Chemical tools for modulating autophagy

Autophagy (or self-eating) is a cellular pathway that regulates the lysosomal degradation and recycling of obsolete organelles, long-lived proteins, protein aggregates and pathogens. This process occurs under basal conditions and has a crucial role in cellular development, differentiation, survival and homeostasis. In this review, an overview will be given on the autophagy modulators described up to now with the main purpose of giving the chemists interested in this research field a broad overview on the existing autophagy modulators and the methods employed for their identification. Most of the methods employed to monitor autophagy rely on the detection of the lipidated autophagy marker LC3-II (MAP1LC3, microtubule-associated proteins 1A/1B light chain 3, see below) employing antibodies recognizing both the non-lipidated LC3-I as well as LC3-II.Peer reviewe

Inhibidores de la dihidroceramida desaturasa

Referencia OEPM: P200003045.-- Fecha de solicitud: 19/12/2000.-- Titular: Consejo Superior de Investigaciones Científicas (CSIC).Inhibidores de la dihidroceramida desaturasa (ver figura en archivo de texto adjunto). Derivados de ciclopropenilceramida como inhibidores de desaturasas caracterizados por la fórmula general (I), en la que R1 puede ser un grupo alquilo, alquenilo, alquinilo, arilo o cualquier heterociclo, n puede tener cualquier valor y estar ramificada o no y contener insaturaciones y R2 y R3 pueden tener el mismo o distinto valor, representan grupos arilo, heteroarilo, alquilo o acilo con una o varias insaturaciones en la cadena, que puede estar ramificada o no, y sustituida con grupos OH. Miembros de esta nueva clase de compuestos han mostrado una extraordinaria actividad como inhibidores de la dihidroceramida desaturasa y de la biosíntesis de ceramida y son de utilidad para el tratamiento de patologías asociadas a aumentos en los niveles de ceramida intracelular.Peer reviewe

Combinatorial Synthesis and Biological Evaluation of Inhibitors of D-Ala-D-Ala-Ligase

Report for the scientific sojourn carried out at the Max Planck Institut of Molecular Phisiology, Germany, from 2006 to 2008.The work carried out during this postdoctoral stage was focused on two different projects. Firstly, identification of D-Ala D-Ala Inhibitors and the development of new synthethic approaches to obtain lipidated peptides and proteins and the use of these lipidated proteins in biological and biophysical studies. In the first project, new D-Ala D-Ala inhibitors were identified by using structural alignments of the ATP binding sites of the bacterial ligase DDl and protein and lipid kinases in complex with ATP analogs. We tested a series of commercially available kinase inhibitors and found LFM-A13 and Tyrphostine derivatives to inhibit DDl enzyme activity. Based on the initial screening results we synthesized a series of malononitrilamide and salicylamide derivatives and were able to confirm the validity of these scaffolds as inhibitors of DDl. From this investigation we gained a better understanding of the structural requirements and limitations necessary for the preparation of ATP competitive DDl inhibitors. The compounds in this study may serve as starting points for the development of bi-substrate inhibitors that incorporate both, an ATP competitive and a substrate competitive moiety. Bisubstrate inhibitors that block the ATP and D-Ala binding sites should exhibit enhanced selectivity and potency profiles by preferentially inhibiting DDl over kinases. In the second project, an optimized synthesis for tha alkylation of cysteins using the thiol ene reaction was establisehd. This new protocol allowed us to obtain large amounts of hexadecylated cysteine that was required for the synthesis of differently lipidated peptides. Afterwards the synthesis of various N-ras peptides bearing different lipid anchors was performed and the peptides were ligated to a truncated N-ras protein. The influence of this differently lipidated N-ras proteins on the partioning and association of N-Ras in model membrane subdomains was studied using Atomic Force Microscopy.Estudi realitzat a partir d’una estada al Max Planck Institut of Molecular Phisiology, Alemanya, entre 2006 i 2008. S’han realitzat dos projectes. En primer lloc la identificació de nous lligands per a proteïnes gràcies a una nova aproximació que combina l'ús de mètodes computacionals amb el desenvolupament de llibreries basades en productes naturals. Segons aquesta aproximació, la similitud estructural existent entre diferents proteïnes pot ser utilitzada com a punt de partida per l'obtenció de nous lligands. Així, un lligand conegut d'una proteïna pot servir com a punt de partida per a l'obtenció de lligands per altres proteïnes que formin part del mateix "cluster". Aquest projecte que requereix l'ús de mètodes multidisciplinaris com la síntesi orgànica, el desenvolupament d'assaigs in vitro i l'expressió i purificació de proteïnes entre d' altres, ha permès trobar nous inhibidors per a l'enzim DD-ligasa en base a la seva similitud estructural amb diferents kinases com p56lck o cdc2. El segon projecte dur a terme durant aquesta estada postdoctoral s´ha basat en el desenvolupament de nous mètodes de síntesi de proteines lipidades per a utilitzar-les posteriorment com a eina química en estudis bioquímics, biofísics o de l'àmbit de la biologia cel•lular. Concretament el projecte s' ha centrat en el desenvolupament de nous mètodes de síntesi de la proteïna Ras lipidada així com el posterior estudi de la distribució de Ras en els diferents microdominis de membranes que componen les cèl•lules, mitjançant espectroscòpia de fluorescència o microscopi de forca atòmica, entre d'altres

Procedimiento para la obtención de compuestos derivados de ciclopropenilesfingosina

Referencia OEPM: P200402456.-- Fecha de solicitud: 15/10/2004.-- Titular: Consejo Superior de Investigaciones Científicas (CSIC).Procedimiento para la obtención de compuestos derivados de ciclopropenilesfingosina. En la presente invención se describe un nuevo procedimiento de síntesis de derivados de ciclopropenilesfingosina, los cuales pueden ser utilizados en la elaboración de composiciones farmacéuticas con distintas aplicaciones.Peer reviewe

