Inhibition of Sema-3A Promotes Cell Migration, Axonal Growth, and Retinal Ganglion Cell Survival

Semaphorin 3A (Sema-3A) is a secreted protein that deflects axons from inappropriate regions and induces neuronal cell death. Intravitreal application of polyclonal antibodies against Sema-3A prevents loss of retinal ganglion cells ensuing from axotomy of optic nerves. This suggested a therapeutic approach for neuroprotection via inhibition of the Sema-3A pathway.Funded by the EU seventh framework program, Grant Agreement #604884.Peer reviewe

Allosteric positive interaction of thymol with the GABA(A) receptor in primary cultures of mouse cortical neurons

11 pages, 8 figures, 2 tables.Thymol is a naturally occurring phenolic monoterpene known for its anti-microbial and anti-oxidant properties. It is used in dental practice and in anaesthetic halothane preparations. Recent studies have reported enhanced GABAA receptor-operated chloride channel activity and increased binding affinity of [3H]flunitrazepam in the presence of thymol. In the present work, we more closely examined the pharmacological action of thymol on the native GABAA receptor by using primary cultures of cortical neurons. Thymol enhanced GABA-induced (5 μM) chloride influx at concentrations lower than those exhibiting direct activity in the absence of GABA (EC50 = 12 μM and 135 μM, respectively). This direct effect was inhibited by competitive and non-competitive GABAA receptor antagonists. Thymol increased [3H]flunitrazepam binding (EC50 = 131 μM) and showed a tendency to increase [3H]muscimol binding. These results confirm that thymol is a positive allosteric modulator of the GABAA receptor. The thymol structural analogues menthol and cymene, which lack an aromatic ring or a hydroxyl group, did not affect [3H]flunitrazepam binding. Using a pharmacophoric model that includes a hydrogen bond donor group as well as an aromatic ring with two aliphatic substituents, we propose to demonstrate the molecular essential features of these compounds to interact with GABAA receptors. Thymol (0–1 mM) did not affect cellular viability.This work was supported by grant SAF 2003-4930 (Spanish Ministry of Science and Technology, cofinanced with FEDER funds). D.A.G. is a member of CONICET (Argentina) and during this work was the receipt of a postdoctoral fellowship from the Fundación Carolina (Spain).Peer reviewe

Structure-guided redesign of D-fructose-6-phosphate aldolase from E. coli: remarkable activity and selectivity towards acceptor substrates by two-point mutation

Structure-guided re-design of the acceptor binding site of D-fructose-6-phosphate aldolase from E. coli leads to the construction of FSA A129S/A165G double mutant with an activity between 5- to >900-fold higher than that of wild-type towards N-Cbz-aminoaldehyde derivatives.Peer reviewe

Studies on toxic oil syndrome: stereoselective hydrolysis of 3-(phenylamino)propane-1,2-diol esters by human pancreatic lipase

7 pages, 5 figures, 2 charts, 1 table.-- PMID: 15257613 [PubMed].-- Printed version published Jul 15, 2004.-- Supporting information available at: http://pubs.acs.org/doi/suppl/10.1021/tx049952zThe ingestion of rapeseed oil batches denatured with aniline and illegally refined and distributed by street vendors was responsible for toxic oil syndrome (TOS), an intoxication episode that took place in Spain in 1981, causing over 400 deaths and affecting more than 20 000 people. Despite the intense research efforts carried out to date, the compounds responsible for that intoxication have not been elucidated. Nevertheless, epidemiological studies have pointed to fatty acid mono- and diesters of 3-phenylamino-1,2-propanediol (PAP) as the biomarkers of those toxic oil batches. The structure of these esters bears common features with that of triglycerides, which suggested that PAP esters could follow the route of lipids metabolism up to a certain extent. The incubation of racemic PAP dioleyl ester with human pancreatic lipase (hPL) led to the formation of the corresponding stereoisomeric monoesters bearing the oleyl residue at C-2, although a kinetic resolution in favor of the (S)-enantiomer was observed. These monoesters are unstable and in equilibrium with their corresponding regioisomers with the acyl residue at C-1, apparently without the intervention of the lipase. Finally, incubations of these latter monoesters with hPL led to the formation of the respective PAP enantiomers. Again, the kinetic resolution of this hydrolytic process favored the formation of the enantiomer with the (S)-configuration. Taken together, these results showed that PAP esters are substrates of hPL and that the two hydrolytic steps exhibit kinetic resolution in favor of the (S)-enantiomers.Financial support from the WHO/TOS Committee is gratefully acknowledged.Peer reviewe

Allopregnanolone and pregnanolone analogues modified in the C ring: Synthesis and activity

