished the anti adduct (i.e., FucA stereochemistry) with high stereoselectivity. Molecular models of aldol products with iPr and iBu substituents and as complexes with the RhuA active site suggest that the anti adducts could be kinetically preferred, while the syn adducts would be the equilibrium products. The polyhydroxylated pyrrolidines generated were tested as inhibitors against seven glycosidases. Among them, good inhibitors of alpha-L-fucosidase (IC50=1-20 microM), moderate of alpha-L-rhamnosidase (IC50=7-150 microM), and weak of alpha-D-mannosidase (IC50=80-400 microM) were identified. The apparent inhibition constant values (Ki) were calculated for the most relevant inhibitors and computational docking studies were performed to understand both their binding capacity and the mode of interaction with the glycosidases.

Promising results of the chaperone effect caused by iminosugars and aminocyclitol derivatives on mutant glucocerebrosidases causing Gaucher disease

Gaucher disease is an autosomal recessive disorder. It is characterized by the accumulation of glucosylceramide in lysosomes of mononuclear phagocyte system, attributable to acid beta-glucosidase deficiency. The main consequences of this disease are hepatosplenomegaly, skeletal lesions and, sometimes, neurological manifestations. At sub-inhibitory concentrations, several competitive inhibitors act as chemical chaperones by inducing protein stabilization and increasing enzymatic activity. Here we tested two iminosugars (NB-DNJ and NN-DNJ) and four aminocyclitols with distinct degrees of lipophilicity as pharmacological chaperones for glucocerebrosidase (GBA). We report an increase in the activity of GBA using NN-DNJ, NB-DNJ and aminocyclitol 1 in stably transfected cell lines, and an increment with NN-DNJ and aminocyclitol 4 in patient fibroblasts. These results on specific mutations validate the use of chemical chaperones as a therapeutic approach for Gaucher disease. However, the development and analysis of new compounds is required in order to find more effective therapeutic agents that are active on a broader range of mutations.

Molecular Basis for β-Glucosidase Inhibition by Ring-Modified Calystegine Analogues

Calystegines, polyhydroxy nortropane alkaloids first isolated from the extracts of Calystegia sepium, represent the most recently discovered members of the azasugar and iminosugar glycomimetic families. 3] Similar to the well-studied polyhydroxypiperidine, indolizidine, pyrrolidine and pyrrolizidine-type iminocyclitols, calystegines exhibit potent glycoside hydrolase (glycosidase) inhibitory properties. Given the broad range of biological events in which glycosidases are involved, inhibitors of these enzymes have potential in therapies that are targeted at, for example, cancer, viral infections, tuberculosis, diabetes and glycosphingolipid storage disorders. The molecular basis for glycosidase inhibition by the calystegines remain poorly understood. Recent structural and mechanistic studies on a b-glucosidase from Thermotoga maritima, TmGH1, which belongs to family 1, clan GH-A, in the CAZy classification, showed unambiguously that calystegine B2 (1; Scheme 1), one of the most powerful representatives, binds at

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

Accumulated Bending Energy Elicits Neutral Sphingomyelinase Activity in Human Red Blood Cells

We propose that accumulated membrane bending energy elicits a neutral sphingomyelinase (SMase) activity in human erythrocytes. Membrane bending was achieved by osmotic or chemical processes, and SMase activity was assessed by quantitative thin-layer chromatography, high-performance liquid chromatography, and electrospray ionization-mass spectrometry. The activity induced by hypotonic stress in erythrocyte membranes had the pH dependence, ion dependence, and inhibitor sensitivity of mammalian neutral SMases. The activity caused a decrease in SM contents, with a minimum at 6 min after onset of the hypotonic conditions, and then the SM contents were recovered. We also elicited SMase activity by adding lysophosphatidylcholine externally or by generating it with phospholipase A(2). The same effect was observed upon addition of chlorpromazine or sodium deoxycholate at concentrations below the critical micellar concentration, and even under hypertonic conditions. A unifying factor of the various agents that elicit this SMase activity is the accumulated membrane bending energy. Both hypo-and hypertonic conditions impose an increased curvature, whereas the addition of surfactants or phospholipase A(2) activation increases the outer monolayer area, thus leading to an increased bending energy. The fact that this latent SMase activity is tightly coupled to the membrane bending properties suggests that it may be related to the general phenomenon of stress-induced ceramide synthesis and apoptosis.

Synthesis and evaluation of hydroxymethylaminocyclitols as glycosidase inhibitors

Four series of C7N aminocyclitol analogues of glucose were synthesized by stereocontrolled epoxide opening of hydroxyl protected forms of the cyclohexane epoxides cyclophellitol and 1,6-epi-cyclophellitol. The resulting hydroxymethyl substituted aminocyclitols were tested as glycosidase inhibitors. Cyclitols having an amino group in an α configuration at a position equivalent to the anomeric in the sugar were found to be low micromolar inhibitors of the α-glucosidase from bakers yeast with Kis near to 2 μM. On the other hand, N-octyl aminocyclitols having the nitrogen substituents in an α or β configuration were found to be good inhibitors of recombinant β-glucocerebrosidase with Ki values between 8.3 and 17 μM, and also inhibited lysosomal β-glucosidase activity in live cells at low-micromolar concentrations. A computational docking study suggests a differential binding among the different series of β-glucocerebrosidase inhibitors. In agreement with the experimental results, the binding poses obtained indicate that the presence of an alkyl lipid substituent in the inhibitor mimicking one of the lipid chains in the substrate is critical for potency. In contrast, the matching of hydroxymethyl substituents in the aminocyclitols and the parent glucosylceramide does not seem to be strictly necessary for potent inhibition, indicating the risk of simplifying structural analogies in sugar mimetic design.

Adamantane substituted aminocyclitols as pharmacological chaperones for Gaucher disease

Gaucher disease (GD), resulting from deficient lysosomal enzyme β-glucosidase (GCase) activity, is the most common lysosomal storage disorder. We have previously shown that aminocyclitol derivatives displayed selective inhibition of GCase and enhanced GCase activity in N370S and L444P at very low concentrations. In the present study, we combined amino-myo-inositol and amino-scyllo-inositol cores with a hydrophobic alkyl adamantyl amide to afford novel small molecules with enhanced ability to increase GCase activity in GD lymphoblasts. The most potent inhibitor, amino-myo-inositol 2, displayed a Ki value of 250 nM in isolated enzyme. This compound produced a maximum increase of GCase activity of 64% in N370S lymphoblasts at 1 μM and 150% in L444P at 100 μM following a 3 day incubation.

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.

Corrigendum to “An unexpected access to 5-epi-cyclophellitol, a new cyclitol member” [Tetrahedron: Asymmetry 18/16 (2007) 1971]

Corrigendum Corrigendum to ‘‘An unexpected access to 5-epi-cyclophellitol, a new cyclitol member” [Tetrahedron: Asymmetry 18/16 (2007) 1971] Pedro Serrano , Meritxell Egido-Gabas , Amadeu Llebaria , Antonio Delgado a,b,* Research Unit on Bioactive Molecules (RUBAM), Department of Biological Organic Chemistry, Chemical and Environmental Research Institute of Barcelona (IIQAB-C.S.I.C), Jordi Girona 18-26, 08034 Barcelona, Spain University of Barcelona, Faculty of Pharmacy, Unit of Pharmaceutical Chemistry (CSIC Associated Unit), Avda. Joan XXIII, s/n, 08028 Barcelona, Spain