Maria Garrido

Azide-tagged sphingolipids: new tools for metabolic flux analysis.

Several diseases involve alterations in sphingolipid metabolism, so the development of tools for the analysis of sphingolipid metabolic fluxes is of interest. In this work, ω‐azidosphingolipids 1–3 have been synthesized and tested as tracers in live cells. The synthesis starts from (S)‐Garners aldehyde and uses bromide or tosyloxy precursors for the introduction of the azido group into the sphingoid base. Studies in HGC‐27 cells showed that probes 1–3 compete with the natural metabolites and are incorporated into sphingolipid pathways without affecting cell viability. The reactivity and bioorthogonality of the terminal azido group have been exploited by means of click reactions with different azadibenzocyclooctyne tags. This allows the mass spectrometric characterization of azidosphingolipidomes in pooled samples from different cell populations after independent treatments, providing proof of concept of the applicability of this technology in sphingolipid metabolic flux analysis.

Efficient Synthesis of Conformationally Restricted Apoptosis Inhibitors Bearing a Triazole Moiety

Apoptosis is a biological process relevant to different human diseases that is regulated through protein-protein interactions and complex formation. Peptidomimetic compounds based on linear peptoids and cyclic analogues with different ring sizes have been previously reported as potent apoptotic inhibitors. Among them, the presence of cis/trans conformers of an exocyclic tertiary amide bond in slow exchange has been characterized. This information encouraged us to perform an isosteric replacement of the amide bond by a 1,2,3-triazole moiety, in which different substitution patterns would mimic different amide rotamers. The syntheses of these restricted analogues have been carried out through an Ugi multicomponent reaction followed by an intramolecular cyclization. The unexpected formation of a β-lactam scaffold prompted us to study the course of the intramolecular cyclization of the Ugi adducts. In order to modulate this cyclization, a small library of compounds bearing both heterocyclic scaffolds has been synthesized and their activities as apoptosis inhibitors have been evaluated.

3-Deoxy-3,4-dehydro analogs of XM462. Preparation and activity on sphingolipid metabolism and cell fate.

Three analogs of the dihydroceramide desaturase inhibitor XM462 are reported. The compounds inhibit both dihydroceramide desaturase and acid ceramidase, but with different potencies depending on the N-acyl moiety. Other enzymes of sphingolipid metabolism, such as neutral ceramidase, acid sphingomyelinase, acid glucosylceramide hydrolase, sphingomyelin synthase and glucosylceramide synthase, are not affected. The effect on the sphingolipidome of the two best inhibitors, namely (R,E)-N-(1-hydroxy-4-(tridecylthio)but-3-en-2-yl)octanamide (RBM2-1B) and (R,E)-N-(1-hydroxy-4-(tridecylthio)but-3-en-2-yl)pivalamide (RBM2-1D), is in accordance with the results obtained in the enzyme assays. These two compounds reduce cell viability in A549 and HCT116 cell lines with similar potencies and both induced apoptotic cell death to similar levels than C8-Cer in HCT116 cells. The possible therapeutic implications of the activities of these compounds are discussed.

Inhibitory effect of positively charged triazine antagonists of prokineticin receptors on the transient receptor vanilloid type-1 (TRPV1) channel.

Four positively charged compounds, previously shown to produce analgesic activity by interacting with prokineticin receptor or T-type calcium channels, were tested for their ability to inhibit capsaicin-induced elevation of intracellular Ca(2+) in HEK-293 cells stably transfected with the human recombinant TRPV1, with the goal of identifying novel TRPV1 open-pore inhibitors. KYS-05090 showed the highest potency as a TRPV1 antagonist, even higher than that of the open-pore triazine inhibitor 8aA. The latter showed quite remarkable agonist/desensitizer activity at the rat recombinant TRPM8 channel. The activity of KYS-05090 and the other compounds was selective because none of these compounds was able to modulate the rat TRPA1 channel. Open-pore inhibitors of TRPV1 may be a new class of multi-target analgesics with lesser side effects, such as loss of acute pain sensitivity and hyperthermia, than most TRPV1 antagonists developed so far.

Synthesis and in vitro, ex-vivo and in vivo activity of hybrid compounds linking a potent ROS and RNS scavenger activity with diverse substrates addressed to pass across the blood-brain barrier

The synthesis of a small library of CR-6 (a potent ROS and RNS scavenger agent) derivatives bearing covalent linkage with different endogen nutrients that have specific transport through the blood-brain barrier (BBB) is reported. The synthetic sequence involved the preparation of a common precursor ester 6 derived from CR-6, which was easily converted into the carboxylic acid 7a or the amino derivative 11, for its further coupling with the required substrates through amide bonds. Antioxidant inxa0vitro (DPPH) and cellular assays (CAA) with the SH-S5SY cell line performed on these library members revealed that the couplings did not affect the antioxidant activity elicited by CR-6 itself. More interestingly, results from the intraperitoneal administration of selected library components in rats showed that compounds 2b, 2c and 2d were able to pass across the BBB. In particular, the amino acid compound 2d was the most penetrating derivative (15.8xa0±xa01.7xa0nmol/g brain with respect to the 4.0xa0±xa01.2xa0nmol/g brain found for the parent CR-6).

In situ synthesis of fluorescent membrane lipids (ceramides) using click chemistry

Ceramide analogues containing azide groups either in the polar head or in the hydrocarbon chains are non-fluorescent. When incorporated into phospholipid bilayers, they can react in situ with a non-fluorescent 1,8-naphthalimide using click chemistry giving rise to fluorescent ceramide derivatives emitting at ≈440 nm. When incorporated into giant unilamellar vesicles, two-photon excitation at 760 nm allows visualization of the ceramide-containing bilayers. This kind of method may be of general applicability in the study of model and cell membranes.

An Unexpected Access to a New Sphingoid Base Containing a Vinyl Sulfide Unit

An unexpected access to a new sphingoid base containing a vinyl sulfide unit is described. The process involves the one-pot regioselective opening of an epoxide with a thiolate, followed by intramolecular acyl transfer, base-promoted elimination, and final hydrolysis. Excess sodium hydride and high dilution conditions are required for optimal yields. The process is amenable to a variety of thiolates. The resulting compounds can be regarded as new hybrid sphingoid bases that combine some structural motifs present in other reported sphingolipid analogues.