El pdf es la versión post-print.(25R)-3β-Hydroxy-5α-spirostan-12-one (hecogenin) and 11α-hydroxypregn-4-ene-3,20-dione (11α-hydroxyprogesterone) were used as starting materials for the synthesis of a series of 11- and 12-substituted derivatives of 5ξ-pregnanolone (3α-hydroxy-5α- pregnan-20-one and 3α-hydroxy-5β-pregnan-20-one), the principal neurosteroid acting via γ-aminobutyric acid (GABA). These analogues were designed to study the structural requirements of the corresponding GABA A receptor. Their biological activity was measured by in vitro test with [3H]flunitrazepam as radioligand in which allopregnanolone and its active analogues stimulated the binding to the GABAA receptor. Analysis of the SAR data suggests dependence of the flunitrazepam binding activity on the hydrophobic-hydrophilic balance of the groups at the C-ring edge rather than on specific interactions between them and the receptor. © 2013 American Chemical Society.This work was supported by Czech Grant 303/12/1464, Research Project of the AS CR RVO 61388963, and the Spanish Grants PI 061212 and PI 10/0453 from the Ministries of Health and of Science and Innovation, Grant 2009/SGR/214 from the Generalitat of Catalunya, and Grant 2006CZ0025 from CSIC.Peer Reviewe

Biosynthesis of 10,12-dienoic fatty acids by a bifunctional Delta11 desaturase in Spodoptera littoralis

8 pages, 4 figures.-- PMID: 16876706 [PubMed].-- Printed version published Aug 2006.In the biosynthetic pathway of Spodoptera littoralis sex pheromone, (E,E)-10,12-tetradecadienoic acid is produced from (Z)-11-tetradecenoic acid by desaturation and concomitant migration of the precursor double bond. With the aim of identifying the enzyme involved in this biotransformation, yeast Δelo1/Δole mutants, which are both elongase 1 and Δ9 desaturase-deficient, were transformed with the S. littoralis Δ11 desaturase gene using a Cu+2 inducible expression vector. The transformants produced a recombinant polyhistidine-tagged Δ11 desaturase that could be detected by immunoblotting from cell lysates. Lipid analysis revealed that besides producing large quantities of C11-monounsaturated fatty acids, mainly (Z)-11-hexadecenoic acid, (E,E)-10,12-tetradecadienoic acid and minor amounts of (E,Z)-10,12-hexadecadienoic acid were also produced, as well as very low quantities of another tetradecadienoate, which was tentatively identified as the (E,Z)-10,12-tetradecadienoic isomer. None of these dienes was detected with the Δ11 desaturase gene of Trichoplusia ni, which does not produce conjugated dienes as pheromone components. We conclude that the Δ11 desaturase of S. littoralis is a bifunctional enzyme with both Δ11 and Δ10,12 desaturation activities. The relationship between the substrate structure and the stereochemical outcome of the reaction is discussed.We thank Shogo Matsumoto and Ken’ichi Moto (Molecular Entomology Laboratory, The Institute of Physical and Chemical Research, Wako, Saitama, Japan) for the 10,12-hexadecadienoate standards, Caterina Holz (Proteinstrukturfabrik, Berlin University of Technology, Berlin, Germany) for the pYEXTHS-BN vector and Douglas C. Knipple (Department of Entomology, New York State Agricultural Experiment Station, Cornell University, Geneva, New York, USA.) for the YEpOLEX-CLR7 plasmid. This work was supported by grants from the Spanish Ministry of Science and Technology (Grant AGF 2001-0585), Generalitat de Catalunya (Grant 2001 SGR-00342) and FEDER funds from EU. A predoctoral fellowship from the Ministry of Science and Technology to M.S. is acknowledged.Peer reviewe

Click Chemistry Approach to New N-Substituted Aminocyclitols as Potential Pharmacological Chaperones for Gaucher Disease

New N-alkylaminocyclitols bearing a 1,2,3-triazole system at different positions of the alkyl chain have been prepared as potential GCase pharmacological chaperones using click chemistry approaches. Among them, compounds 1d and 1e, with the shorter spacer (n = 1) between the alkyltriazolyl system and the aminocyclitol core, were the most active ones as GCase inhibitors, revealing a determinant effect of the location of the triazole ring on the activity. Furthermore, SAR data and computational docking models indicate a correlation between lipophilicity and enzyme inhibition and suggest “extended” and “bent” potential binding modes for the compounds. In the “bent” mode, the most active compounds could establish a hydrogen-bond interaction between the triazole moiety and enzyme residue Q284. Such an interaction would be precluded in compounds with a longer spacer between the triazole and the aminocyclitol core.Partial financial support from the “Ministerio de Ciencia e Innovación”, Spain (Project CTQ2008-01426/BQU), and “Generalitat de Catalunya” (Grant 2009SGR-1072) is acknowledged. L.D. is grateful to CSIC for predoctoral research training support within the JAE-Predoc program. The authors thank Dr. Meritxell Egido-Gabas for technical assistance, Núria Guillem and Laura Planas for experimental contributions, Eva Dalmau for HRMS analysis, and Genzyme Corp. for a generous supply of imiglucerase (Cerezyme). Finally, the authors acknowledge the “Centre de Supercomputació de Catalunya” (CESCA) for allowing the use of its software and hardware resources.Peer reviewe