Analogs of the dihydroceramide desaturase inhibitor GT11 modified at the amide function: synthesis and biological activities

6 pages, 3 figures, 2 schemes, 1 table.-- PMID: 16211106 [PubMed].-- Printed version published Sep 2005.Dihydroceramide desaturase is the last enzyme in the biosynthesis of ceramide de novo. The cyclopropene-containing sphingolipid GT11 is a competitive inhibitor of dihydroceramide desaturase. The biological effects of chemical modification of the GT11 amide linkage are reported in this article. Either N-methyl substitution or replacement of the amide -carbonyl methylene by oxygen result in inactive compounds. In contrast, both urea (3) and thiourea (4) analogs of GT11, as well as three -ketoamides (5–7), did inhibit the desaturation of N-octanoylsphinganine to N-octanoylsphingosine, although with significantly lower potency than GT11. Furthermore, the -ketoamides 5–7 inhibit the acidic ceramidase with similar potencies (IC50 52–83 µM). Inhibition of the neutral/alkaline ceramidase by these compounds requires around 20-fold higher concentrations. Structure–activity relationships and the biological interest of these compounds are discussed.This work was supported in part by MCYT (grants BQU2002-03737). GT thanks Genzyme for partial support of a CSIC I3P contract and CB thanks MECD for a predoctoral FPU fellowshipPeer reviewe

Palmitoylation as a Key Regulator of Ras Localization and Function

Ras proteins require membrane association for proper function. This process is tightly regulated by reversible palmitoylation that controls not only the distribution over different subcellular compartments but also Ras compartmentalization within membrane subdomains. As a result, there is a growing interest in protein palmitoylation and the enzymes that control this process. In this minireview, we discuss how palmitoylation affects the localization and function of Ras proteins. A better understanding of the regulatory mechanism controlling protein lipidation is expected to provide new insights into the functional role of these modifications and may ultimately lead to the development of novel therapeutic approaches.Financial support from the Caixa Foundation under the agreement LCF/PR/HR20/52400006 and the Ministerio de Ciencia e Innocación (RTI2018-096323-b-100) are gratefully acknowledged.Peer reviewe

Utilización de derivados de ciclopropenilesfingosina para la elaboración de una composición farmacéutica moduladora de la actividad de ceramidasa, y sus aplicaciones

Referencia OEPM: P200302767.-- Fecha de solicitud: 25/11/2003.-- Titular: Consejo Superior de Investigaciones Científicas (CSIC).Utilización de derivados de ciclopropenilesfingosina para la elaboración de una composición farmacéutica moduladora de la actividad de ceramidasa, y sus aplicaciones. La presente invención describe la actividad moduladora de los derivados de ciclopropenilesfingosina sobre las enzimas ceramidasas. Así, un objeto de la invención es el uso de dichos compuestos en la elaboración de una composición farmacéutica útil para el tratamiento de distintas enfermedades (enf. de Faber, enfermedades cardiovasculares, inflamatorias y cáncer). Además, forma parte de la invención el uso de dichas composiciones farmacéuticas en el tratamiento de las mencionadas enfermedades.Peer reviewe

Synthesis of cyclopropene analogues of ceramide and their effect on dihydroceramide desaturase

9 pages, 3 figures, 6 schemes, 2 charts.-- PMID: 14682684 [PubMed].-- Printed version published Dec 26, 2003.-- Supporting information available at: http://pubs.acs.org/doi/suppl/10.1021/jo030141uThe synthesis of several analogues of the N-[(1R,2S)-2-hydroxy-1-hydroxymethyl-2-(2-tridecyl-1-cyclopropenyl)ethyl]octanamide (GT11), the first reported inhibitor of dihydroceramide desaturase, as well as their effects on this enzyme, are described. Modifications of the parent structure include variations on the cyclopropene ring, the N-acyl chain length, the configuration of the stereocenters, and the hydroxyl group at C1. The key intermediates for the synthesis are the products resulting from the addition of suitable organolithium compounds to either Garner's aldehyde or its enantiomer. The final products are obtained by TMSTf-induced cleavage of the protecting groups and N-acylation, both under specific conditions. An alternative method for N-Boc deprotection is also reported that allows us to obtain the cyclopropene analogue of sphingosine 12a, which can be transformed into GT11 upon acylation. The procedure consists of the conversion of the Garner aldehyde addition products into the bicyclic dihydrooxazolo[3,4,0]oxazol-3-ones 19 by transesterification in basic medium of the tert-butyl group with the hydroxyl function at C3. Mild cleavage of the N,O-isopropylidene cyclic acetal present in 19 affords the oxazolidin-2-one 20, which gives 12a upon saponification. Furthermore, compound 20 is also the key intermediate in the synthesis of the terminal deoxy, methoxy, and fluoro derivatives 9, 10, and 11, respectively. Determination of dihydroceramide desaturase activity in vitro showed that GT11 was a competitive inhibitor (Ki = 6 μM) and that its analogues with N-hexanoyl (6) and N-decanoyl (7) moieties inhibited the enzyme with similar potencies (IC50 = 13 and 31 μM, respectively). No decrease in dihydroceramide desaturase activity was observed with any of the other compounds tested.This work was supported by Ministerio de Ciencia y Tecnología (Projects BQU2002-03737 and BQU2002-00786) and Departament d'Universitats, Recerca i Societat de la Informació, Generalitat de Catalunya (Grant No. 2001SGR00342 and Predoctoral fellowship to G.T.).Peer reviewe