Our group has developed several chemical probes and biochemical tools to monitor the activity of some of the enzymes of the sphingolipid metabolism. Please, contact us for a quotation.
1) Fluorogenic substrates to determine enzyme activity
Chemical probes for sphingosine-1-phosphate lyase (S1PL) activity
Sphingosine-1-phosphate lyase (SPL) is the last enzyme in the catabolism of sphingolipids. It catalyzes the retroaldol cleavage of long chain base phosphates into phosphoethanolamine and a fatty aldehyde. We have developed a new fluorescent substrate for this enzyme by incorporating a fluorescent reporter as part of the sphingoid base chain.
Sanllehí, P.; Casasampere, M.; Abad, J.-L.; Fabriàs, G.; López, O.; Bujons, J.; Casas, J.; Delgado, A. The First Fluorogenic Sensor for Sphingosine-1-Phosphate Lyase Activity in Intact Cells. Chem. Commun. 2017, 53 (39), 5441–5444.
Chemical probes for ceramidases
Ceramidases (CDases) are lipolytic amidohydrolases that catalyze the cleavage of ceramides into sphingosine and fatty acids. According to their optima pH, ceramidases are classified into acidic, neutral and alkaline, with different tissue distribution, subcellular localization and substrate specificity. We have developed a probe for the determination of acid CDase activity with applicability for the diagnosis of Farber disease, a rare disease resulting for the deficiency of this enzyme
Bedia, C.; Camacho, L.; Abad, J. L.; Fabrias, G.; Levade, T. A Simple Fluorogenic Method for Determination of Acid Ceramidase Activity and Diagnosis of Farber Disease. J Lipid Res 2010, 51 (12), 3542–3547.
Bedia, C.; Casas, J.; Garcia, V.; Levade, T.; Fabrias, G. Synthesis of a Novel Ceramide Analogue and Its Use in a High-Throughput Fluorogenic Assay for Ceramidases. ChemBioChem 2007, 8 (6), 642–648.
We have enlarged the scope of applicability of these probes with the discovery of RBM14C24:1 (selective for NC) and RBM15C18:1 selective for ACER1 and ACER2 with very low NC activity). These results represent a valuable addition to the available toolkit of fluorogenic CDases substrates for efficient HTS protocols for the screening of potential selective inhibitors of this relevant group of amidohydrolases.
Casasampere, M. et al, in J. Lipid Res. (Methods), 2019, jlr.D092759.
2) Azide-Tagged Sphingolipids for cellular trafficking studies
Investigation of the biological functions, localization and traffi cking of SLs requires their detection in their natural compartments, either the cell membranes or the inner organelle. In addition to the current available probes that incorporate a fluorescent reporter as part of the sphingoid backbone, we have developed minimally modified azidosphingolipids that allow a biorthogonal reaction with a suitable reporter at a later stage of the experiment.
Garrido, M.; Abad, J. L.; Alonso, A.; Goni, F. M.; Delgado, A.; Montes, L.-R. In Situ Synthesis of Fluorescent Membrane Lipids (Ceramides) Using Click Chemistry. J. Chem. Biol. 2012, 5 (3), 119–123.
Azido-tagged sphingolipids can also be used for the simultaneous, quantitative sphingolipidomics from different cell populations, as illustrated in this reference:
Garrido, M.; Abad, J. L.; Fabrias, G.; Casas, J.; Delgado, A. Azide-Tagged Sphingolipids: New Tools for Metabolic Flux Analysis. ChemBioChem 2015, 16 (4), 641–650.
3) Biochemical tools
Bodipy-SOCLAC: An activity-based imaging probe for acid ceramidase
Acid ceramidase (AC) hydrolyses ceramides into sphingoid bases and fatty acids. The enzyme is over-expressed in several types of cancer and Alzheimer’s disease, and its genetic defect causes different incurable disorders. Our group has developed Bodipy-SOCLAC, a fluorescent probe that allows the specific visualisation of catalytically active AC in intracellular compartments, which is crucial for the diagnosis and follow-up of therapeutic strategies in diseases linked to altered AC activity.
More details in: Ordóñez, Y. F.; et al. Activity-Based Imaging of Acid Ceramidase in Living Cells. J. Am. Chem. Soc. 2019, 141 (19), 7736–7742.
HPA12: CerT inhibitor
CerT is a cytosolic protein that mediates the transport of ceramide from the ER to the Golgi. HPA12 is a useful biochemical tool in cell biology studies to block the transport of this important lipid
For a review of the chemistry and the biology of HPA-12, see:
Berkeš, D.; Daïch, A.; Santos, C.; Ballereau, S.; Génisson, Y. Chemistry and Biology of HPAs: A Family of Ceramide Trafficking Inhibitors. Chem. – A Eur. J. 2016, 22 (49), 17514–17525.
We can also provide deuterium-labelled and other tagged sphingosines and ceramides upon request. The synthesis and characterization of other small molecules can also be undertaken. Please, contact us for additional information.