Highly efficient aldol additions of DHA and DHAP to N-Cbz-amino aldehydes catalyzed by L-rhamnulose-1-phosphate and L-fuculose-1-phosphate aldolases in aqueous borate buffer

Aldol addition reactions of dihydroxyacetone (DHA) to N-Cbz-amino aldehydes catalyzed by L-rhamnulose-1-phosphate aldolase (RhuA) in the presence of borate buffer are reported. High yields of aldol adduct (e.g. 70–90%) were achieved with excellent (>98 : 2 syn/anti) stereoselectivity for most S or R configured acceptors, which compares favorably to the reactions performed with DHAP. The stereochemical outcome was different and depended on the N-Cbz-amino aldehyde enantiomer: the S acceptors gave the syn (3R,4S) aldol adduct whereas the R ones gave the anti (3R,4R) diastereomer. Moreover, the tactical use of Cbz protecting group allows simple and efficient elimination of borate and excess of DHA by reverse phase column chromatography or even by simple extraction. This, in addition to the use of unphosphorylated donor nucleophile, makes a useful and expedient methodology for the synthesis of structurally diverse iminocyclitols. The performance of aldol additions of dihydroxyacetone phosphate (DHAP) to N-Cbz-amino aldehydes using RhuA and L-fuculose-1-phosphate aldolase (FucA) catalyst in borate buffer was also evaluated. For FucA catalysts, including FucA F131A, the initial velocity of the aldol addition reactions using DHAP were between 2 and 10 times faster and the yields between 1.5 and 4 times higher than those in triethanolamine buffer. In this case, the retroaldol velocities measured for some aldol adducts were lower than those without borate buffer indicating some trapping effect that could explain the improvement of yields.This work was supported by the Spanish MICINN projects CTQ2009-07359 and CTQ2009-08328, Generalitat de Catalunya (2009SGR00281), and ESF project COST CM0701. X. Garrabou acknowledges the CSIC for an I3P predoctoral scholarship.Peer reviewe

Optimizing the control of apoptosis by amide/triazole isosteric substitution in a constrained peptoid

Apoptosis is a biological process relevant to several human diseases that is strongly regulated through protein–protein complex formation. We have previously reported a peptidomimetic compound as potent apoptotic modulator. Structural studies of this compound showed the presence of cis/trans isomers of the exocyclic tertiary amide bond in slow exchange. This information encouraged us to perform an isosteric replacement of the amide bond by a 1,2,3-triazole moiety, where different substitution patterns would mimic different amide rotamers. The syntheses of these restricted analogs have been carried out using the Ugi multicomponent reaction followed by an intramolecular cyclization. Unexpectedly, for one of the proposed structures, a novel β -lactam compound was formed. All compounds showed to efficiently inhibit apoptosis, in vitro and in cellular extracts, with slight differences for the corresponding regioisomers. We propose the binding to Apaf-1 as the inhibition mechanism.This work was supported by grants from the Spanish Ministry of Science and Innovation (SAF2008-00048, SAF30542-C01-01 and SAF2010-15512) and a fellowship to M. Corredor from CSIC JAE program.Peer reviewe

New Glucocerebrosidase Inhibitors by Exploration of Chemical Diversity of N-Substituted Aminocyclitols Using Click Chemistry and in Situ Screening

A library of aminocyclitols derived from CuAAC reaction between N-propargylaminocyclitol 4 and a series of azides [1−25] is described and tested against GCase. Azides have been chosen from a large collection of potential candidates that has been filtered according to physical and reactivity constraints. A synthetic methodology has been optimized in order to avoid the use of protecting groups on the aminocyclitol scaffold. Because the reaction can be carried out in an aqueous system, the resulting library members can be screened in situ with minimal manipulation. From the preliminary GCase inhibition data, the most potent library members have been individually resynthesized for further biological screening and complete characterization. Some of the library members have shown biochemical data (IC50, Ki, and stabilization ratio) similar or superior to those reported for NNDNJ. Docking studies have been used to postulate ligand−enzyme interactions to account for the experimental results.Partial financial support from the Ministerio de Ciencia e Innovación, Spain (project CTQ2008-01426/BQU) and Generalitat de Catalunya (Grant 2009SGR-1072) is acknowledged. Lucía Díaz is grateful to CSIC for predoctoral research training support within the JAE-Predoc program. The authors thank Dr. Meritxell Egido-Gabas for technical assistance, Eva Dalmau for HRMS analysis, and Genzyme Corporation for a generous supply of Imiglucerase (Cerezyme). We also acknowledge the Centre de Supercomputació de Catalunya (CESCA) for allowing the use of their software and hardware resources.Peer reviewe